Immunoassay factors affecting

EE Howell, J Nasser, KJ Schray. Coated tube immunoassay Factors affecting sensitivity and effects of reversible protein binding to polystyrene. J Immunoassay 2 205, 1981. 

VALIDATION PARAMETERS AND FACTORS AFFECTING THE PERFORMANCE OF IMMUNOASSAYS... 

Many of the coagulation factors measured by global coagulation tests have limited stability, and the time and temperature of storage of sample will affect their measurements. Concepts of analyte stability and half-life in plasma extend to markers measured by immunoassay. Markers of platelet activation are affected by artifactual activation in vitro upon collection of the blood specimen. This section will highlight some of the nonanalytical variables that, if uncontrolled, can lead to spurious results and thus affect the interpretation of laboratory data. 

As with any analytical technique, generation of a reproducible standard curve with minimal error is critical. An assay calibration consists of several steps during which the value of the primary standard is transferred to the calibrators used in the final assay [22]. Immunoassay optimization is usually difficult due to protein heterogeneity and matrix effects and these factors, heterogeneity and matrix effects, will also affect MIP based assays [22]. 

Like in the case of immunoassays, the MIP-ILAs will be governed by the Law of Mass Action when working at equilibrium so that the reagents are under equilibrium conditions and subject to temperature fluctuations. Shaking can also affect the local concentration of reagents and the reaction rate these factors must be controlled to improve the assay precision. 

Immunoassays for Bik, based on polyclonal antibodies (pAb), are affected by cross-reaction with Tamm-Horsfall protein (THP). This problem can lead to the generation of false positive results in cases of proteinuria [14], In contrast, immunoassays that utilize plasma suffer from cross-reactivity to Iof [23]. The cross-reactivity with THP is due to complexed N-linked glycan, whereas cross-reactivity with Iof is due to bound Bik [14]. Cross-reaction with a-1-glycoprotein (AGP) also does not appear to be a significant factor in blood. 

Polyclonal antibodies are widely used in clinical laboratories for the measurement of plasma protein concentrations. However, immunoassays are often sensitive to the nature of the antibody used. The development of polyclonal antibodies is affected by several factors, such as the purity and dose of the antigen used, the species of host animal, and the immunization procedure. Monoclonal antibodies are viewed as a viable alternative to alleviate these problems. However, the expression of particular epitopes varies with the hpoprotein particles and among individuals in addition, the apohpoproteins themselves are polymorphic in nature. Therefore the use of a single monoclonal antibody might not detect a particular variant. If a monoclonal antibody is used in the determination of an apohpoprotein, it should he directed to an epitope that is expressed on all polymorphic forms of that particular apoprotein. Furthermore, the epitope should be equally reactive to the antibodies regardless of which hpoprotein class contains it. Alternatively a mixture of monoclonal antibodies directed at different epitopes of the apohpoprotein may also be used. Such mixtures are referred to as panmonoclonal antibodies. 

To ensure the reliability of analytical techniques, they need to be validated. Validation provides information on the overall performance of the assay as well as on individual parameters and factors that can be used to estimate the degree of uncertainty associated with an assay (Ellison et al., 2000). An adequate validation procedure assesses, and therefore ensures, that the immunoassay performs within an acceptable range of established criteria. Parameters used to evaluate the performance of the assays may be affected by (1) factors inherent to the analytical technique, such as antibody specificity and antibody cross-reactivity, and (2) external factors such as environmental conditions (temperature) and type of sample (matrix, processed food vs. raw ingredients). A... 

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