Antibody, double immunoassay

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. 

This is a non-competitive immunoassay using ferrocene carboxylic acid 7 as the redox mediator of horseradish peroxidase (POD), which is itself attached to the detection antibody (Scheme 8.21) (91). When an anode potential is applied, a catalytic current is generated at the surface of the electrode, with an intensity proportional to the quantity of POD located in close proximity to the surface of the electrodes, and thus proportional to the quantity of captured analyte. Applying several antibodies sequentially on the same electrode permitted a double immunoassay of LH and FSH with detection limits respectively of 2.1 and 1.8 lU L. ... 

As described above, the dendrimer-coupled antibody conjugates show uniquely enhanced properties compared to the classical double antibody systems. Important characteristics such as complete solubility in aqueous buffers, flexibility in immunoassay format, ability to improve assay sensitivity, consistent, reproducible manufacturing and favorable stability has driven the utilization of these dendrimer-based reagents in Stratus CS, the latest member of the Stratus family of immunochemistry analyzers. In this new analyzer system, the primary... 

Mild conjugation reactions have been used in an enzyme-linked immunoassay for detecting cephalexin residues in milk, hen tissues, and eggs. The assay was a double-antibody separation procedure based on use of a rabbit antiserum... 

In 1982, the first enzyme immunoassay of clenbuterol was described (134). It was used to determine clenbuterol levels in plasma of human patients treated by oral route with this drug. It was a highly sensitive double-antibody and heterologous immunoassay based on a competition for binding to a clenbuterol-specific antibody between a diazotized clenbuterol analogue labeled with -galactosidase and unlabeled standard or sample clenbuterol. The antibody-bound enzyme hapten was separated from free hapten by anti-rabbit IgG immobilized to a polystyrene ball. The assay could detect levels as low as 0.5 pg clenbuterol per tube. 

Fig. 6. Comparison of immunoassays using clonotype antibodies. The EDso values (thyroxine concentration corresponding to 50% of bound form at zero concentration of thyroxine) in thyroxine immunoassays using clonotype antithyroxine antibody prepared by fast protein liquid chromatography (FPLC). RIA, double-antibody radioimmunoassay FLA, fluorescence polarization immunoassay EIA, enzyme immunoassay. [Cited from Miyai et al. (M5).]...

The TRH radio-immunoassay was developed as a double antibody assay, with modifications mainly depending on the antiserum used, and on the conditions of incubation. Addition of an enzyme inhibitor to the assay tubes is essential to avoid degradation of TRH in samples and of radioiodinated TRH. 

The assay described below in more detail uses the principle of a competitive immunoassay with a double antibody precipitation (Chard 1990). The antibody (from guinea pig) bound tracer is separated from the free tracer by the second antibody present in the precipitating reagent (goat anti guinea pig antibody). After... 

In addition to ELISA and Western blots for detecting host-cell protein impurities there is a third immunoassay now available to the bioanalyst for this purpose the immunoligand assay (ILA). Like the ELISA, it is configured as a double-antibody sandwich. The anti-host-cell protein antibodies are separately conjugated to biotin and to fluorescein. A tripartite immune complex is formed between host-cell protein impurities and these two anti-... 

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. 

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)]. 

Fig. 1. Schematic representation of double-antibody solid phase enzyme immunoassay using cross-linked second antibody particles for separation. Encircled E, enzyme label.

Although most immunoassays have used polyclonal antibodies as the critical binding reagents, development of monoclonal antibodies by Kohler and Milstein in 1975,has resulted in their widespread use, particularly in assays for macromolecules. Their unique epitope specificity conveys advantages in double antibody immunoassays for proteins, where one monoclonal antibody may be used to capture the protein by a specific subunit or epitope, and another, directed against a... 

In contrast to competitive immunoassays, the number of theoretical models describing the kinetics and thermodynamics of non-competitive immunoassays is considerably lower and most of them are derived from double-site immunometric configurations. The dose-response curve for such assays is sigmoid the signal increasing with the analyte concentration with a plateau value reached when the capture antibody becomes saturated. 

We have also applied ELISA to several biological pesticides including the endotoxin of Bacillus thurineiensis kurstaki (Btk). In this application to a macromolecular analyte, we have used a double antibody sandwich ELISA for Btk to measure the amount of ELISA reactive material in formulations of the pesticide. Figure 7 shows the use of an ELISA standard curve of gel purified Btk endotoxin to measure the immunoreactive material in dilutions of two Btk formulations. It has been demonstrated that ELISA can serve as a quick quality control check for formulations of Bacillus thurineiensis lsraelensis (44). Such examples indicate that immunoassays will be increasingly important as biologicals and products of recombinant DNA research impact our field (M) ... 

Figure 7. Standard curve of gel purified 60 kd protein endotoxin of Btk was generated using a double antibody sandwich ELISA. The arrows indicate dilutions of two Btk formulations absorbance values were used to determine the endotoxin concentrations of the formulations, based on the standard curve. The formulation dilutions gave curves that were virtually superimposable on the standard curve. Such similarity in shape and slope indicate that the antibody is likely binding to a specific determinant common to the purified Btk and the Btk in the formulation. In general immunoassays for biopolymers are much easier to develop than assays for small molecules. However, only recently has an interest in trace analysis of such materials begun to develop in the environmental field. Thus, sample cleanup and handling is not as sophisticated as with small molecules.

Heterogeneous immunoassay in which a piece of single- or double-stranded DNA is used as a label for an antibody in a sandwich assay. Bound DNA label is amplified using the polymerase chain reaction (PCR). The amplified DNA product is separated by gel electrophoresis and quantitated by densitometric scanning of an ethidium stained gel. 

Panteghini M, Pagani F. Diagnostic value of measuring pancreatic isoamylase with a double-monoclonal antibody immunoassay in serum of hospitalized hyperamylasemic patients. J Clin Lab Analysis 1990 4 449-52. 

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. 

In general, kits for the measurement of estradiol in sahva are not commercially available. However, Tamate and coworkers have reported the use of a direct enzyme immunoassay that shows promise for the measurement of salivary estradiol. This assay uses monoclonal anti-estradiol-coated microtiter plates and an estradiol-peroxidase conjugate, No extraction is required. These authors reported a sahvary estradiol peak of 15 to 31 pmol/L in the preovulatory phase and from 9 to 33 pmol/L in the midluteal phase. Lu and co-workers have described a double-antibody RIA using I-labeled estradiol conjugate for the determination of salivary estradiol. ... 

Since its introduction in medical research in 1971, various types of enzyme immunoassays have been developed (21) The double antibody sandwich ELISA (DAS-ELISA) introduced in plant pathology in 1976 (23) has become the major test system for plant indexing in the Netherlands This technique can be applied to a wide range of plant pathogens Figure 1 shows the principle for the complex antigen situation for detecting bacteria (23) ... 

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. 

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