Immunoassay double-antibody method

Heterogenous fluorescence immunoassays can be carried out with the aid of the same separation procedures used in radioimmunoassay. The more expedient homogenous fluorescence immunoassays require quenching, enhancement, polarization, or shifting of the fluorescence of the label upon binding of the labeled analyte to its antibody. Occasionally, a second antibody, directed at the anti analyte antibody, will be used in a double-antibody method to precipitate the bound labeled and unlabeled analyte or to alter the optical properties of the label in such a way as to make the analysis more sensitive. 

Although unlike RIA, enzyme immunoassays can be carried out in homogeneous systems without a separation step (based on the change in enzyme activity during the immune reaction), in practice, heterogeneous (enzyme-linked immunosorbent assay) methods are much more frequently used. The antibody, or in the case of the double-antibody method the second antibody, is immobilized, either covalently or by coating enzyme multiplied immunoassay technique (EMIT). This can be done on the walls of microtiter plates. After the immunogenic reaction, the enzyme activity, which is the equivalent of radioactivity in RIA systems, can be measured by suitable photometric methods on the microtiter plates themselves. 

These four methods have been combined to develop a number of EIAs for quantifying antigens, antibodies, and haptens. For example, the enzyme multiplied immunoassay technique (EMIT ) is a commercially available homogeneous and competitive EIA for the determination of a variety of drugs. In addition, double antibody methods that utilize a heterogeneous and competitive EIA system are available. In this case, enzyme-labeled antigen and sample antigen are mixed... 

In 1976, we developed a double-antibody enzyme immunoassay of TSH using alkaline phosphatase, but this method was not very sensitive (M8). As shown in Table 10, several other methods were later reported (A3, 12, K2, K3, M7) and two methods have been used to measure TSH in dried blood samples in screening for neonatal hypothyroidism in Japan. 

Bovine serum albumin (BSA) and cyclic AMP (cAMP) are determined by a competitive binding enzyme immunoassay (315). With urease as label, an ammonia gas-sensing electrode is used to measure the amount of urease-labeled antigen bound to a double-antibody solid phase by continuously measuring the rate of ammonia produced from urea as substrate. The method yields accurate and sensitive assays for proteins (BSA less than 10 ng/mL) and antigens (cAMP less than 10 nM), with fairly good selectivity over cGMP, AMP, and GMP. 

Traditionally, circulating concentrations of Tg have been measured using double-antibody immunoassays. Commercial RIA kits based on sequential addition techniques have been developed for routine use. These methods involve the preliminary incubation of serum with primary antibody. After a number of hours (2 to 72), the Tg tracer is added and allowed to compete for antibody binding sites. The longer the preincubation the more sensitive the method. Separation of bound from free Tg is then accompHshed by precipitation with a second antibody. 

Aggerbeck, H., Norgaard Pedersen, B., and Heron, I. (1996) Simultaneous quantitation of diphtheria and tetanus antibodies by double antigen, time resolved fluorescence immunoassay. Journal of Immunological Methods, 190, 171 183. 

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