What is ELISA?

ELISA, or enzyme-linked immunosorbent assay, is an immunoassay technique involving the reaction of antigen and antibody in vitro. ELISA is a sensitive and specific assay for the detection and quantitation of antigens or antibodies. ELISA tests are usually performed in microwell plates. Both direct and indirect ELISA can be used for antigen (or antibody) detection, but the indirect ELISA is more common.

What is EIA?

EIA, or enzyme immunoassay, is a generic term used to refer to all ELISA-like assays including those designed to detect nucleic acid targets as well as antibodies or antigens

What is direct ELISA? What are its advantages and disadvantages?

The direct ELISA uses the method of directly labeling the antibody itself. Microwell plates are coated with a sample containing the target antigen, and the binding of labeled antibody is quantitated by a colorimetric, chemiluminescent, or fluorescent end-point. Since the secondary antibody step is omitted, the direct ELISA is relatively quick, and avoids potential problems of cross-reactivity of the secondary antibody with components in the antigen sample. However, the direct ELISA requires the labeling of every antibody to be used, which can be a time-consuming and expensive proposition. In addition, certain antibodies may be unsuitable for direct labeling. Direct methods also lack the additional signal amplification that can be achieved with the use of a secondary antibody.

What is indirect ELISA? What are its advantages and disadvantages?

The indirect ELISA utilizes an unlabeled primary antibody in conjunction with a labeled secondary antibody. Since the labeled secondary antibody is directed against all antibodies of a given species (e.g. anti-mouse), it can be used with a wide variety of primary antibodies (e.g. all mouse monoclonal antibodies). The use of secondary antibody also provides an additional step for signal amplification, increasing the overall sensitivity of the assay.

What InnoGenex™ kits should I choose for ELISA or other EIA?

The InnoGenex™ UniMAK™ Kits with chemiluminescent substrates (CDP-Star®, LumiBrite™, and LumiBrite™ ULTRA) and those with colorimetric substrates that produce colored solutions (TMB and pNPP) can be used for ELISA and other EIA applications. [NOTE: Kits with colorimetric substrates that produce water-insoluble, colored precipitates (AEC, DAB, BCIP/NBT, and Fast Red) CANNOT be used for ELISA or EIA applications.] For nucleic acid EIA, choose the kits that include Anti-Biotin (for biotinylated probes) or Anti-Fluorescein (for fluoresceinated probes).

Why does my negative control have strong signal? It seems that there is too much background.

The excessive background signal can be caused by inadequate rinsing of plates, reagents not sufficiently diluted, inadequate blocking of plates or non-specific binding of enzyme conjugate. The appearance of color in negative control wells may also indicate cross-reactivity of secondary antibody with components in the antigen sample.

Why does my ELISA have weak signal?

• Wash buffer not adequately drained after every wash step.
• Inadequate incubation times.
• Detection reagents too dilute. Perform checkerboard titrations.
• Enzyme conjugate defective or inhibited by contaminant.
• Substrate defective or contaminated.
• Microwell plates poorly coated.
• Loss of capture antibody during blocking/washing. Decrease or eliminate use of Tween-20.

Why does my positive control have no signal?

• Microwell plates not coated properly.
• Reagents applied in wrong order or step omitted.
• Secondary antibody not matched to the species of primary antibody.
• Enzyme conjugate defective or inhibited by contaminant.
• Detector antibody not compatible with capture antibody (for sandwich assays).

What references could you provide if I want to learn more about ELISA?

Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (1994) Current Protocols in Molecular Biology. John Wiley & Sons.

Bowers GN Jr, McComb RB (1966) A continuous spectrophotometric method for measuring the activity of serum alkaline phosphatase. Clin Chem 12(2): 70-89.

Coons A.A., Creech H.J., jones, R.N., and Berliner,E.(1942), J.Immunol. 45, 159-170.

Crowther JR (1995) Methods in Molecular Biology, Vol 42 ? ELISA: Theory and Practice. Humana Press, Totowa, New Jersey.

Neumann H, van Vreedendaal M (1967) An improved alkaline phosphatase determination with p-nitrophenyl phosphate. Clin Chim Acta 17(2): 183-7.

Tominaga K, Miura Y, Arakawa T, Kobayashi K, Mitsuhashi M (1996) Colorimetric ELISA measurement of specific mRNA on immobilized-oligonucleotide-coated microtiter plates by reverse transcription with biotinylated mononucleotides. Clin Chem 42(11): 1750-7.

Weller, T.H. and Coons, A.H. (1954), Proc.Soc.Exp.Biol. 86, 789-794.

Zreiqat H, Sungaran R, Howlett CR, Markovic B (1998) Quantitative aspects of an in situ hybridization procedure for detecting mRNAs in cells using 96-well microplates. Mol Biotechnol 10(2): 107-13.