Double monoclonal antibody assay

A double monoclonal antibody assay is commercially available that uses the synergistic action of two immunoin-hibitory monoclonal antibodies to S-AMY. After the S-AMY activity is inhibited by the addition of the antibodies, the uninhibited P-AMY activity is measured using EPS-4-NP-G7 as a substrate. The test has been the object of a number of evaluations that have described its characteristics, such as practicability even in emergency conditions, possibility of full automation, and high specificity for P-AMY.Falsepositive P-AMY results have been reported in subjects with macroamylasemia, in whom the immunoglobulin com-plexed to AMY forms diminishes or voids the ability of monoclonal antibodies included in the test to efficiently inhibit S-AMY. 

Tietz NW, Burlina A, Gerhardt W, Junge W, Malfertheiner P, Murai T, et al. Multicenter evaluation of a specific pancreatic isoamylase assay based on a double monoclonal-antibody technique. Clin Chem 1988 34 2096-102. 

Valdivieso-Garcia, A. Riche, E. Abubakar, O. Waddell, T. E. Brooks, B. W. A double antibody sandwich enzyme-linked immunosorbent assay for the detection of Salmonella using biotinylated monoclonal antibodies. J. Food Prot. 2001, 64,1166-1171. 

Fig. 7. Schematic representation of the principle of TUNEL assay. The enzyme TdT catalyzes a template-independent addition of bromolated deoxyuridine triphosphates (Br-dUTP) to the 3 -OH ends of double- and single-stranded DNA. After Br-dUTP incorporation, DNA break sites are identified by an FITC-labeled anti-BrdU monoclonal antibody.

It is not always possible to use exactly the same format for screening as will be used in the final assay but it is advisable to attempt to ensure that the position that the monoclonal antibodies (MAb) will occupy in the final format is the position that is used for screening. For example MAbs which will eventually be conjugated to enzymes and used in Double Antibody Sandwich (DAS) ELISA should be screened for using TAS ELISA. This ensures that the epitope-binding portion of the antibody is in the same position as it will be in the final test format with the analyte bound to the coating antibody. 

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... 

Generally, polyclonal antibodies are easier to produce, and high-affinity polyclonal antibodies can be obtained. Monoclonal antibodies are more specific to a certain epitope. They provide continuous production of exactly the same defined reagent and are more preferable for excess-reagent assays. The double sandwich technique has used two antibodies from monoclonals or combinations of mono- and polyclonals, with specificity against two different epitopes of the analyte. One antibody functions as a capturing antibody for the analyte and the other as the label carrier (118). 

The purified anti-TKSNVYGK monoclonal antibody was used to quantify E. coli OmpA throughout the process of purification of P40. A sequential double-sandwich ILA assay, based on the use of two specific anti-... 

Dissociation constants for polymer-template complexes were obtained from competitive assay experiments, using a double reciprocal plot. These values were compared to those obtained for polyclonal and monoclonal antibodies selective for... 

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. ... 

Many of the early ELISA methods devised for botulism neurotoxin detection, like most of the in vitro tests,suffered from a lack of specificity, due to impurities in the antigen preparation used to produce the antitoxins. More purified toxins are now available for the production of better quality antitoxins. The most sensitive ELISA protocols use an indirect assay sometimes referred to as the sandwich assay. In the basic procedure, a specific antitoxin is first adsorbed to the surface of the wells of a plastic plate. The toxin added to the wells is then bound by these antibodies and detected with a second antitoxin which is conjugated to an enzyme or other labeling molecule. The amount of label is measured by supplying the enzyme substrate, which is converted to a colored product that is measured colorimetrically. Some ELISA protocols use a polyclonal antitoxin on one side of the sandwich and a monoclonal on the other side. Other assays use the same antibody for both sides but label the antibody the second time it is used. A modification of the sandwich assay is the double sandwich ELISA, which employs a third antibody that is conjugated to an enzyme and is directed against the second antitoxin it is an anti-antibody such as rabbit anti-horse IgG. The steps in a typical application of this assay for botulism toxin are shown in Figure 2. 

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