RIA Methodology Evaluation

RIA Methodology Evaluation

The reagents used in radioimmunoassay are mostly biological products, which are susceptible to external environmental factors. Factors such as experimental operation and instrument measurement errors will affect the accuracy and precision of the entire experimental result. To enhance the comparability of results, the RIA methodology needs to be evaluated. In addition to the conventional sensitivity, precision (CV%), accuracy, specificity and stability indicators, the following indicators should also be noted:

(1) Reliability (validity)

Also called soundness, it is an index to evaluate whether the immune activity of the test substance and the standard crystal are the same. The reliability of the method is judged by the parallel analysis of the standard curve and the sample dilution curve. Good parallelism is reliable.

(2) Dose-response curve

The labeled immunity experiment is to draw a dose-response curve by using a standard with a known concentration and corresponding response parameters. The quantification of the analyte is determined by calculating the concentration value of the standard for the response parameter on the dose-response curve. Therefore, when fitting the dose-response curve, we must pay attention to the selection of appropriate mathematical models for different types of labeled immune response, so that this dose-response curve has a good correlation (r should not be less than 0.9900). In addition, the effective dose value (ED2s, Ea () \ ED7 (i) should be set between the highest and lowest concentration points of the dose-response curve, and there should be sufficient drop.

(3) High-dose hook effect

The high-dose hook effect (highdosehookeffect, HOOK effect) refers to the phenomenon that when the concentration of the analyte in the specimen exceeds the upper limit of the linear range, the result obtained is instead reduced or negative. The HOOK effect is common in the two-site IRMA. In a one-step experiment, the reason for its occurrence is that a large amount of excess antigen competes with the captured antigen for binding to a limited amount of labeled secondary antibody. In the two-step experiment, it is mostly related to the "quality" of the antigen (the number of epitopes and the number of repeated expressions): the solid-phase primary antibody captures the antigen that is excessive or has repeated expression of the epitope, and is saturated; Overlapping combination produces a three-dimensional effect, making the antigen "isomeric" and weakening the affinity with the primary antibody. During washing, the complex formed by the labeled secondary antibody and the antigen is detached from the solid phase. The HOOK effect can be improved or eliminated by selecting high affinity antibodies or diluting such specimens and measuring them.


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