ELISA Handbook

Posted by Jack on November 24, 2022
Table of Contents

    Introduction

    ELISA stands for "enzyme-linked immunosorbent assay." ELISA kits are used to detect the presence of an antibody or antigen in a sample by measuring a specific type of protein called an enzyme. The enzyme reacts with an antibody that's attached to a solid surface, like a piece of plastic or silicon. For example, let's say you have a substance in your blood that's made up of molecules called antibodies. With this kit, we can test whether those antibodies react with certain antigensproteins from germs like bacteria and viruses. If they do react (and this reaction is measured), then we know that those germs might be present!

    ELISA kits

    ELISA kits are available for many different purposes. They can be used to detect antibodies in blood or body fluids, such as:

    • Serum
    • Urine
    • Cerebrospinal fluid (CSF)

    Wash buffer

    • Wash buffer: This is a solution of pH 7.4, 0.15 M sodium phosphate and 0.01 M sodium chloride (NaCl) at room temperature. The wash buffer can be prepared by dissolving 1 g of NaCl in 100 mL of distilled water at room temperature.
    • Antibody dilution buffer: To prepare antibody dilution buffer you will need to mix 20 mg/mL anti-β-tubulin monoclonal antibody with 5% glycerol at a ratio of 2 parts glycerol to 1 part antibody. Then add 10 mL PBS+0.1% Tween 20 and incubate for 30 minutes at room temperature before adding 50 μL α-MEM medium containing 0–5% fetal bovine serum (FBS).

    Blocking buffer

    Blocking buffer is used to prevent the binding of nonspecific proteins to your ELISA plate. It also improves the sensitivity of your assay by reducing non-specific binding. Blocking buffer is made up of BSA (bovine serum albumin) and phosphate buffered saline (PBS). The amount of BSA used in making blocking buffer will vary depending on how much protein you want to immobilize. Some people use 1% w/v, while others use 0.5% w/v or even less! The PBS helps keep your sample from clumping together during centrifugation and prevents precipitation when adding certain reagents like antibodies or antigens.

    When it comes time to add blocking buffer, simply pour enough onto each well until it covers all wells completely with no gaps visible between them (Figure 2). If you have any leftover after adding this amount then simply discard it as there will be plenty left over for future uses if needed!

    After adding blocking solution leave plates overnight at room temperature without agitation so that all surfaces are equally covered by the solution (Figure 3).

    Sample treatment and dilution

    Sample treatment: The sample needs to be treated before it is applied to the microtiter plate. Sample dilution: The sample may need to be diluted with a dilution buffer if it contains high concentrations of protein or cells. Sample application: After treatment and/or dilution, the sample is transferred from your pipette onto the wells of your microtiter plate (see below). If you are performing an ELISA experiment in which multiple samples are applied to one plate, this step will involve pipetting different solutions into different wells on the same plate.

    Sample incubation: Once all of the samples have been added to their respective wells, they must sit for a period of time that allows for binding between antibodies and antigens on cell surfaces and capture antibodies attached to beads inside each well (see below). This incubation period can vary depending upon how long you want it take for two things—firstly, for any non-specific binding between antibody molecules not recognizing antigens on cell surfaces; secondly, so there's enough time for those specific binders between Biotin-labeled capture antibody molecules that do recognize antigen targets on cell surfaces (if using biotinylated capture antibody) with fluorophore-labeled conjugate probes attached via streptavidin bridge (if using fluorescent detection methods like APC® anti-IgM+ anti human IgM FITC).

    Wash steps: After incubation has occurred some number of times over several hours depending upon what kind of detection method is being used along with other factors such as whether live cells vs dead cells were used etcetera...the microtiter plates must undergo multiple wash steps in order remove excess unbound material from each well before going through another round(s)

    Plate coating antibody

    • The antibody solution is usually made up of a mixture of monoclonal and polyclonal antibodies.
    • You need to dilute the antibody so that it disperses well over the plate, otherwise you will get a hazy background on your final ELISA plate.
    • A table top centrifuge should be used for this step to avoid introducing air bubbles into the solution or having it stick to the side of your tube during mixing.

    Diluted standard

    A diluent is prepared to match the sample type. This is done in most cases by mixing the sample with a preservative, such as 1% Bovine Serum Albumin (BSA). The preservative prevents evaporation of small molecules that would normally occur when adding fresh serum to an antibody conjugate. If you are working with mouse blood or mouse tissues, then PBS 0.1% Tween 20 should be used instead of BSA because these samples contain antibodies against human antigens and would be cross-reactive if exposed to BSA.

    Because it's important not to dilute out your results, here are some tips:

    • Use a suitable diluent for your sample type; if you're working with serum from mice, use PBS 0.1% Tween 20 rather than 1% Bovine Serum Albumin (BSA) because mice have antibodies against human antigens
    • Use a compatible detection antibody; for example, R-phycocyanin conjugated anti-mouse IgG antibodies require buffers with low pH values (4-5), whereas alkaline phosphatase conjugated anti-mouse IgG requires buffers around 7-8

    Detection antibody

    The detection antibody is specific for the capture antibody and is conjugated to an enzyme. The detection antibody can be a secondary or a primary antibody. Secondary antibodies are used when there is too much sample protein or when you want to detect bound analyte (for example, an ELISA with low sensitivity). Primary antibodies are used if there is too little sample protein or if you want to detect unbound analyte (for example, an ELISA with high sensitivity).

    The detection reaction involves the addition of substrate that reacts with the enzyme-antibody conjugate. The substrate may generate a colorimetric reaction or fluorescence.

    Immediately evaluate results!

    The results of ELISA experiments can be analyzed immediately. If you have the expected result, then great! You are done with this experiment and can move on to analyzing your data. However, if there is a problem or an unexpected result it is important to keep in mind that ELISA experiments sometimes fail for reasons that are not understood. It could be due to a false positive reaction, which means that something other than what you expected was detected. This is generally caused by impurities in reagents or equipment used for the assay. If this happens, it's best not to panic and try troubleshooting your experiment before repeating it again with a different kit or protocol using different materials (e.g., plates)

    Conclusion

    We hope this guide has helped you to understand how ELISAs work and how to perform them. This is a very powerful technique which can be used for many types of research!

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