By John R. Crowther
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Extra resources for Elisa: Theory and Practice (Methods in Molecular Biology) (НЕ полн.)
Example text
Addition of the antispecies enzyme conjugate quantifies the bound antibodies. of pretitrated antibody should be about 70% of the maximal reaction (solidphase antigen excess). Competition is reflected by a decrease in the expected color obtained without competitor. The scheme is illustrated in Fig. 10. 6. Choice of Assays The most difficult question to answer when initiating the use of ELISAs is which system is most appropriate. This section will attempt to 51 Choice of Assays C D Fig. 11. Basic ELISA methods.
However, where coating solutions contain relatively small amounts of required antigen(s), the amount of this attaching to a well is reduced according to its proportion in the mixture. The other contaminating proteins will take up sites on the plastic. Since the plastic has a finite saturation level, use of relatively crude antigens for coating may lead to poor assays. Care must be taken to assesseffects of binding proteins at different concentrations, since the actual density of binding may affect results.
Another alternative, as shown in Fig. 14B, is available if the rabbit serum can be conjugated with enzyme. 3. SITUATION 3 Here we have all the possibilities of the first two situations plus the production of a second species (guinea pig) serum against the purified antigen (seeScheme2). This allows the development of competitive assays as in Fig. 15B using either the rabbit or guinea pig as capture serum or detector with the relevant antispecies conjugate. Different species may have better properties in acting as capture reagents and also show varying specificities.