Interference in immunoassay

TH Weber, KI Kapyaho, P Tanner. Endogenous interference in immunoassays in clinical chemistry. A review. Scand J Clin Lab Invest 50(suppl 201) 77, 1990. 

There are proficiency testing programs that are geared toward clinical sensitivity or specificity by seeking to determine whether a disease can be detected versus other types of controls that are use to test sensitivity, selectivity, and most importantly, reproducibility and precision. With mass spectrometry, the controls are and should be no different than those used for other assays, with one interesting exception. Quality assurance materials prepared for MS/MS may not be useful in other assays that are less selective. The example is newborn screening where quality assurance/control QA/QC materials have a mixture of compounds present in the blood specimens. However, in less selective immunoassays, the mixture creates interferences. In addition, material is used to spike a blood sample is key and one should ensure there is no enzyme activity. We have encountered such a problem with a d/1 mixture of metabolites where one form was degraded in the prepared blood. 

Nanoparticles, e.g., silicon, gold, silver (Dequaire et al., 2000), are often used as materials for protein labeling in immunoassays, especially in lateral flow device (LFD), where they are responsible for visualization of the results. Their advantage lies in the fact that they enhance the optical signal, and reduce the background interference (Schneider et al., 2000 Lochner et al., 2003 Matveeva et al., 2005 Chumbimuni-Torres et al., 2006 Li et al., 1999 Peng et al., 2007a). 

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)]. 

Endogenous enzymic activity wifi probably not interfere in a solid phase enzyme immunoassay where the activity bound to the solid phase is measured. It is evident that endogenous enzymes can interfere in EMIT-type assays. 

Chemiluminescence offers yet another sensitive detection system, which is easily implemented with simple instrumentation, but suffers to some extent from background interference in complex matrices. A recent example of an enzyme immunoassay with chemiluminescence as the detection system is the assay for 8-oxoguanine in DNA, which uses a secondary antibody conjugated with peroxidase-anti-peroxidase complex and a substrate solution containing hydrogen peroxide, luminol and p-iodophenol. 

Enzymes are nowadays the labels that are most used in immunoassay. Their main disadvantage is their susceptibility to interferences due to changes in assay conditions, e.g., pH, ionic strength, content of organic solvents. The addition of anti-microbial agents such as azides or mercury derivatives also can affect the enzyme activity when using peroxidase labels [96]. The presence of other catalysts in the sample can also have an effect on the enzyme immunoassay result, e.g., Cu(II) ions promote luminol chemiluminescence in the presence of hydrogen peroxide [16]. 

ST107 Sacks, D.B., Lim, M.M., Parvin, C.A. and Kessler, G. (1990). Interference in an automated radial partition fluorescent immunoassay of thyrotropin associated with liver-function abnormalities. Clin. Chem. 36, 1343-1345. 

Figure 6.11. Cross-reactivity of an interferent in a competitive immunoassay.

Figure 6.14. Chemical stmctures and immunoassay responses of 1-pg/mL concentrations of triazine interferents in an atrazine immunoassay kit.

This kind of interference is called cross-reactivity, or specific interference, and is usually quantitated by assaying for the interferent in the absence of analyte. Validation procedures involve screening of a large number of potential interferents at concentrations higher than their expected levels in real samples. Cross-reactivity methods for immunoassays have been described in Chapter 6. 

LM Boscato, MC Stuart. Incidence and specificity of interference in two-site immunoassays. Clin Chem 32 1491, 1986. 

M Adamczyk, et al. Immunoassay reagents for psychoactive drugs. Part 3. Removal of phenothiazine interferences in the quantification of tricyclic antidepressants. Ther Drug Monit 15 436, 1993. 

Ismail, A.A. (2005) A radical approach is needed to eliminate interference from endoge nous antibodies in immunoassays. Clinical Chemistry, 51, 25 26. 

Matrix effects are particularly troublesome in immunoassay methods. Possible matrix interference and non-specific binding must be evaluated and documented in a number of different ways during the method vahdation ... 

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... 

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