How to eliminate interference due to immunoglobulins in samples in competitive EIA?

How to eliminate interference due to immunoglobulins in samples in competitive EIA ?

Enzyme ImmunoAssays (EIA) are generally performed in 96-well microtiter plates. Those wells are pre-coated with a monoclonal anti-body. This second antibody binds the complex constituted with the specific antibody linked to the molecules to be assayed (or to the tracer), in competitive assay. This second antibody permits to maintain the complexes formed to the well and thus to eliminate the free reagents (i.e. tracer and molecule to be assayed), during the washing.

Thus, it is very important that this binding is not perturbed by various interference. Now, it is well known that antibodies present in biological samples can compete with the complex to bind the second antibody. The effect of this competition is a dramatic drop of the specific bound complex and thus a decrease of the signal (partial or global).

As an example, in rabbit inflammatory model, large amount of rabbit immunoglobulin G (IgG) is produced. CAYMAN cGMP EIA involves a rabbit antibody against cGMP as well as a mouse monoclonal antibody anti-rabbit IgG used as second antibody. Thus, in this model, it is crucial to eliminate the rabbit IgG.

This can be solved by extracting or diluting the samples. However, extracting procedures are time, cost consuming, and do not allow to eliminate totally those immunoglobulins in large amount. Besides, it is hardly possible to carry out a dilution since the molecules to be assayed are so few.

We have developed an original approach prior to the assay, which is low time and cost consuming. The technique we propose is to link covalently the specific antibody to the second antibody before deposing the samples in the wells. The reagents used for this technique have been previously employed for coupling proteins or haptens with antibodies. It has allready been tested for cGMP and LTC4 EIAs. It can be extended to any other assays using antibodies from various species (mouse, sheep, and rabbit). To validate this approach, we added up to 1,6 mg/ml of interfering immunoglobulins in the standard. For cGMP and the LTC4 measurement, the signal was recovered following the procedure below.

The operating procedure is as follows:

unpack the pre-coated microtiter plates;
wash the plate according to the instruction booklet of the kit;
dilute the antiserum supplied with the kit in a volume of water twice as superior as indicated in the kit;
add 100 µl (against 50 µl normally) of antiserum in the "standard", "Bo" and "sample" wells;
add 100 µl of EIA buffer in the "NSB" wells;
incubate overnight at 4°C;
wash the plate according to the procedure proposed in the kit;
depose in the whole wells, 100 µl of glutaraldehyde at 0.25 % (mother solution at 25 %) in a 0.1 M phosphate buffer pH 7.4;
let it react during 10 minutes at room temperature while shaking;
dispatch 250 µl of NaBH4 (borohydrure of sodium) at 4 mg/ml in ultra pure water in every well;
agitate it for 10 minutes at room temperature (step that allows to deactivate the gluturaldehyde functions);
wash the plate according to the normal procedure and continue the assay as mentioned in the protocol.

For all topics presented above further information are available on simple request such as instruction booklets or product information sheets. Please, do not hesitate to contact Dr. Morge for any further information.

| Back to Scientific Section Page |