Radioimmunoassay reagent preparation

The costs of any radioimmunoassay system lie largely in labor reagents and capital equipment make only a small contribution. However, costs of reagents should always be taken into account, and in the case of the separation system can vary widely. Chemical precipitations are the cheapest of all—a fraction of a cent per tube. The most expensive systems are those that are labor intensive (e.g., electrophoresis) or that require considerable effort for the original preparation of reagents (e.g., solid-phase systems). 

In immunoradiometric assays the use of labeled antibody as the reagent for antigen measurement imposes the need for a very high degree of purity of the antibody. This situation is the converse of that posed by specificity in a radioimmunoassay. In the latter the antiserum used may be markedly heterogeneous provided only that the purity of the labeled antigen selects from the mixture of antibodies present an immune reaction that has the specificity required. Therefore, in an immunoradiometric assay in order to achieve sufficient purity of the antibody it is essential to prepare immunoadsorbent from a highly purified polypeptide. 

If the antibody is immobilised on Sepharose , the supernatant containing the free, radioactive peptide can be separated easily and assayed in a gamma counter. With a standard curve drawn for known amounts of peptide subjected to assay under exactly the same conditions, unknown amounts of peptide can be determined by interpolation on the standard curve. There are two potential problems with this type of radioimmunoassay. First, the peptide to be assayed perhaps does not contain Tyr. If it contains His, however, this may suffice since His can be iodinated, especially by an enzymic procedure described below. Alternatively, the peptide is allowed to react with the Bolton and Hunter reagent (Bolton and Hunter, 1973), prepared by iodina-tion of the ester of 3-(4 -hydroxyphenyl)propionic acid and /V-hydroxysuccinimide. Any free amino group can be acylated by this reagent. Secondly, reaction of a peptide with Nal and chloramine-T can cause oxidation of Met, Cys and even Tyr residues, which can interfere with complexation of the iodinated peptide with antibodies raised to the un-iodinated peptide. An alternative method (Holohan et al., 1973) of iodination uses lactoperoxidase in the presence of H202. As pointed out above, this procedure is applicable to the iodination of His residues. This method avoids modification of the side-chains of Met, Cys and Tyr. 

Precision. The radioimmunoassay obeys the same laws of precision as any other chemical reaction, and therefore, under the same conditions, with the same standard and antibody preparation, and with the labeled hormone freshly purified, the results should be identical from day to day and from week to week. Variation is due to errors of technique and can be minimized. However, when a large number of tubes are being prepared with the addition of several reagents in micro... 

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