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What is the advantage of indirect ELISA over direct one?

What is the advantage of indirect ELISA over direct one?


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I guess the answer is about indirect one giving less error due to selectivity but how exactly does that happen?


You are correct, the selectivity advantage of an indirect or sandwich ELISA comes from the fact that two antibodies are employed - one to capture the analyte, the other to detect it.

Here is the classic illustration of how this type of ELISA works. First ([1]), the capture antibody is coated onto the plate and bound via one of a number of different types of chemistry. The plate is then washed (this occurs between all steps).[2], the analyte is added, sometimes in a homogenous solution (i.e., pure recombinant protein), other times in a matrix like serum, cell lysate, etc. The plate-bound antibody has a certain specificity for the analyte - let's say it binds the wrong epitope one time in 1000.[3]The detection antibody is added, and let's say it has the same specificity of 1/1000. Finally,[4]the secondary antibody (with the enzyme linked to it) is added, and[5]the enzyme's substrate is added, producing the color or light output.

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If we were to use a direct ELISA, the error rate would be 1/1000. However, by combining two antibodies, our error rate is now 1000 times lower - 1 in 1 million. Since both antibodies are required to bind properly in order to get a signal, this makes the results much more reliable.


It's often not necessarily an advantages vs disadvantages question, but one determined by available reagents and what question is being asked. For example, if you are developing a test for the antibody response to an antigen following immunization, you likely use a protocol like this:

  1. coat plates with antigen of interest (assume block / wash as appropriate going forward)
  2. add diluted serum from test animals / subjects
  3. add a labelled secondary antibody specific for this animal/subject

    • a labelled secondary antibody must be used because the animal's own antibody is not (of course) labelled.

if you are making your own labelled antibody in the lab and you have done this before you might do both a direct and indirect ELISA to verify that you have labeled the antibody and to determine the quality of the labeling. Assume you made a biotinylated mouse antibody.

do one direct ELISA (to quantitate label): 1. coat with target antigen 2. add dilution series of your antibody-biotin 3. add Streptavadin-Alkaline phosphatase to detect

a second indirect ELISA(to quantitate antibody): 1. coat with target antigen 2. add dilution series of your antibody-biotin 3. add biotinylated anti-mouse antibody 4. add Streptavadin-Alkaline phosphatase to detect

Now you know how much antibody (from the indirect ELISA) will give you how much signal (from the direct ELISA) against known amounts of your coating antigen.

One time you might actually have the option to do a direct or indirect assay is when you have all the reagents you need, but you need to amplify your signal sufficiently to detect low amounts. In that case a direct ELISA only has one antibody's worth of label, while coming in with a secondary (e.g. anti-heavy chain) can result in many antibodies worth of label per target.

Thermo has a nice page on this.


I agree with the other answers and the biggest difference is indeed the specificity. An indirect ELISA is indeed more specific, but also for a reason which isn't described here yet: Using indirect ELISA means your plate is coated with the primary antibody. Since this primary AB is attached to the well surface with its heavy chain, the 2 light chains (= the parts which bind antigens, in this case the secondary ABs ) are available to bind the secondary AB. This means that each 1 of the primary ABs has the potential to bind 2 secondary ABs, thus increasing the specific signal considerably.


Advantages of SPR over ELISA

ELISA, which stands for enzyme-linked immunosorbent assay, has been the gold standard for the quantification and detection of antibodies, peptides, proteins, and other biomolecules for the past 50 years. There are 3 main types of ELISA assays: direct, indirect, and sandwich. All of these approaches rely on a secondary reaction that generates a measurable signal to be detected by either a standard absorbance plate reader, spectrophotometer, fluorometer, or a luminometer. The main advantages of ELISA are high sensitivity and specificity. Take a standard sandwich ELISA assay, for instance. The use of avidin or streptavidin chemistry allows multiple enzymes (e.g. horseradish peroxidase) to be bound to the detection antibody, which leads to signal amplification of the biomolecule of interest. However, a sandwich ELISA has the following disadvantages:

Has long washing and incubation steps – takes hours to days to get results

Need to choose capture and detection antibodies wisely to prevent cross-reactivity

Need for labels or enzymes and substrates for indirect detection

Does not provide kinetic data – it has an endpoint detection

May wash away any low-affinity interactions of interest

Surface plasmon resonance (SPR), on the other hand, is an optical detection technique which is equally as sensitive and specific [1-3] that can address these issues. This blog will highlight the main advantages of SPR technology (Fig. 1) over sandwich ELISA (Fig. 2).

Figure 1 . Schematics of an SPR experiment.

Fig. 2 . Schematics of an ELISA experiment.


Custom ELISA Kits Services

What is ELISA? Enzyme-linked immunosorbent assay (ELISA) is a biochemical technique used to detect the presence of a specific antigen or antibody in a sample. The ELISA has maintained its popularity for over 40 years due to its high specificity, sensitivity, and reliability – characteristics that customers consistently cite for using the method.

ELISA Services and Test Process

ELISA Services and Test Methods

Direct ELISA

The direct version of the ELISA assay uses monoclonal antibodies to test for a specific antigen. This form of ELISA testing is used primarily in the immunohistochemical staining of tissues and cells.

Direct ELISA, when compared to other forms of ELISA testing, is performed quickly because only one antibody is employed. This assay can be used to test specific antibody-to-antigen reactions, and helps avoid complications due to cross-reactivity between other antibodies. Disadvantages The primary antibody must be labeled individually, which can be time-consuming and arduous when performing multiple experiments. Also, the signal is not as amplified in direct ELISA, compared with the indirect approach, which can be a disadvantage in some applications involving trace analyte detection.

Indirect ELISA Test

The indirect detection method uses a labeled secondary antibody for detection and is the most popular format for ELISA. The secondary antibody has specificity for the primary antibody. In a sandwich ELISA, it is critical that the secondary antibody is specific for the detection of the primary antibody only (and not the capture antibody) or the assay will not be specific for the antigen. Generally, this is achieved by using capture and primary antibodies from different host species (e.g., mouse IgG and rabbit IgG, respectively). For sandwich assays, it is beneficial to use secondary antibodies that have been cross-adsorbed to remove any antibodies that have affinity for the capture antibody. The most powerful ELISA assay format is the sandwich assay. This type of capture assay is called a “sandwich” assay because the analyte to be measured is bound between two primary antibodies – the capture antibody and the detection antibody. The sandwich format is used because it is sensitive and robust.

Competitive ELISA Test

The competitive ELISA is used to quantify antigen using a competition method. Briefly, the free antigen and antibody are incubated to form antigen-antibody complex and then the complex is added to an antigen-coated surface in the assay plate. The unbound antibody-antigen complex is washed off before adding enzyme-linked secondary antibody against the primary antibody. The substrate is then added and the antigen concentration is subsequently determined by the signal strength elicited by the enzyme-substrate reaction. In this assay, the enzyme-linked secondary antibody competes with the sample antigen, which is associated with the primary antibody.

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Four Types of ELISA

ELISA, short for enzyme-linked immunosorbent assay, is a very mature method for the detection of various targets. One advantage of ELISA is that it's quick and simple to carry out, so it is often used for both diagnostic and research purposes.

As its name implies, ELISA involves the use of enzymes and the specific binding of antibody and antigen. According to how it works, ELISA can be divided into four major types: direct, indirect, sandwich, and competitive. Let's see them one by one.

In direct ELISA, only an enzyme-labeled primary antibody is used, meaning that secondary antibodies are not needed. The enzyme-labeled primary antibody "directly" binds to the target (antigen) that is immobilized to the plate (solid surface). Next, the enzyme linked to the primary antibody reacts with its substrate to produce a visible signal that can be measured. In this way, the antigen of interest is detected.

In indirect ELISA, both a primary antibody and a secondary antibody are used. But in this case, the primary antibody is not labeled with an enzyme. Instead, the secondary antibody is labeled with an enzyme.

The primary antibody binds to the antigen immobilized to the plate, and then the enzyme-labeled secondary antibody binds to the primary antibody. Finally, the enzyme linked to the secondary antibody reacts with its substrate to produce a visible signal that can be measured.

In direct and indirect ELISA, it is the antigen that is immobilized to the plate. In sandwich ELISA, however, it is the antibody that is immobilized to the plate, and this antibody is called capture antibody. In addition to capture antibody, sandwich ELISA also involves the use of detection antibodies, which generally include the unlabeled primary detection antibody and the enzyme-labeled secondary detection antibody.

Firstly, the antigen of interest binds to the capture antibody immobilized to the plate. Secondly, the primary detection antibody binds to the antigen. Thirdly, the secondary detection antibody binds to the primary detection antibody, and then the enzyme reacts with its substrate to produce a visible signal that can be measured.

More details about direct ELISA protocol, indirect ELISA protocol, and sandwich ELISA protocol, please check it here.

Compared with the three ELISA types above, competitive ELISA is relatively complex because it involves the use of inhibitor antigen, so competitive ELISA is also known as inhibition ELISA. In fact, each of the three formats, direct, indirect, and sandwich, can be adapted to the competitive format. In competitive ELISA, the inhibitor antigen and the antigen of interest compete for binding to the primary antibody. Here is a procedure of competitive ELISA:

Firstly, the unlabeled primary antibody is incubated with the sample containing the antigen of interest, leading to the formation of antigen-antibody complex (Ag-Ab). In this step, the antibody is excessive compared with the antigen, so there are free antibodies left.

Secondly, the Ag-Ab mixture is added to the plate coated with inhibitor antigen that can also bind to the primary antibody. The free primary antibody in the mixture binds to the inhibitor antigen on the plate, while the Ag-Ab complexes in the mixture do not and are therefore washed off.

Thirdly, the enzyme-labeled secondary antibody is added to the plate and binds to the primary antibody bound to the inhibitor antigen on the plate.

Finally, a substrate is added to react with the enzyme and emit a visible signal for detection.

Through this procedure, you may find that the final signal is inversely associated with the amount of the antigen of interest in the sample, meaning that the more antigen in the sample, the weaker the final signal. This is because primary antibodies bound to sample antigen will be washed off, while free primary antibodies left will be captured by inhibitor antigen immobilized to the plate and be measured by an enzymatic reaction.

Competitive ELISA described here is based on antibody capture, in which the plate is coated with antigen. There is another type of competitive ELISA that is based on antigen capture, in which the plate is coated with unlabeled antibody. Furthermore, competitive ELISA generally uses a labeled antibody for detection, but sometimes it uses labeled antigen instead of a labeled antibody.

Comparison of direct, indirect, sandwich, and competitive ELISA

Now we know how the four most common types of ELISA work, but how to choose the right type for your experiment? To find the answer, you need to understand the advantages and disadvantages of each ELISA type.

Table 1. Advantages and disadvantages of each ELISA type

  1. Simple protocol, time-saving, and reagents-saving.
  2. No cross-reactivity from secondary antibody.
  1. High background.
  2. No signal amplification, since only a primary antibody is used and a secondary antibody is not needed.
  3. Low flexibility, since the primary antibody must be labeled.
  1. Signal amplification, since one or more secondary antibodies can be used to bind to the primary antibody.
  2. High flexibility, since the same secondary antibody can be used for various primary antibodies.
  1. Complex protocol compared with direct ELISA.
  2. Cross-reactivity from secondary antibody.
  1. High flexibility.
  2. High sensitivity.
  3. High specificity, since different antibodies bind to the same antigen for detection.
  1. The antigen of interest must be large enough so that two different antibodies can bind to it at different epitopes.
  2. It's sometimes difficult to find two different antibodies that recognize different epitopes on the antigen of interest and cooperate well in a sandwich format.
  1. High flexibility.
  2. High sensitivity.
  3. Best for the detection of small antigens, even when they are present in low concentrations.
  1. Relatively complex protocol.
  2. Needs the use of inhibitor antigen.

In addition to the four most common ELISA types above, there are other ELISA types that help meet the various demands of experiments. Here are two examples:

ELISPOT, short for enzyme-linked immunospot assay, is used to measure the frequency of protein-secreting cells at the single-cell level. The technique that ELISPOT uses is very similar to that of sandwich ELISA.

In-cell ELISA is used to measure the levels of the target protein within cells that are fixed on the plate. It also involves the use of the technique used by sandwich ELISA.

First, cells are fixed to the plate and are permeablized. Next, a primary antibody is added to react with the target protein within the cells. Finally, a labeled secondary antibody is added to react with the primary antibody. In this way, the target protein within cells is detected.

Quantitative and qualitative ELISA

On the basis of whether ELISA can quantify the level of the target molecule, ELISA can be divided into two types, qualitative and quantitative. Qualitative ELISA provides a simple positive or negative result for a sample, while quantitative ELISA reflects the concentration of the target molecule in a sample via a standard curve. So, if you want to quantify the target molecule level, choose quantitative ELISA.

ELISA is used for both diagnostic and research purposes. Diseases detected by ELISA include HIV, HBV, influenza, Hemolytic Anemia, Lyme disease, food allergy, and so on. Currently, there is a large number of ELISA kits supplied by manufacturers worldwide. But some ELISA kits are only used in research and cannot be used in diagnosis. Cusabio is one of the manufacturers offering ELISA kits for research use. How to choose the right ELISA kit for your research? Please read this article: 11 tips for choosing your right ELISA kit.

You can also develop your own ELISA if there are no ELISA kits commercially available for your research. During ELISA development, the antibody selection is of critical importance. Many factors such as the affinity, specificity, and titer of the antibody must be taken into consideration.

Besides that, here are some common problems of ELISA for you, and we hope that can give you help.


The Key Benefits of Indirect Detection

Even though the direct detection method is becoming more popular for immunofluorescence (IF) and flow cytometry experiments, the indirect detection method still remains the preferred choice for many other applications. In direct detection, the labeled primary antibody is responsible for both binding and detection of the antigen of interest. In indirect detection, this process is broken down into at least two distinct steps – (i) an unconjugated primary antibody forms a complex with the antigen, (ii) a labeled secondary antibody, interacting with the constant region of the primary antibody, facilitates detection.

HAI-1 was detected in paraffin-embedded sections of human lung cancer using goat anti-human HAI-1 ectodomain antigen affinity-purified polyclonal antibody (Catalog # AF1048) followed by anti-goat IgG VisUCyte HRP polymer antibody (Catalog # VC004). Tissue was stained with DAB (brown) and counterstained with hematoxylin (blue).

Enhanced Sensitivity
One of the main reasons for using the indirect method is an increase in the lower limit of detection. Since two or more labeled secondary antibodies are able to bind a single primary antibody, the result is an amplification in signal and an increase in assay sensitivity. This is particularly important for low abundance antigens which may require additional signal amplification steps to generate an appreciable signal over background staining. One option for signal amplification using chromogenic detection is an HRP polymer conjugated secondary antibody in which multiple HRP molecules are associated with each secondary antibody. Learn more about signal amplification methods from our IHC Detection page.

Greater Flexibility
With the indirect method, researchers aren&rsquot restricted by the range of commercially available conjugated primary antibodies nor are they limited to using a particular label for an experiment. Instead of worrying about choosing the best label, e.g. FITC or PE, for the primary antibody, the primary antibody can be paired with different conjugated secondary antibodies from the same species. You can also easily shift from a colorimetric to a fluorescent assay when working with an unconjugated primary antibody.

Studying a novel antigen or using a new antibody introduces a number of unknowns such as antigen abundance and successful antigen-antibody binding. The indirect method of detection may be a more suitable starting point for your experimental design. Your lab may already have an inventory of conjugated secondary reagents that you can use for preliminary testing.

Conjugated secondary antibodies enrich the detection of low abundance target molecules and allow greater freedom in choosing detection reagents. Novus Biologicals has an extensive list of conjugated secondary antibodies available for your indirect detection needs.


ELISA advantages and disadvantages

Advantages

  • High sensitivity and specificity: it is common for ELISAs to detect antigens at the picogram level in a very specific manner due to the use of antibodies.
  • High throughput: commercial ELISA kits are normally available in a 96-well plate format. But the assay can be easily adapted to 384-well plates.
  • Easy to perform: protocols are easy to follow and involve little hands-on time.
  • Quantitative: it can determine the concentration of antigen in a sample.
  • Possibility to test various sample types: serum, plasma, cellular and tissue extracts, urine, and saliva among others.

Disadvantages

  • Temporary readouts: detection is based on enzyme/substrate reactions and therefore readout must be obtained in a short time span.
  • Limited antigen information: information limited to the amount or presence of the antigen in the sample.

What is the advantage of indirect ELISA over direct one? - Biology

Once proteins have been separated by gel electrophoresis and transferred onto a blotting membrane, Western blotting can be performed in either one or two steps.

Direct – One step method

In the direct detection method (Figure 1 (A)), a primary antibody directly conjugated to a reporter enzyme or fluorescent dye is used to detect the protein antigen on the blotting membrane after a single incubation step. Although this one step method is quicker, it is not widely used. Figure 1(B) illustrates one of the problems of directly conjugating the primary antibody, whereby the reporter molecule can occlude the antigen binding region of the primary antibody, preventing good recognition of the antigen and leading to reduced sensitivity. Excess primary may compensate for this effect but may lead to poor reproducibility and increased background.

Indirect – Two step method

The two step, indirect detection method of Western blotting avoids such interference with antigen detection. An unlabeled primary antibody forms a complex with the antigen bound to the blot membrane. After washing, the blot is incubated with a secondary antibody conjugated with the reporter enzyme or fluorophore. Multiple secondary antibodies bind to epitopes on the primary antibody, creating a labeled antigen-antibody complex (Figure 1(C)). Although the indirect method requires one more wash and incubation step, it presents numerous advantages over the direct method, including amplification of signal and flexibility.

Figure 1: (A) Directly conjugated primary antibody binds antigen bound to membrane. (B) Directly conjugated primary antibody binds poorly to antigen bound to membrane, since reporter enzyme or fluorophore can conjugate into antigen-binding region and reduce affinity of the antibody for its target. Using a secondary eliminates this. (C) Indirect method – multiple conjugated secondary antibodies bind to an unconjugated primary antibody.

The advantages and disadvantages of the direct and indirect detection methods are detailed in the table below. They apply to Western blotting but may also be relevant to other techniques, such as IHC/ICC, ELISA and Flow Cytometry.

Table 1: Advantages and disadvantages of direct and indirect Western blotting methods.

Direct Detection Method Indirect detection method
Disadvantages Advantages Disadvantages Advantages
Time Labeling individual primary antibodies is time-consuming. One incubation step means that direct detection methods can be rapid. The addition of the secondary antibody incurs extra steps. Many reliable commercially available labeled secondaries reduce time consuming optimization steps.
Flexibility and availability The number of conjugated primary antibodies is limited. Secondary antibodies offer a wide range of conjugate options and antibody specificities.
Sensitivity The signal from direct method may appear weaker than indirect method. Signal amplification – each primary antibody can accommodate multiple secondary antibodies, increasing the number of molecules available to produce signal.
Immunoreactivity Primary antibodies may have reduced immunoreactivity due to conjugate interference with antigen binding. Immunoreactivity of the primary antibody is preserved, and signal is provided by the conjugated secondary.
Cost Conjugated primary antibodies may be expensive, and more antibody may be required to counteract loss of sensitivity. One secondary antibody can be used to detect any primary antibody raised in the same host species, reducing cost.
Signal enhancement options Sample concentration may be required for low abundance proteins prior to loading. Secondary antibodies can be used to enhance signal in numerous ways.

Alberts B et al (1994) Molecular biology of the Cell. 3rd Ed. Garland press. London


What is Competitive ELISA?

Competitive ELISA measures the antigen concentration in a sample through the detection of signal interference. Here, the assay uses an inhibitor antigen. Hence, it is a type of inhibition ELISA. During this procedure, the antigens present in the sample compete with the selected reference antigen for binding to a specific amount of labeled antibody. Furthermore, this procedure initiates with the incubation of the sample, with the excess amount of labeled antibody. Also, the reference antigen should be pre-coated on a multiple well assay plate. Then the sample mixture should be added to the assay plate which contains the reference antigen. Free antibodies will bind to the reference antigen depending on the amount of antigen in the sample. Hence, if more sample antigen is present, less reference antigen will be detected. Thus, it creates a weaker signal.

Figure 01: ELISA

In contrast, when the sample contains less amount of antigen, more and more reference antigen will occupy the antibodies and give a strong signal. Since competitive ELISA gives a stronger signal when the sample contains a low amount of antigens, competitive ELISA is a very sensitive assay, even for samples with a small number of antigens.

In some competitive ELISA kits, a labeled antigen is used instead of a labeled antibody. Here, the labeled antigen and the sample antigen will compete for binding to the primary antibody. Similarly, when the amount of antigen in the sample is low, the amount of labeled antigen that binds to antibodies will be higher and create a stronger signal.


Immunofluorescence protocol (IF protocol)

Immunofluorescence is one of the widely used techniques in modern biology and medicine, and it is developed by Coons et al. (1950), and it is a combination of immunofluorescence technique and morphological technology to develop immune fluorescent cells (or tissue). The development of fluorescence immunoassay technology is very fast in recent years, especially in the field of medical, biological and environmental studies, it is widely used in the determination of endocrine hormones, growth factors, proteins, nucleic acids, neurotransmitters, receptors, in vivo drug and infectious sources, and so on, these subjects have been developed. According to the detection of different samples can be divided into three major categories.
There are two different immunofluorescence assay which include indirect immunofluorescence assay and direct immunofluorescence assay.For indirect immunofluorescence assay, the protocol mainly include tissue or cell preparation, tissue or cell fixation, serum blocking, primary antibody incubation, marked second antibody incubation, staining, result judgment and imaging. For direct immunofluorescence assay, there are only marked primary antibody been incubated without second antibody and other steps are same.
For direct immunofluorescence assay, specific fluorescent antibody was prepared by the combination of specific antibodies and fluorescein. It is the most simple and fast method for the examination of cell or tissue antigen. This method is highly specific and is commonly used in renal biopsy and pathogen examination. Its disadvantage is that a fluorescent antibody can only examine one kind of antigen, which is less sensitive. For indirect immunofluorescence assay, specific antibodies against the corresponding antigen, fluorescein labeled anti - antibody (anti - specific IgG fluorescent antibody) and the primary antibody
Although the basic steps and principles of immune fluorescence are the same, but because of the specific conditions are not the same, the detailed operation steps of each laboratory will not be exactly the same. For example, the use of the solution, the fixed liquid and antibody dilution liquid will be slightly different. Here to give a more common method, the detailed use of the operation can be based on the steps to adjust and change, and ultimately determine the most appropriate method for youself.

Indirect Immunofluorescence / IF

1. Prepare tissue or culture cells
2. Prepare tissue section or cells coverlip
3. Wash samples two times with PBS
4. Fix amples with 4% paraformaldehye in PBS for 15 min at room temperature(Note: Paraformaldehye is toxic, use only in fume hood)
5. Aspirate fixative, rinse two times in PBS for 5 min each
6. Permeabilize samples with 0.1-0.5% triton x-100 in PBS for 10 min(Note: Permeabilization is only required when the antibody needs access to the inside of the cells to detect the protein. These include intracellular proteins and transmembrane proteins whose epitopes are in the cytoplasmic region.
7. Aspirate triton x-100, rinse two times in PBS for 5 min each
8. Incubate samplels in 10% normal goat serum in PBS for 30 min at room temperature
9. Aspirate goat serum, incubate sections with primary antibody at appropriate dilution in PBS overnight at 4°C or 1 hour at 37°C(optimal condition should be confirmed in different laboratory)
10. Rinse three times in PBS for 5 min each
11. Incubate samplels with fluorochrome-conjugated secondary antibody at appropriate dilution in PBS for 1 hour at 37°C in dark(optimal condition should be confirmed in different l aboratory)
12. Rinse three times in PBS for 5 min each in dark
13. Incubate samples with 1 μg/ml DAPI
14. Mount samples with a drop of mounting medium

Direct Immunofluorescence / IF

1. Prepare tissue or culture cells
2. Prepare tissue section or cells coverlip
3. Wash samples two times with PBS
4. Fix amples with 4% paraformaldehye in PBS for 15 min at room temperature(Note: Paraformaldehye is toxic, use only in fume hood)
5. Aspirate fixative, rinse two times in PBS for 5 min each
6. Permeabilize samples with 0.1-0.5% triton x-100 in PBS for 10 min(Note: Permeabilization is only required when the antibody needs access to the inside of the cells to detect the protein. These include intracellular proteins and transmembrane proteins whose epitopes are in the cytoplasmic region.
7. Aspirate triton x-100, rinse two times in PBS for 5 min each
8. Incubate samplels in 10% normal goat serum in PBS for 30 min at room temperature
9. Aspirate goat serum, incubate sections with fluorochrome- conjugated primary antibody at appropriate dilution in PBS overnight at 4°C or 1 hour at 37°C(optimal condition should be confirmed in different laboratory)
10. Rinse three times in PBS for 5 min each in dark
11. Incubate samples with 1 μg/ml DAPI
12. Mount samples with a drop of mounting medium


ELISA Assays: Indirect, Sandwich, and Competitive

Source: Whitney Swanson 1,2 , Frances V. Sjaastad 2,3 , and Thomas S. Griffith 1,2,3,4
1 Department of Urology, University of Minnesota, Minneapolis, MN 55455
2 Center for Immunology, University of Minnesota, Minneapolis, MN 55455
3 Microbiology, Immunology, and Cancer Biology Graduate Program, University of Minnesota, Minneapolis, MN 55455
4 Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455

Enzyme-linked immunosorbent assay (ELISA) is frequently used to measure the presence and/or concentration of an antigen, antibody, peptide, protein, hormone, or other biomolecule in a biological sample. It is extremely sensitive, capable of detecting low antigen concentrations. The sensitivity of ELISA is attributed to its ability to detect the interactions between a single antigen-antibody complex (1). Moreover, the inclusion of an enzyme-conjugated antigen-specific antibody permits the conversion of a colorless substrate into a chromogenic or fluorescent product that can be detected and easily quantitated by a plate reader. When compared to the values generated by titrated amounts of a known antigen of interest, the concentration of the same antigen in the experimental samples can be determined. Different ELISA protocols have been adapted to measure antigen concentrations in a variety of experimental samples, but they all have the same basic concept (2). Choosing the type of ELISA to perform, indirect, sandwich, or competitive, depends on a number of factors, including the complexity of the samples to be tested and the antigen-specific antibodies available to use. The indirect ELISA is frequently used to determine the outcome of an immunological response, such as measuring the concentration of an antibody in a sample. The sandwich ELISA is best suited for analyzing complex samples, such as tissue culture supernatants or tissue lysates, where the analyte, or antigen of interest, is part of a mixed sample. Finally, the competitive ELISA is most often used when there is only one antibody available to detect the antigen of interest. Competitive ELISAs are also useful for detecting a small antigen with only a single antibody epitope that cannot accommodate two different antibodies due to steric hinderance. The protocol will describe the basic procedures for the indirect, sandwich, and competitive ELISA assays.

The indirect ELISA assay is commonly used to measure the amount of antibodies in serum or in the supernatant of a hybridoma culture. The general procedure for the indirect ELISA assay is:

  1. Coat wells with antigens
  2. Add serum or hybridoma culture supernatant containing antibody (primary or 1° antibody)
  3. Incubate and wash
  4. Add secondary (or 2°) enzyme-conjugated antibody
  5. Incubate and wash
  6. Add substrate

The sandwich ELISA assay differs from the indirect ELISA assay in that the method does not involve coating the plates with a purified antigen. Instead, a "capture" antibody is used to coat the wells of the plate. The antigen is "sandwiched" between the capture antibody and a second "detection" enzyme-conjugated antibody - where both antibodies are specific for the same antigen, but at different epitopes (3). By binding to the capture antibody/antigen complex, the detection antibody remains in the plate. Either monoclonal antibodies or polyclonal antisera can be used as the capture and detection antibodies. The main advantage of the sandwich ELISA is that the sample does not have to be purified before analysis. Moreover, the assay can be quite sensitive (4). Many commercially available ELISA kits are of the sandwich variety and use tested, matched pairs of antibodies. The general procedure for the sandwich ELISA assay is:

  1. Coat wells with capture antibody
  2. Add test samples containing antigen
  3. Incubate and wash
  4. Add enzyme-conjugated detection antibody.
  5. Incubate and wash
  6. Add substrate

Most commercially available sandwich ELISA kits come with enzyme-conjugated detection antibodies. In cases where an enzyme-conjugated detection antibody is not available, a secondary enzyme-conjugated antibody specific for the detection antibody can be used. The enzyme on the secondary antibody performs the same role, which is to convert the colorless substrate to a chromogenic or fluorescent product. The above-mentioned secondary enzyme-conjugated antibody would more like to be used in a "homemade" sandwich ELISA developed by an investigator who has generated their own monoclonal antibodies, for example. One drawback to using a secondary enzyme-conjugated antibody is to be sure it only binds to the detection antibody, and not the capture antibody bound to the plate. This would result in a measurable product in all wells, regardless of the presence or absence of antigen or detection antibody.

Finally, the competitive ELISA assay is used to detect soluble antigens. It is simple to perform, but it is only suitable when the purified antigen is available in a relatively large amount. The general procedure for the competitive ELISA assay is:

  1. Coat wells with antigen
  2. Incubate and wash
  3. Preincubate test sample with enzyme-conjugated primary antibodies
  4. Add mixture to well
  5. Incubate and wash away any unbound enzyme-conjugated primary antibody
  6. Add substrate

The "competition" in this assay comes from the fact that more antigen in the test sample used in step 3 will result in less antibody available to bind to the antigen coating the well. Thus, the intensity of the chromogenic/fluorogenic product in the well at the end of the assay is inversely related to the amount of antigen present in the test sample.

A key component in any type of ELISA is the titrated standards of known concentrations that will allow the user to determine the antigen concentration present in the test samples. Typically, a series of wells are designated for creating a standard curve, where known amounts of a purified recombinant protein are added to the wells in decreasing amounts. When these wells are processed at the same time as the test samples, the user can then have a reference set of absorbance values obtained from a microplate reader for known protein concentrations to go along with the absorbance values for the test samples. The user can then calculate a standard curve to which the test samples can be compared for determining the amount of protein of interest present. The standard curve can also determine the degree of precision of the user's dilution making.

Finally, the last step in each of the ELISA types listed above calls for the addition of a substrate. The degree of conversion of the substrate to product is directly related to the amount of enzyme present in the well. Horseradish peroxidase (HRP) and alkaline phosphatase (AP) are the most common enzymes found conjugated to antibodies. As expected, there are a number of substrates available specific for either enzyme that produce a chromogenic or fluorescent product. Moreover, substrates are available in a range of sensitivities that can increase the overall sensitivity of the assay. The user must also take into consideration the type of instrumentation available for reading the plate at the end of the experiment when picking the type of substrate to use, along with its corresponding enzyme-conjugated antibody.

Commonly used chromogenic substrates for HRP include 2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS) and 3,3',5,5'-tetramethylbenzidine (TMB), whereas p-Nitrophenyl Phosphate (PNPP) is used for AP. ABTS and TMB produce water-soluble green and blue colored reaction products, respectively. The green ABTS product has two major absorbance peaks, 410 and 650 nm, while the blue TMB product is best detected at 370 and 652 nm. The colors of ABTS and TMB change to yellow upon the addition of an acidic stop solution, which is best read at 450 nm. Color development for ABTS is slow, while it is fast for TMB. TMB is more sensitive than ABTS, and may produce a higher background signal if the enzymatic reaction proceeds too long. PNPP produces a yellow water-soluble product after AP conversion that absorbs light at 405 nm.

Procedure

1. Indirect ELISA

An indirect ELISA is one where the primary antigen-specific antibody is recognized by a secondary conjugated antibody. The following protocol is an example of an indirect ELISA method, where the serum samples of of influenza A virus (IAV)-infected mice are tested for the presence of IAV-specific IgG antibody. One strength of this example is that different secondary antibodies can be used that recognize all antibody isotypes or specific isotypes (e.g., IgG).

Coating antigen to the microplate

  1. Coat the wells of a 96-well ELISA plate with purified antigen by pipetting 50 µL of purified antigen (2 mg/mL of purified A/PR/8 Influenza A virus in 0.05M Tris-HCl buffer (pH 9.5)) into each well of the plate.
  2. Cover the plate with an adhesive cover and incubate it overnight at 4°C to allow the antigen to bind to the plate.
  3. Upon incubation completion, remove the coating solution by flicking the plate over a sink.
  1. Block the remaining protein-binding sites in the coated wells by adding 200 µL blocking buffer, 5% donkey serum in 1X PBS is used here, per well. Alternative blocking reagents include 5% non-fat dry milk or BSA in PBS or normal serum from an animal in which the secondary antibody was generated.
  2. Incubate for at least 2 hours at room temperature or overnight at 4°C.
  3. Following the incubation, remove the blocking buffer by flicking the plate and then wash plate with PBS containing 1% Tween-20.

Incubation with the primary antibody

  1. Prepare a serial dilution of the serum sample, which contains the primary antibody, to obtain a dilution range of 1 to 204,800, using 1X PBS. To do this, first dilute the serum 1:12.5 and then perform a 4X dilution (dilution range - 1:12.5 to 1:204,800).
  2. Add 100 µL of the serially-diluted serum samples to the wells.
  3. Cover plate with adhesive cover and incubate at room temperature for 1-2 h.
  4. Following the incubation, flick the plate over a sink and wash plate with PBS containing 1% Tween-20.

Incubation with the secondary antibody

  1. Add 100 µL of an enzyme-conjugated secondary antibody, horseradish peroxidase, HRP-conjugated donkey anti-mouse secondary in this experiment, to each well.
  2. Incubate the plate for 1 hour at room temperature.
  3. Following the incubation, flick the plate over a sink and then wash plate with PBS containing 1% Tween-20.
  1. Add 100 µL of the indicator substrate (3,3',5,5'-tetramethylbenzidine (TMB)) at a concentration of 1 mg/mL to each well.
  2. Incubate the plate with the substrate for 5-10 min at room temperature.
  3. After 10 min, stop the enzymatic reaction by adding 100 µL 2N Sulfuric acid (H2SO4).
    Within 30 min of adding the stop solution, read the plate using a microplate reader at 405 nm to determine the absorbance of the wells.

2. Sandwich ELISA

In this ELISA version, the experimental sample is "sandwiched" between an unconjugated capture antibody and a conjugated detection antibody, both of which are specific to the same protein but at different epitopes. In the following sandwich ELISA example, concentration of human TNFα was determined in unknown sample using a standard curve generated from 2.5X serial dilution of a known standard, recombinant human TNFα (stating at concentration of 75 pg/mL).

Coating capture antibody to the microplate

  1. Coat the wells of a 96-well ELISA plate with purified capture antibody by adding 100 µL of capture antibody (1-10 µg/mL range) to each well of the plate.
  2. Cover plate with an adhesive plate cover and incubate it overnight at 4°C.
  3. After incubation, remove the coating solution from plate by flicking the plate over a sink.
  1. Block the remaining protein-binding sites in the antibody coated wells by adding 200 µL blocking solution, 5% nonfat dry milk containing PBS, to the wells.
  2. Incubate for at least 2 h at room temperature or overnight at 4°C.
  3. Following the incubation, remove the blocking buffer by flicking the plate and then wash plate with PBS containing 1% Tween-20.

Add antigen containing test samples

  1. Add 100 µL of the test sample to the wells. Seal the plate with an adhesive cover.
  2. Incubate for 1-2 h at room temperature or overnight at 4°C.
  3. After incubation, remove the samples by flicking the plate over the sink and then wash the wells with 200 µL 1X PBS containing 1% Tween-20.

Add enzyme-conjugated detection antibody

  1. Add 100 µL of enzyme-conjugated detection antibody to the wells at a preoptimized concentration.
  2. Seal the plate with an adhesive cover and incubate at room temperature for 2 h.
  3. Remove the unbound detection antibody by flicking the plate over a sink and wash the wells with 200 µL 1X PBS containing 1% Tween-20.
  1. Add 100 µL of the indicator substrate at a concentration of 1 mg/mL. Any bound enzyme-conjugated detection antibody will convert the substrate to a detectable signal.
  2. Incubate the plate for 5-10 min at room temperature.
  3. After 5-10 min, stop the enzymatic reaction by adding 100 µL 2N H2SO4 to the wells. Within 30 min of adding the stop solution, read the plate using a microplate reader to determine the absorbance of the wells.

3. Competitive ELISA

The steps of a competitive ELISA are different from those used in indirect and sandwich ELISA, with the main difference being the competitive binding step between the sample antigen and the "add-in" antigen. The sample antigen is incubated with the unlabeled primary antibody. These antibody-antigen complexes are then added to the ELISA plate, which has been pre-coated with the same antigen. After an incubation period, any unbound antibody is washed away. There is an inverse correlation between the amount of free antibody available to bind the antigen in the well and the amount of antigen in the original sample. For example, a sample with abundant antigen would have more antigen-primary antibody complexes, leaving little unbound antibody to bind to the ELISA plate. An enzyme-conjugated secondary antibody specific to the primary antibody is then added to the wells, followed by the substrate.

Coating antigen to the microplate

  1. Coat the wells of a 96-well ELISA plate with 100 μL of purified antigen at a concentration of 1-10 μg/mL.
  2. Cover plate with an adhesive plate cover and incubate the plate overnight at 4°C.
  3. Following incubation, remove the unbound antigen solution from the wells by flicking the plate over a sink.
  1. Block the remaining protein-binding sites in the coated wells by adding 200 μL of blocking buffer to each well, which can be either 5% non-fat dry milk or BSA in PBS.
  2. Incubate the plate for at least 2 h at room temperature or overnight at 4°C.

Incubation sample (antigen) with the primary antibody

  1. While blocking the wells, prepare the antigen-antibody mixture by mixing 150 μL sample antigen and 150 μL of primary antibody for each well in the assay.
  2. Incubate this mixture for 1 h at 37°C.

Add antigen-antibody mixture to the well

  1. Now, remove the blocking buffer from the wells by flicking the plate over a sink.
  2. Then, wash the wells with 1X PBS containing Tween-20.
  3. Add 100 μL of the sample antigen-primary antibody mixture.
  4. Incubate the plate at 37°C for 1 h.
  5. Remove the sample mixture by flicking the plate over a sink.
  6. Then, wash the wells with 1X PBS containing 1% Tween-20 to remove any unbound antibody.

Add the secondary antibody

  1. Add 100 μL of an enzyme conjugated secondary antibody, which in this case is AP-conjugated antibody, to each well.
  2. Incubate the plate for 1 h at 37°C.
  3. Following incubation, wash the plate with 1X PBS containing 1% Tween-20.
  1. Add 100 μL of the substrate solution to each well.
  2. Wait for 5-10 min.
  3. After 10 min, stop the enzymatic reaction by adding 100 μL 2N sulfuric acid to the wells. Then, measure the absorbance in a microplate reader within 30 min of adding the stop solution

Enzyme-linked Immunosorbent Assay, or ELISA is a highly sensitive quantitative assay commonly used to measure the concentration of an analyte like cytokines and antibodies in a biological sample. The general principle of this assay involves three steps: starting with capture, or immobilization, of the target analyte on a micro plate, followed by the detection of the analyte by target-specific detection proteins, and lastly, enzyme reaction, where a conjugated enzyme converts its substrate to a colored product. Based on different methods of capture and detection, ELISA can be of four types: direct, indirect, sandwich, and competitive.

For direct ELISA, the target antigen is first bound to the plate, and is then detected by a specific detection antibody. This method is commonly used for screening antibodies for a specific antigen. Indirect ELISA is used for detecting antibodies in a sample in order to quantify immune responses. The plate is first coated with a specific capture antigen, which immobilizes the target antibody, and this antigen-antibody complex is then detected using a second antibody.

In the case of sandwich ELISA, the target analyte is an antigen, which is captured on the plate using a capture antibody and then detected by the detection antibody, hence forming an antibody-antigen-antibody sandwich. This method is useful for measuring the concentration of an antigen in a mixed sample.

Competitive ELISA is used when only one antibody is available for a target antigen of interest. The plate is first coated with the purified antigen. Meanwhile, the sample containing the antigen is pre-incubated with the antibody and then added to the plate, to allow any free antibody molecules to bind to the immobilized antigen. The higher the signal from the plate, the lower the antigen concentration in the sample. In all of the four types of ELISA, direct, indirect, sandwich, and competitive, the detection antibody is either directly conjugated to the enzyme or can be indirectly linked to it through another antibody or protein.

The enzymes commonly used for the reaction are horseradish peroxidase or alkaline phosphatase with their respective substrates, both producing a soluble, colored product that can be measured and quantified using a plate reader. In this video, you will observe how to perform indirect ELISA, sandwich ELISA, and competitive ELISA, followed by examples of quantification of the target analyte from the indirect and sandwich ELISA methods.

The first experiment will demonstrate how to use indirect ELISA to determine the presence of anti-influenza virus antibodies in serum obtained from influenza-infected mice.

To begin, add 50 microliters of purified antigen - in this case, 2 milligrams per milliliter of purified A/PR/8 Influenza A virus- to each well of a 96-well ELISA plate. Next, cover the plate with an adhesive cover and incubate it overnight at 4 degrees celsius to allow the antigen to bind to the plate. The following day, remove the coating solution by flicking the plate over a sink. Next, block the remaining protein-binding sites in the coated wells by adding 200 microliters of a blocking buffer- here, 5% donkey serum in 1X PBS- to each well. Leave the plate to incubate for at least 2 hours at room temperature. Following the incubation, remove the blocking buffer and then wash the plate by adding 200 microliters of 1X PBS containing 1% Tween-20. Flick the plate over the sink once more to remove the wash.

Then, prepare the test samples by adding 460 microliters of PBS to a fresh tube, and then adding 40 microliters of serum to make a 1 to 12.5 dilution. Then, add 300 microliters of PBS to a second tube, and then add 100 microliters of the first dilution. Continue this serial dilution range until obtaining a final sample with a dilution of 1 to 204,800. Add the serially diluted serum samples in triplicate to the wells. Cover the plate with an adhesive cover and incubate at room temperature for an hour. Next, remove the samples by flicking the plate into the sink and then wash the plate by adding 200 microliters of 1X PBS containing 1% Tween-20. Once again, flick the plate to remove the wash.

Now, add 100 microliters of an enzyme-conjugated secondary antibody, which in this experiment is a horseradish peroxidase, or HRP, conjugated donkey anti-mouse secondary, to each well. Incubate the plate for one hour at room temperature, and flick the plate to remove any excess liquid. Wash the plate with 1X PBS containing 1% Tween-20 and then apply 100 microliters of the indicator substrate at a concentration of one milligram per milliliter to each well. Incubate the plate with the substrate for 5 to 10 minutes at room temperature. In this example, the colorless 3,3', 5,5' - tetramethylbenzidine, or TMB, substrate turns a blue color when HRP is present. After 10 minutes, stop the enzymatic reaction by adding 100 microliters of 2N sulfuric acid. The samples will turn a yellow color.

Within 30 minutes of adding the stop solution, insert the plate into a microplate reader and read the plate at the appropriate wavelength for the substrate to determine the absorbance of the wells.

To begin the sandwich ELISA, the plate must be coated with purified capture antibody. To do this, add 100 microliters of the capture antibody at a concentration within the 1-10 microgram per milliliter range, to each well of a 96-well ELISA plate. Next, cover the plate with an adhesive plate cover and then incubate the plate overnight at 4 degrees celsius. After the incubation, remove the coating solution by flicking the plate over a sink.

Now, block the remaining protein- binding sites in the coated wells by adding 200 microliters of 5% nonfat dry milk to the wells. Incubate the plate at room temperature for at least 2 hours. Next, remove the blocking buffer, and then wash the wells with 1X PBS containing 1% Tween-20. Remove the wash by flicking the plate over the sink. Now, add 100 microliters of the test sample to the wells, seal the plate with an adhesive cover, and then incubate it at room temperature for 2 hours. After incubation, remove the samples by flicking the plate over the sink and then wash the wells with 200 microliters of 1X PBS containing 1% Tween-20. Flick the plate over the sink to remove the wash and then add 100 microliters of enzyme-conjugated detection antibody to the wells.

Seal the plate with an adhesive cover. Leave the plate to incubate at room temperature for 2 hours. After the incubation, remove the unbound detection antibody by flicking the plate over a sink and wash the wells with 200 microliters of 1X PBS containing 1% Tween-20. Next, add 100 microliters of the indicator substrate at a concentration of 1 milligram per milliliter, and incubate the plate for 5 to 10 minutes at room temperature. After 10 minutes, stop the enzymatic reaction by adding 100 microliters of 2N sulfuric acid to the wells and then read the plate within 30 minutes of adding the stop solution in a microplate reader.

To perform a competitive ELISA, first coat the wells of a 96-well ELISA plate with 100 microliters of purified antigen at a concentration of 1-10 micrograms per milliliter. Cover the plate with an adhesive plate cover and then incubate overnight at 4 degrees celsius. Following this, remove the unbound antigen solution from the wells by flicking the plate over a sink.

Next, block the remaining protein-binding sites in the coated wells by adding 200 microliters of blocking buffer to each well- here, 5% nonfat dry milk in PBS. Incubate the plate for at least 2 hours at room temperature. While blocking the wells, prepare the antigen-antibody mixture in a 1. 5 milliliter tube by adding 150 microliters of sample antigen to 150 microliters of primary antibody for each well in the assay. Incubate this mixture for 1 hour at 37 degrees celsius. Now, remove the blocking buffer from the wells by flicking the plate over a sink. Then, wash the wells with 1X PBS containing Tween 20 and then add 100 microliters of the sample antigen- primary antibody mixture.

Leave the plate to incubate at 37 degrees celsius for one hour. Next, remove the sample mixture by flicking the plate over a sink and then wash the wells with 1X PBS containing 1% Tween-20 to remove any unbound antibody. Add 100 microliters of an enzyme-conjugated secondary antibody to each well and incubate the plate for one hour at 37 degrees celsius. Following this, wash the plate with 1X PBS containing 1% Tween-20 and then add 100 microliters of the substrate solution to each well. Wait for 5-10 minutes. After 10 minutes, stop the enzymatic reaction by adding 100 microliters of 2N sulfuric acid and then measure the absorbance in a microplate reader within 30 minutes of adding the stop solution.

For the semi-quantitative indirect ELISA assay, the presence of influenza A virus antibodies in serially diluted samples of serum from influenza A- infected mice was determined by reading the absorbance of each well at 405 nanometers in a plate reader. This raw data is exported to a spread sheet for calculation purposes. In this experiment, the serially diluted serum samples, which range from 1 - 12.5, to 1 - 204,800, were repeated in triplicate.

To analyze the data, the mean absorbance value is therefore calculated for each set of triplicates by adding all the values for each dilution and dividing the sum by 3. Once the mean for each set of triplicates is determined, the mean OD450 readings are plotted against the serial dilutions. The OD readings decrease as the serum is diluted, indicating that less antibodies are found in the more diluted samples. In the quantitative sandwich ELISA, dilutions of known standard, in this case recombinate Human TNFalpha, were added to a 96-well plate and read along with the unknown samples.

To create the standard curve, the mean absorbance value for each set of readings of the known concentrations was calculated. Then, the mean absorbance value was plotted on the y-axis, against the known protein concentrations on the x-axis. A best fit curve is added through the points in the graph.

Once the standard curve is generated, the amount of TNFalpha protein in the test sample can be determined by first calculating the mean absorbance value for the test sample. In this example, the test samples gave OD450 readings of 0.636 and 0. 681. Adding these values and dividing the sum by 2 gives an average of 0.659. From the y-axis on the standard curve graph, extend a horizontal line from this absorbance value to the standard curve. At the point of intersection, extend a vertical line to the x-axis and read the corresponding concentration which, in this test sample, corresponds to a TNFalpha concentration of 38.72 picograms per milliliter.

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Results

In the following example of an indirect ELISA, the presence of influenza A virus (IAV)-specific IgG in the serum of IAV-infected mice was determined. C57Bl/6 mice were infected with influenza A virus (A/PR/8 10 5 PFU in 100 µL PBS i.p.) and serum was collected 28 days later. To quantitate the amount of IAV-specific IgG in the serum, 96-well ELISA plates were coated with purified A/PR/8 Influenza A virus (50 µL/well of 2 mg/ml PBS virus) overnight at 4°C. Coated plates were blocked for 1 hour at room temperature with 5% normal donkey serum in PBS, followed by incubation with diluted serum samples from IAV-challenged mice overnight at 4°C. The serum was initially diluted 1:12.5, followed by 1:4 dilutions (dilution range - 1:12.5 to 1:204,800). After washing, plates were incubated with an alkaline phosphatase (AP)-conjugated donkey anti-mouse IgG for 1 h. The plates were washed, and then p-Nitrophenyl Phosphate (PNPP 1 mg/mL, 100 µL/well) was added. The colorless PNPP solution turns to a yellow color when AP is present. After 5-10 min, the enzymatic reaction was stopped by adding 100 µL/well 2N H2SO4. The plate was read on a microplate reader at 405 nm. The results obtained are shown in Table 1 and Figure 1.

Sample Wells OD405 Mean
Serum 1:12.5 A1 2.163 2.194
B1 2.214
C1 2.204
Serum 1:50 A1 1.712 1.894
B1 2.345
C1 1.624
Serum 1:200 A1 1.437 1.541
B1 1.73
C1 1.456
Serum 1:800 A1 1.036 0.957
B1 0.912
C1 0.923
Serum 1:3200 A1 0.579 0.48
B1 0.431
C1 0.429
Serum 1:12800 A1 0.296 0.281
B1 0.312
C1 0.236
Serum 1:51200 A1 0.308 0.283
B1 0.299
C1 0.243
Serum 1:204800 A1 0.315 0.303
B1 0.298
C1 0.297

Table 1: Indirect ELISA assay data. Serum dilutions (from 1:12.5 to 1:204,800), of influenza A virus (IAV)-infected mice containing IAV-specific IgG, optical density (OD) (405 nm) values and mean OD405 values.


Figure 1: Indirect ELISA assay scatter plot of mean OD405 values(+ S. D.) and serum dilutions (from 1:12.5 to 1:204,800), of influenza A virus (IAV)-specific IgG in the serum of IAV-infected mice. The OD405 values can be inversely correlated to the serum dilutions.

In the following example of a sandwich ELISA, a 1:2.5 dilution of recombinant human TNFα standards (starting at a concentration of 75 pg/mL) was added to the indicated wells of a 96-well flat-bottom plate. These standards led to a corresponding 2.5-fold change in the absorbance readings.

Sample Concentration (pg/mL) Wells Values Mean Value Back Concentration Calculation Average
Standard 1 75 A1 1.187 1.169 76.376 75.01
A2 1.152 73.644
Standard 2 30 B1 0.534 0.52 30.827 29.962
B2 0.506 29.098
Standard 3 12 C1 0.23 0.217 12.838 12.105
C2 0.204 11.372
Standard 4 4.8 D1 0.09 0.084 5.055 4.726
D2 0.078 4.398
Standard 5 1.92 E1 0.033 0.031 1.941 1.86
E2 0.03 1.778
Standard 6 0.768 F1 0.009 0.011 0.626 0.764
F2 0.014 0.901
Standard 7 0.307 G1 0.002 0.004 0.238 0.377
G2 0.007 0.516

Table 2: TNFα Sandwich ELISA standard curve data. A 1:2.5 dilution of recombinant human TNFα standards (75 to 0.3 pg/mL), OD (450 nm) values, mean OD450 values, back concentration calculations and their averages.


Figure 2: Standard Curve for TNFα sandwich ELISA. A 1:2.5 dilution of recombinant human TNFα standards (75 to 0.3 pg/mL) was analyzed using sandwich ELISA.The OD450 values can be directly correlated to the standard dilution concentrations. The amount of TNFα protein in the test sample was determined using the standard curve, which corresponds to a concentration of 38.72 pg/mL.

Once the standard curve was generated, the amount of TNFα protein in the test sample was determined. In this sandwich ELISA example, the test samples gave OD450 readings of 0.636 and 0.681, which give an average of 0.6585. When plotting this OD450 reading on the above chart, this corresponds to a TNFα concentration of 38.72 pg/ml.

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Applications and Summary

As demonstrated, a range of immunoassays (with slight variation in protocols) fall within the ELISA technique family. Determining which version of ELISA to use depends on a number of factors, including what antigen is being detected, the monoclonal antibody available for a particular antigen, and the desired sensitivity of the assay (5). Some strengths and weaknesses of the different ELISAs described herein are:

ELISA Strengths Weaknesses
Indirect 1) High sensitivity due to the fact that multiple enzyme-conjugated secondary antibodies can bind to the primary antibody 1) High background signal may occur because the coating of the antigen of interest to the plate is not specific (i.e., all proteins in the sample will coat the plate)
2) Many different primary antibodies can be recognized by a single enzyme-conjugated secondary antibody giving the user the flexibility of using the same enzyme-conjugated secondary antibody in many different ELISA (regardless of the antigen being detected)
3) Best choice when only a single antibody for the antigen of interest is available
Sandwich 1) The use of antigen-specific capture and detection monoclonal antibody increases the sensitivity and specificity of the assay (compared to the indirect ELISA) 1) Optimizing the concentrations of the capture and detection monoclonal antibodies can be difficult (especially for non-commercial kits)
2) Best choice for detecting a large protein with multiple epitopes (such as a cytokine)
Competitive 1) Impure samples can be used 1) Requires a large amount of highly pure antigen to be used to coat plate
2) Less sensitivity to reagent dilution effects
3) Ideal for detecting small molecules (such as a hapten)

Table 3: Summary. A summary of the strengths and weaknesses of the different ELISA techniques.

While a simple and useful technique, there are also some drawbacks to any ELISA. One is the uncertainty of the amount of the protein of interest in the test samples. If the amount is too high or too low, the absorbance values obtained by the microplate reader may fall above or below the limits of the standard curve, respectively. This will make it difficult to accurately determine the amount of protein present in the test samples. If the values are too high, the test sample can be diluted prior to adding to the wells of the plate. The final values would then need to be adjusted according to the dilution factor. As mentioned, homemade kits often require careful optimization of the antibody concentrations used to yield a high signal-to-noise ratio.

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References

  1. Porstmann, T. and Kiessig S.T. Enzyme immunoassay techniques. An overview. Journal of Immunological Methods.150 (1-2), 5-21 (1992).
  2. Suleyman Aydin. A short history, principles, and types of ELISA, and our laboratory experience with peptide/protein analyses using ELISA. Peptides, 72, 4-15 (2015).
  3. Gan. S. D. and Patel K. R. Enzyme Immunoassay and Enzyme-Linked Immunosorbent Assay. Journal of Investigative Dermatology, 133 (9), 1-3 (2013).
  4. Kohl, T. O. and Ascoli C.A. Immunometric Antibody Sandwich Enzyme-Linked Immunosorbent Assay. Cold Spring Harbor Protocols, 1 (6), (2017).
  5. Sakamoto, S., Putalun, W., Vimolmangkang, S., Phoolcharoen, W., Shoyama, Y., Tanaka, H., and Morimoto S. Enzyme-linked immunosorbent assay for the quantitative/qualitative analysis of plant secondary metabolites. Journal of natural medicines, 72 (1), 32-42 (2018).

Transcript

Please note that all translations are automatically generated.

Enzyme-linked Immunosorbent Assay, or ELISA is a highly sensitive quantitative assay commonly used to measure the concentration of an analyte like cytokines and antibodies in a biological sample. The general principle of this assay involves three steps: starting with capture, or immobilization, of the target analyte on a micro plate, followed by the detection of the analyte by target-specific detection proteins, and lastly, enzyme reaction, where a conjugated enzyme converts its substrate to a colored product. Based on different methods of capture and detection, ELISA can be of four types: direct, indirect, sandwich, and competitive.

For direct ELISA, the target antigen is first bound to the plate, and is then detected by a specific detection antibody. This method is commonly used for screening antibodies for a specific antigen. Indirect ELISA is used for detecting antibodies in a sample in order to quantify immune responses. The plate is first coated with a specific capture antigen, which immobilizes the target antibody, and this antigen-antibody complex is then detected using a second antibody.

In the case of sandwich ELISA, the target analyte is an antigen, which is captured on the plate using a capture antibody and then detected by the detection antibody, hence forming an antibody-antigen-antibody sandwich. This method is useful for measuring the concentration of an antigen in a mixed sample.

Competitive ELISA is used when only one antibody is available for a target antigen of interest. The plate is first coated with the purified antigen. Meanwhile, the sample containing the antigen is pre-incubated with the antibody and then added to the plate, to allow any free antibody molecules to bind to the immobilized antigen. The higher the signal from the plate, the lower the antigen concentration in the sample. In all of the four types of ELISA, direct, indirect, sandwich, and competitive, the detection antibody is either directly conjugated to the enzyme or can be indirectly linked to it through another antibody or protein.

The enzymes commonly used for the reaction are horseradish peroxidase or alkaline phosphatase with their respective substrates, both producing a soluble, colored product that can be measured and quantified using a plate reader. In this video, you will observe how to perform indirect ELISA, sandwich ELISA, and competitive ELISA, followed by examples of quantification of the target analyte from the indirect and sandwich ELISA methods.

The first experiment will demonstrate how to use indirect ELISA to determine the presence of anti-influenza virus antibodies in serum obtained from influenza-infected mice.

To begin, add 50 microliters of purified antigen - in this case, 2 milligrams per milliliter of purified A/PR/8 Influenza A virus- to each well of a 96-well ELISA plate. Next, cover the plate with an adhesive cover and incubate it overnight at 4 degrees celsius to allow the antigen to bind to the plate. The following day, remove the coating solution by flicking the plate over a sink. Next, block the remaining protein-binding sites in the coated wells by adding 200 microliters of a blocking buffer- here, 5% donkey serum in 1X PBS- to each well. Leave the plate to incubate for at least 2 hours at room temperature. Following the incubation, remove the blocking buffer and then wash the plate by adding 200 microliters of 1X PBS containing 1% Tween-20. Flick the plate over the sink once more to remove the wash.

Then, prepare the test samples by adding 460 microliters of PBS to a fresh tube, and then adding 40 microliters of serum to make a 1 to 12.5 dilution. Then, add 300 microliters of PBS to a second tube, and then add 100 microliters of the first dilution. Continue this serial dilution range until obtaining a final sample with a dilution of 1 to 204,800. Add the serially diluted serum samples in triplicate to the wells. Cover the plate with an adhesive cover and incubate at room temperature for an hour. Next, remove the samples by flicking the plate into the sink and then wash the plate by adding 200 microliters of 1X PBS containing 1% Tween-20. Once again, flick the plate to remove the wash.

Now, add 100 microliters of an enzyme-conjugated secondary antibody, which in this experiment is a horseradish peroxidase, or HRP, conjugated donkey anti-mouse secondary, to each well. Incubate the plate for one hour at room temperature, and flick the plate to remove any excess liquid. Wash the plate with 1X PBS containing 1% Tween-20 and then apply 100 microliters of the indicator substrate at a concentration of one milligram per milliliter to each well. Incubate the plate with the substrate for 5 to 10 minutes at room temperature. In this example, the colorless 3,3', 5,5' - tetramethylbenzidine, or TMB, substrate turns a blue color when HRP is present. After 10 minutes, stop the enzymatic reaction by adding 100 microliters of 2N sulfuric acid. The samples will turn a yellow color.

Within 30 minutes of adding the stop solution, insert the plate into a microplate reader and read the plate at the appropriate wavelength for the substrate to determine the absorbance of the wells.

To begin the sandwich ELISA, the plate must be coated with purified capture antibody. To do this, add 100 microliters of the capture antibody at a concentration within the 1-10 microgram per milliliter range, to each well of a 96-well ELISA plate. Next, cover the plate with an adhesive plate cover and then incubate the plate overnight at 4 degrees celsius. After the incubation, remove the coating solution by flicking the plate over a sink.

Now, block the remaining protein- binding sites in the coated wells by adding 200 microliters of 5% nonfat dry milk to the wells. Incubate the plate at room temperature for at least 2 hours. Next, remove the blocking buffer, and then wash the wells with 1X PBS containing 1% Tween-20. Remove the wash by flicking the plate over the sink. Now, add 100 microliters of the test sample to the wells, seal the plate with an adhesive cover, and then incubate it at room temperature for 2 hours. After incubation, remove the samples by flicking the plate over the sink and then wash the wells with 200 microliters of 1X PBS containing 1% Tween-20. Flick the plate over the sink to remove the wash and then add 100 microliters of enzyme-conjugated detection antibody to the wells.

Seal the plate with an adhesive cover. Leave the plate to incubate at room temperature for 2 hours. After the incubation, remove the unbound detection antibody by flicking the plate over a sink and wash the wells with 200 microliters of 1X PBS containing 1% Tween-20. Next, add 100 microliters of the indicator substrate at a concentration of 1 milligram per milliliter, and incubate the plate for 5 to 10 minutes at room temperature. After 10 minutes, stop the enzymatic reaction by adding 100 microliters of 2N sulfuric acid to the wells and then read the plate within 30 minutes of adding the stop solution in a microplate reader.

To perform a competitive ELISA, first coat the wells of a 96-well ELISA plate with 100 microliters of purified antigen at a concentration of 1-10 micrograms per milliliter. Cover the plate with an adhesive plate cover and then incubate overnight at 4 degrees celsius. Following this, remove the unbound antigen solution from the wells by flicking the plate over a sink.

Next, block the remaining protein-binding sites in the coated wells by adding 200 microliters of blocking buffer to each well- here, 5% nonfat dry milk in PBS. Incubate the plate for at least 2 hours at room temperature. While blocking the wells, prepare the antigen-antibody mixture in a 1. 5 milliliter tube by adding 150 microliters of sample antigen to 150 microliters of primary antibody for each well in the assay. Incubate this mixture for 1 hour at 37 degrees celsius. Now, remove the blocking buffer from the wells by flicking the plate over a sink. Then, wash the wells with 1X PBS containing Tween 20 and then add 100 microliters of the sample antigen- primary antibody mixture.

Leave the plate to incubate at 37 degrees celsius for one hour. Next, remove the sample mixture by flicking the plate over a sink and then wash the wells with 1X PBS containing 1% Tween-20 to remove any unbound antibody. Add 100 microliters of an enzyme-conjugated secondary antibody to each well and incubate the plate for one hour at 37 degrees celsius. Following this, wash the plate with 1X PBS containing 1% Tween-20 and then add 100 microliters of the substrate solution to each well. Wait for 5-10 minutes. After 10 minutes, stop the enzymatic reaction by adding 100 microliters of 2N sulfuric acid and then measure the absorbance in a microplate reader within 30 minutes of adding the stop solution.

For the semi-quantitative indirect ELISA assay, the presence of influenza A virus antibodies in serially diluted samples of serum from influenza A- infected mice was determined by reading the absorbance of each well at 405 nanometers in a plate reader. This raw data is exported to a spread sheet for calculation purposes. In this experiment, the serially diluted serum samples, which range from 1 - 12.5, to 1 - 204,800, were repeated in triplicate.

To analyze the data, the mean absorbance value is therefore calculated for each set of triplicates by adding all the values for each dilution and dividing the sum by 3. Once the mean for each set of triplicates is determined, the mean OD450 readings are plotted against the serial dilutions. The OD readings decrease as the serum is diluted, indicating that less antibodies are found in the more diluted samples. In the quantitative sandwich ELISA, dilutions of known standard, in this case recombinate Human TNFalpha, were added to a 96-well plate and read along with the unknown samples.

To create the standard curve, the mean absorbance value for each set of readings of the known concentrations was calculated. Then, the mean absorbance value was plotted on the y-axis, against the known protein concentrations on the x-axis. A best fit curve is added through the points in the graph.

Once the standard curve is generated, the amount of TNFalpha protein in the test sample can be determined by first calculating the mean absorbance value for the test sample. In this example, the test samples gave OD450 readings of 0.636 and 0. 681. Adding these values and dividing the sum by 2 gives an average of 0.659. From the y-axis on the standard curve graph, extend a horizontal line from this absorbance value to the standard curve. At the point of intersection, extend a vertical line to the x-axis and read the corresponding concentration which, in this test sample, corresponds to a TNFalpha concentration of 38.72 picograms per milliliter.


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