Antigen Assay choice

The issue of which antibody to select for an assay is not a new problem. Certainly anyone involved in the development of an immunoassay has been faced with this choice. Consider attempting to create a multianalyte, microarray-based micro-ELISA of modest density (10 to 100 analytes) and determining which capture antibodies to use based upon their affinities, stabilities, and cross-reactivities. For a sandwich assay, add in the 10 to 100 analyte-specific secondary (reporter) antibodies and determine their levels of cross-reactivity with each other and with the specified antigens and capture antibodies. In other words, achieving high performance for all analytes with a microarray immunoassay is indeed a formidable challenge. 

Choice of Radioligand. A 11+C radiolabel will probably exist for most pesticides which will be considered for radioimmunoassay development. Such an intrinsic radiolabel will prove very valuable in titering antisera and possibly in numerous other steps from antigen synthesis through assay development. Unfortunately, for the actual assay, the commonly available 11+C radiolabels may not be of high enough specific activity. The theoretical limit on the specific activity of a single carbon atom is 63 mCi/mmole, and few pesticides have a specific activity of over 50 mCi/mmole even when they are labeled in... 

The specific activity of proteins assayed by direct immunochemical methods or those used as standards in radioimmunoassays profoundly affects the resolution attainable by these techniques. Therefore, the nature of the radioactive label to be used in such experiments must be considered carefully. Table 8-6 lists the isotopes available for this purpose along with the number of atoms of each isotope that must be incorporated to produce an arbitrary counting rate. As can be seen here, 557 atoms of H and 261,672 atoms of C must be incorporated into every molecule of protein to yield the same number of disintegrations per minute as only one I or 11 S molecules. S-methionine is often the isotope of choice for many direct immunochemical procedures since it is relatively inexpensive to prepare at high specific activity. On the other hand, the relative ease with which radioactive iodine may be incorporated into a purified antigen makes it the isotope of choice for radioimmunoassay methods. Of the two iodine isotopes available, is most often used because of its longer half-life. This is an important consideration since it usually takes more than 1 week to prepare and test a labeled antigen prior to its experimental use. 

The choice of the type of assay to be used for such measurements depends on the expected concentration of the protein to be measured. For example, in the case of albumin, the assay chosen must be precise at the upper limit of the reference interval (for maximal clinical effectiveness), while also being able to ensure that antigen excess does not occur at high pathological concentrations. In practice, this cannot be achieved in a direct aggregation assay, where the antibody-created particles are aggregated in the presence of... 

With appropriate sample dilution, the light-scattering immunoassays will provide precise results within an interval of O.lmg/L to 20g/L. Choice of an immunoinhibition assay wiU ensure that the antigen excess does not lead to an erroneously reported result (see Chapter 9), although sample dilution wiU be required to give an accurate result at high protein concentration. Values for imprecision of less than 5% within run and 8% between runs are attainable over a concentration range of 0.1 mg/L to 20 g/L with the appropriate choice of reaction conditions. 

We may require an enzymatic reaction in the assay, and therefore will need an antispecies conjugate (commercial most probably) or will have to label an antigen-specific serum with enzyme (are there facilities to do this ). We must decide which commercial conjugate to buy. This will depend on the desired specificity of the conjugate (anti Cwhole molecule IgG, anti-H-chain IgG, anti-H-chain IgM, and so on). The choice is somewhat determined by the aims of the assay and its design. Thus, we may wish to determine the IgM response of cattle to our antigen, which will require an anti-IgM (specific) somewhere in the ELISA protocol. 

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