Enzyme-linked immunosorbent assays (ELISAs) that are intended to quantitate peptides or proteins in biological fluids must be very sensitive and have minimal cross-reactivity with non-target molecules. One ELISA format that is capable of satisfying both of these requirements is the capture, or sandwich, ELISA. For this type of assay, an antibody against the antigen to be quantitated is immobilized in the bottoms of microplate wells. After blocking exposed sites on the plastic, the fluid containing the antigen (e.g. serum, urine etc.) is then pipetted into the wells where the immobilized antibody can capture it from the solution. This has the effect of concentrating the antigen, which may be at very low levels, and separating it from everything else in the fluid. After washing away unbound protein, the captured antigen is detected with a second antibody against the same antigen and conventional horseradish-peroxidase (HRP)- or alkaline phosphatase (AP)-based colorimetric or fluorescent methods.
The one downside to the capture ELISA is that 2 antibodies recognizing distinct epitopes on the antigen must be used. The reason being that if both the capture and detecting antibodies recognize the same epitope, that epitope would be blocked after interaction with the capture antibody. The combination that is frequently used to avoid this problem is a monoclonal capture antibody and a polyclonal detecting antibody. Another possibility is to use 2 different polyclonal antibodies for capture and detection although the requirement that they recognize distinct epitopes still holds. This format, however, requires a modification to the detecting antibody since an anti-IgG-HRP or anti-IgG-AP secondary antibody would also recognize the capture antibody. This situation can be overcome by conjugating the detecting antibody directly to HRP or AP, or by conjugating it to biotin. If the latter method is used, quantitation of bound detecting antibody is with streptavidin-conjugated HRP or AP.
Since an antibody-biotin conjugate can be used for more applications, such as immunoprecipitation or affinity purification, than an antibody-enzyme conjugate, it may be prudent to conjugate the detecting antibody to biotin. From my experience, one of the easiest and most reliable methods is the EZ-Link™ Sulfo-NHS-LC-Biotinylation Kit from Pierce (Rockford, IL, USA). The kit comes with the biotinylation reagent (sulfo-NHS-LC-biotin), an envelope of PBS mix, a desalting column, plus 2 reagents for determination of moles conjugated biotin/moles protein.
I have used the kit to biotinylate purified mouse and rabbit IgG with equally good results. The procedure is very simple. You simply add the water-soluble sulfo-NHS-LC-biotin to the IgG in PBS, let it incubate for 30 min at RT or 2 h on ice, and then separate the unreacted reagent from the biotinylated protein. I initially used the desalting column for unreacted reagent removal, monitoring the eluate by absorbance at 280 nm. However, since the unreacted reagent peak appeared before all of the IgG had eluted, it seemed like the separation was less than optimal. In addition, since the IgG eluted in about 5 mls, the concentration was less than I had wanted. Since this necessitated an additional step to concentrate the IgG, I have since switched to spin concentrators for both unreacted reagent removal and concentration, eliminating the desalting column altogether. Although I have yet to quantitate the moles of conjugated biotin/mole IgG, ELISAs have shown that the IgG is indeed biotinylated.
Overall, this is a very useful product. The ability to biotinylate IgG, or any protein, quickly and efficiently is a valuable asset. However, the kit is probably not necessary for most people. It is much more economical to buy the sulfo-NHS-LC-biotin from Pierce and download the instruction manual from their website. The only other thing needed is a method to get rid of the unreacted reagent and concentrate the biotinylated protein. A centrifugal concentrator works well for both procedures. After concentrating, simply bring the volume back up to the original level, or higher, and re-spin. Repeat if necessary.
Michael Campa, Ph.D.
Asst. Research Professor of Radiology
Duke University Medical Center