Protein Protocols Protein Protocols

1088 Jordan
a suitable plate is by trial and error, or using a recommended make known to bind the
protein you intend to coat.
4. Although the HRP–TMB system represents a good, reliable, and sensitive combination,
HRP has a number of alternative substrates that can be used (2). These include
o-phenylene diamine (OPD) or 2,2′-azino-di(3-ethylbenzthiazoline-6-sulfonic acid
(ABTS). There are also a number of options for the enzyme used other than HRP, such as
alkaline phosphatase (AP) which can be used in combination with the substrate
p-nitrophenyl phosphate (p-NPP) or phenolphthalein monophosphate (PMP). The choice
of enzyme–substrate system depends on a number of factors including price, sensitivity,
and whether a filter is available for the substrate specific wavelength to be measured.
5. The use of biotinylated secondary antibodies (or detection antibody in the indirect sandwich
ELISA) in conjunction with enzyme-conjugated streptavidin (or avidin, extravidin,
etc.) both increase sensitivity and save time in that a further step is eliminated from the
assay and therefore another step of optimization is eliminated.
6. If the major aim of the ELISA is to obtain quantitation of substances present in extremely
low concentrations a number of adaptations to the technique can be used. Such techniques
often use AP enzyme systems which can be utilized, for example, to lock into a circular
redox cycle producing an end product such as red formazan which is greatly amplified in
comparison to standard amplification methods (3). Chemiluminescent amplified ELISA
principles have also been shown to give very high sensitivity (4) and can be optimized to
measure as little as 1 zeptomol (about 350 molecules!) of AP (5). Although extremely
sensitive, such techniques are time consuming to set up and optimize, and are far more
expensive than the simple colormetric ELISAs described in this chapter.
7. In some cases, molecules present in a sample are masked by the solution that they are in.
This problem can sometimes be solved by diluting the samples in PBS–Tween–10% FCS.
If this is performed, remember to make similar adjustments to the solution used for the
standards. Possible interference molecules within samples such as soluble receptors for the
antigen can also cause a problem. Commercially available matched antibody pairs for molecules
should have been pretested and guaranteed against being affected by such problems.
References
1. Engvall, J. R. and Perlamann, P. (1971) Enzyme-linked immunosorbent assay (ELISA).
Quantitative assay of immunoglobulin G. Immunochemistry 8, 871–874.
2. Roberts, I. M., Solomon, S. E., Brusco, O. A., Goldberg, W., and Bernstein, J. J. (1991) A
comparison of the sensitivity and specificity of enzyme immunoassays and time-resolved
fluoromimmunoassay. J. Immunol. Meth. 143, 49–56.
3. Self, C. H. (1985) Enzyme amplification—a general method applied to provide an
immunoassisted assay for placental alkaline phosphatase. J. Immunol. Meth. 76, 389–393.
4. Bronstein, I., Voyta, J. C., Thorpe, G. H., Kricka, L. J., and Armstrong, G. (1989) Chemiluminescent
assay of alkaline phosphatase applied in an ultrasensative enzyme immunoassay
of thyrotropin. Clin. Chem. 35, 1441–1446.
5. Cook, D. B. and Self, C. H. (1993) Determination of one-thousandth of an attomole
(1 zeptomole) of alkaline phosphatase: application in an immunoassay of proinsulin. Clin.
Chem. 39, 965–971.