Immunoassay data processing

Dudley RA, Edwards P, Ekins RP, et al. Guidelines for immunoassay data processing. Clin. Chem. (1985) 31 1264-1271. 

In this section we provide some practical considerations to chemists not familiar with the use of immunoassays for food contaminants. We focus primarily on the use of 96-well microtiter ELISA. Regardless of the type of sample and analysis, good laboratory practices (GLPs) and international standards organization (ISO) standards, where they apply, need to be followed to ensure the quality of results and the minimization of variability. Like any other analytical protocol, the analysis of contaminants by immunoassay is a combination of three sequential steps sample collection and preparation, sample analysis, and data processing followed by the interpretation of results. 

The evaluation process can be summarized by the following key milestones 1) an identified EPA need, 2) submission of an immunoassay with accompanying documentation to the EMSL-LV, 3) data review, 4) Agency evaluation studies to provide additional performance data, 5) report addressing applications and limitations of the method, and 6) implementation of the immunoassay into routine monitoring programs. 

The ability to perform quantitative assays on complex mixtures with little sample clean-up is perhaps the most attractive feature of immunoassays for application to agricultural chemistry. A large portion of the cost and labor involved in pesticide residue analysis is invested in sample extraction and clean-up steps to remove substances which may interfere with subsequent chemical analysis. Since most preparatory steps are not required prior to performing an immunoassay, samples can be analyzed much less expensively. This will permit the vast number of data points required for pesticide registration to be gathered in a more timely and cost-effective manner. Studies which were prohibitively expensive because they would have required large numbers of expensive assays can be completed using immunoassay procedures. Such studies may include analysis of pesticide movement from application areas and the rate of dissipation of pesticide from crop tissue, soils, and processed foods. 

Theoretical considerations and existing experimental data indicate that under certain conditions, the rate of photoisomerization strongly depends on the microviscosity around the isomerized molecule and upon the effect of steric hindrance. In a viscous medium, the apparent rate constant of trans-cis photoisomerization ki o is controlled by the reorganization rate of the process in the medium (Equations 4.2 and 4.3). This method was used for the measurement of fluidity of biological membranes and microviscosity of a specific site of a protein. On this theoretical basis, fluorescence-photochrome immunoassay (FPHIA)] were used. 

Major vitamin Bi2-dependent metabolic processes include the formation of methionine from homocysteine, and the formation of succinyl coenzyme A from methylmalonyl coenzyme A. Thus, apart from directly determining vitamin B12 concentration in serum, elevated levels of both methylmalonic acid and homocysteine may indicate a vitamin B12 deficiency. Serum cobalamine concentration is often determined by automated immunoassays using an intrinsic factor as binding agent. These assays have mainly replaced the microbiological methods. Literature data about vitamin B12 concentration in serum varies. Values <110-150pmoll are considered to reflect deficiency, whereas values >150-200pmoll represents an adequate status. 

One of the simplest possible applications of the LAPS/microflow chamber combination is the measurement of enzyme activity. One way to demonstrate this is to immobilize an enzyme in a chamber, and provide it with its substrate in the flow medium. As an example, let s consider acetyl cholinesterase, which catalyzes the hydrolysis of acetylcholine to acetate and choline, liberating protons in the process. Acetyl cholinesterase-coated agarose beads (Sigma) were immobilized between two thin polycarbonate membranes in a Cytosensor [4] chamber, and the pH response measured when an acetylcholine-containing medium is flowed through. Figure 5 shows the data from this experiment. Rates of about 120 pV/s are obtained. Note that the presence of the membranes slows down the time constant of the return to baseline of the pH during the flow-on periods. This demonstrates the measurement of enzyme activity, with possible applications to immunoassays. 

---