Sensitive ELISA Method for Quantitating Antibodies to Specific Protein Epitopes

The detection and quantitation of antibodies to specific peptide epitopes can be a useful technique in many areas of biological research. One application of this technique is in the production of anti-peptide antibodies, a popular method for producing antibodies to single epitopes of proteins, cloned but unpurified proteins or difficult-to-purify proteins. In this report, we describe a simple, quick and sensitive method to quantitate the production of anti-peptide antibodies with an enzyme-linked immunosorbant assay (ELISA) utilizing biotinylated peptides attached to streptavidin-coated plates (Figure 1) (1). The protocol presented here is easily transferable, without significant modification, to many systems. To illustrate the technique, we present data from an experiment where male BALB/c mice were immunized with a peptide having the amino acid sequence of sperm whale myoglobin (SWM) 110–121 (AIIHVLHSRHPG), a classic antigen in immunology research (2). In addition to being an immunodominant T cell epitope of SWM, this peptide has the property of being able to induce an antibody response in BALB/c mice without being coupled to a carrier protein. At the time when the peptide was synthesized (Quality Controlled Biochemicals, Hopkinton, MA, USA), a small amount of the material was biotinylated, for use in the described ELISA. For the immunizations, the unbiotinylated peptide was dissolved in phosphate-buffered saline (PBS) at 2 mg/mL and then combined with an equal portion of complete Freund’s adjuvant (CFA). The mixture was made into an emulsion by repeatedly extruding it through a blunted hypodermic needle. Each mouse was injected with a total of 100 μL of the emulsion, containing 100 μg of peptide, in a divided dose subcutaneously in each rear outer flank. Boosts were prepared in the same manner except that incomplete Freund’s adjuvant (IFA) was used. Animals were bled by nicking the tail vein with a scalpel or by retro-orbital bleeding, while under general anesthesia. Blood was collected with heparinized capillary tubes. Plasma was collected and frozen until assayed. Pre-blocked streptavidin-coated ELISA plates were prepared as follows: One hundred microliters of 5 μg/mL streptavidin (Catalog No. 43-4302; Zymed Laboratories, South San Francisco, CA, USA) in PBS was dispensed into each well of 96-well microplates with removable strips (Catalog No. 950-2920-00P; LabSystems, Needham, MA, USA), sealed and incubated at room temperature overnight. Following aspiration of the contents of each well, the plates were washed and then blocked with 300 μL of PBS containing 1% bovine serum albumin (BSA), 5% sucrose, 0.05% Tween®-20 and 0.1% gentamicin sulfate at room temperature overnight. The following day, the wells were emptied, allowed to dry overnight before storing, sealed with desiccant, at 4°C, until use. Alternatively, streptavidin-coated plates may be purchased from several sources, including American Qualex (San Clemente, CA, USA). The biotinylated peptide was freshly diluted to 1.0 μg/mL from a frozen aliquot at 1.0 mg/mL in PBS containing 10% BSA, and 100 μL of the solution was put into each well of the streptavidin-coated microplate, except for the wells used for the standard curve. Since a true standard curve for an anti-peptide antibody is generally difficult to obtain, we used an alternative that can give an approximation of the quantity of anti-peptide antibodies. This alternative (Figure 1B) utilized an irrelevant antibody, which had been biotinylated, following standard protocols that came with biotinylation reagents (Pierce Chemical, Rockford, IL, USA). This method has the advantages that (i) antibody levels can be assigned an approximate numerical value, rather than a relative titer value, and (ii) one standard curve can be used for most anti-peptide antibody responses to be assayed, allowing easier comparisons. The biotinylated antibody was aliquoted into single assay volumes and stored at -70°C until use. The thawed biotinylated antibody solution was diluted, then serially diluted, to give a standard curve in a volume of 100 μL (Figure 2). While the linear portion of this particular curve is from approximately 25 to 1000 ng/mL, the sensitivity of the assay is dependent on a number of factors