Immunoassay nonspecific interferences

Specific extraction methods are used to prepare the analyte for immunoassay by freeing the analyte fromboth specific and nonspecific interferences. Supercritical fluid extraction has been used to decrease the amount of solvent waste generated. Solid-phase extraction has gained popularity, and many different supports are available. One promising extraction and concentration method is immunoaffinity chromatography, which will be addressed later. 

Immunoassays established to measure the very low (ng/ml) concentrations of cytokines in body fluids are uniquely susceptible to nonspecific interference from plasma or serum and from interfering antibodies or proteins that may cross-react with those used in the assay. Rheumatoid factors are well known to cause interference in solid phase assays such as ELISA (B8). Heterophile antibodies that react with animal immunoglobulins present a problem in assays in which serum or plasma is used at low dilution (V8) (Table 7). Serum contains a complement, which may interfere with solid phase immunoassays unless inactivation has taken place (B55). In designing assays controls must be employed to test for these effects... 

The advantages of homogenous immunoassays are simple formats and rapid data output producing user-friendly and cost-effective products. Technical challenges to consider, however, are the necessity to remove or minimize background interference from the reagents and nonspecific binding reactions. 

The analytical response generated by an immunoassay is caused by the interaction of the analyte with the antibody. Although immunoassays have greater specificity than many other analytical procedures, they are also subject to significant interference problems. Interference is defined as any alteration in the assay signal different from the signal produced by the assay under standard conditions. Specific (cross-reactivity) and nonspecific (matrix) interferences may be major sources of immunoassay error and should be controlled to the greatest extent possible. Because of their different impacts on analyses, different approaches to minimize matrix effects and antibody cross-reactivity will be discussed separately. 

Since FPIAs are conducted as homogeneous immunoassays, they are susceptible to effects from endogenous fluorophores and from intersample variations. Such problems and others due to the sample matrix are largely avoided by sample dilutions of several hundredfold. Low-affinity, nonspecific binding of tracers to sample proteins, when present in sufficiently high concentrations, can result in a falsely elevated polarization signal. Interference from sample proteins can be eliminated when warranted, by proteolytic hydrolysis with pepsin.(46)... 

Immunoglobulin Levels in Serum.Similar immunoassays were developed for human IgM and IgE. Levels of these Igs were determined in the sera of normal individuals and are shown in Table VI. The IgE analyses were performed on samples that were absorbed with PA-Sepharose as described above to remove components responsible for nonspecific (lipids) and specific (IgG) interference in the assay. The IgE levels were comparable to values obtained for the same samples by double-antibody RIA. The concentration of IgG in these sera (Table VI) was determined by the procedure described above [Eq. (1)]. 

Matrix effects can be a problem for immunoassays, especially for a method without any prior sample clean-up. It can be caused by either nonspecific or specific interferences from the sample matrix and reagents. Possible matrix effects can... 

Selected plasma samples were analyzed by enzyme immunoassay and by a validated radioimmunoassay. An excellent correlation (r = 0.97 P < 0.01) was observed between the values obtained by both types of assays. The sensitivity of the assay as determined by the smallest amount of unlabeled hormone that was significantly different from zero was 10.0 pg per assay tube. In order to determine if nonspecific materials in the sample interfere with an accurate determination, 50, 100 and 200 pi of plasma samples from two males were assayed in triplicate. The parallelism exhibited by the groups had a correlation coefficient of 0.99 (P < 0.01). Mean concentrations of plasma testosterone were 4.4, 4.1 and 4.4 ng/mL for 50, 100, and 200 pi of plasma, respectively. Aliquots of a pool sample from a bull were assayed in the same assay (intraassay) and different assays (interassay). The intraassay and interassay coefficients of variation for 9 assays were 5.8% and 9.7%, respectively. The accuracy of the assay was tested by adding known amounts of testosterone to a constant volume (100 pi) of a low pool sample before extraction. The percentage recovery was 110%. Pool samples assayed both at the front and end of the assays averaged 3.3 and 3.5 ng/mL, respectively. 

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