Optimizing Enzyme Immunoassays in Plates
Enzyme immunoassays are powerful immunochemical techniques which combine specificity and sensitivity to detect, quantitate, and characterize antigens or antibodies. However, unless properly designed and optimized, an enzyme immunoassay may create more questions than it provides answers. Many parameters can affect the performance of an enzyme immunoassay, including the concentration of antibodies and antigens, choice of buffers, chemistry of microtiter plates, and incubation times and temperatures.
A major goal of an enzyme immunoassay is to specifically measure an analyte with sensitivity and minimal background. To accomplish this task, the following parameters should be determined:
- Type of microtiter plate (or other medium) used for the EIA.
Capture antibody/antigen concentration, coating buffer, coating temperature, and length of incubation.
Concentration and composition of blocking reagents.
Influence of pipetting and plate washers on assay variability.
Concentration of antibody-enzyme conjugate which gives good signal and minimal background (i.e., optimal signal to noise ratio). Positive to negative signal ratio should ideally be 10:1, but never lower than 3:1
- Sensitivity and range of assay and typical working concentration of analyte.
Microtiter Plate Choices
Though an enzyme immunoassay can be performed in several formats, none is as widely used as the 96 well microtiter plate. These plates are made from a variety of polymers or glass, with modified polystyrene being most popular for EIAs. Polystyrene is a hydrophobic polymer which is readily derivatized by the manufacturer. Microtiter plates for EIAs are usually made from: 1) unmodified polystyrene which will bind proteins by hydrophobic interactions, 2) modified polystyrene which has an ionized and hydrophobic surface, or 3) polystyrene with a hydrophilic or low binding surface. Depending upon the antibodies and antigens in a use, any one of these surfaces may provide the best binding, and thus signal and lowest background for a given assay. The choice of surface must be tested empirically. These traditional surfaces all passively bind antibodies and antigens.
Microtiter plates with chemically reactive, or activated, surfaces can be used so to covalently couple antigens or antibodies to the plates. One popular surface has active esters, such as N-hydroxysuccinimide ester, which can react with primary amines on proteins. The benefit of covalent coupling is that proteins can not be washed off the well; however, such plates tend to be more expensive.
Coating the Well
The first antigen/antibody used in a typical EIA is adsorbed to the surface of a microtiter well. Several variables affect the consistency and effectiveness of coating with the capture molecule, and these include buffer composition, binding time, and incubation temperature. Two main buffers used for coating are phosphate buffered saline and 0.1 M sodium carbonate (Na2CO3).
Antigen/antibody coating conditions are determined by:
- Coating three rows of wells with antigen/antibody concentrations between 0.1-10 μg/ml, e.g., 1000 ng/100 μl serially diluted 1:2 to 16 ng/100 μl over seven rows with the eighth row serving as a negative control. This coating should be repeated with both PBS and carbonate coating buffers and bound overnight at 4°C.
Aspirate the coating solution and add 3% BSA in PBS for 1 hr. This will block all remaining sites. Be aware that the different fractions and lots of BSA will block differently, a variable to examine later.
Measure the amount of coated antibody/antigen by binding anti-Ab/Ag antibody-conjugate. The conjugate should be sufficiently dilute so as not to create background, but should still generate a signal at least 10X background. This dilution initially is empirically determined, but a starting dilution could be 1:15,000 in 3% BSA in PBS. Allow this conjugate to bind for 1 hour at 37°C.
Wash to remove conjugate and add substrate. Following incubation, read the plate.
- Examine the optical densities, comparing the signal between coating buffers and concentrations. The best buffer and Ab/Ag concentration is that which is most dilute but gives a signal equivalent to higher concentrations of coating reagent.
The amount of antigen/antibody which can be assayed depends partly on the type of microtiter plate used. It is advisable to test plates for binding capacity when designing an assay.
The addition of blocking agents is used several times in a typical EIA, once after coating, during primary antibody binding, and if applicable, during secondary antibody binding. Blocking solutions are usually PBS with 1-3% casein or BSA. As noted above, BSA can be variable depending upon the fraction and batch. Furthermore, Tween 20 can be added at 0.05-0.10% which may enhance the blocking.
Pipetting and Washing
Inaccurate and poor pipetting practices can add tremendous variability to EIAs. Foremost, ensure that the pipettes are accurate and that the proper tips are used. The type of tips used will make a difference. Secondly, two practices which will aid in accurate pipetting include prewetting the tips before use (capillary action pulls more liquid up when the tips are dry) and reverse pipetting (pulling up more liquid than is necessary and then dispensing what is needed). A third point is to use the correct tool for the job, i.e., make use of multichannel pipettes or pipetting stations if available.
The means by which reagents are removed from the wells will also affect the variability of the assay. Hand washing wells with a squirt bottle is an undesirable method of washing as there is little consistency between wells. Washing with a hand held aspirator/dispenser is better, but may still be inconsistent. The best results are obtained from a plate washer which aspirates effectively, but does not dry the well.
Primary and Secondary Antibodies
An important aspect of designing an immunoassay is determining the working concentrations of the various components of the assay. As noted above with the coating antibody/antigen, the primary antibody and the secondary enzyme conjugate, if applicable, all must be titrated to optimal concentrations.
A simple method for assessing the best concentrations involves creating a checkerboard matrix with two key components. This technique involves serially diluting one component of an assay, e.g., the primary antibody, in one direction along a microtiter plate while a second component, e.g., the secondary antibody-enzyme conjugate, would be serially diluted in a cross direction. In this example, the antigen would be bound to the plate in a non-limiting concentration.
Select a concentration of the reagents which minimizes background created by either reagent independently. A concentration of both reagents should be chosen which yields an optical density between 1.0 and 2.0.
Sensitivity and Range
It is important to match the EIA to the task of measuring the analyte. Prior to using the assay, it is important to determine the assay's degree of sensitivity and range of detection. By diluting the control analyte over a wide range, e.g., 1 pg/ml to 100 ng/ml, the lower detection limit, range of detection (low to high), and linearity of the assay can be determined. It is important that these parameters are established and matched to the expected analyte concentrations found in the samples, i.e., the concentrations of the unknown analytes will be adequately measured by the assay.