Antibodies immunoassay development

Direct and indirect competition formats, illustrated in Figure 1, are widely used for both qualitative and quantitative immunoassays. Direct competition immunoassays employ wells, tubes, beads, or membranes (supports) on to which antibodies have been coated and in which proteins such as bovine semm albumin, fish gelatin, or powdered milk have blocked nonspecific binding sites. Solutions containing analyte (test solution) and an analyte-enzyme conjugate are added, and the analyte and antibody are allowed to compete for the antibody binding sites. The system is washed, and enzyme substrates that are converted to a chromophore or fluorophore by the enzyme-tracer complex are added. Subsequent color or fluorescence development is inversely proportionate to the analyte concentration in the test solution. For this assay format, the proper orientation of the coated antibody is important, and anti-host IgG or protein A or protein G has been utilized to orient the antibody. Immunoassays developed for commercial purposes generally employ direct competition formats because of their simplicity and short assay times. The price for simplicity and short assay time is more complex development needed for a satisfactory incorporation of the label into the antibody or analyte without loss of sensitivity. 

CK-2 levels are measured in numerous ways. The present discussion will concentrate on the immunoassays that use monoclonal anti-CK-2 antibodies.Immunoassays developed in recent years have improved on the analytical and chnicai sensitivity and specificity of the earlier immuno inhibition and immunoprecipitation assays. These assays now (1) measure CK-2 directly and provide mass measurements, (2) are easily automated, and (3) are rapid (<30 minutes). This allows laboratories the ability to offer... 

Whereas MABs appear to be the choice for use in immunoassays, a majority of immunoassay developers and suppHers use polyclonal antibodies. 

Alternatively, competitive ELISA can be used to estimate the hapten density if an antibody that specitically recognizes the hapten is available. At first observation this approach seems circular because the immunoassay developed is used to determine hapten density on proteins used for immunization. However, if a small molecule mimic of the protein conjugate is used as a standard, the method can be accurate. For example, a hapten containing a carboxylic acid can be coupled to phenethylamine or tyramine, its structure confirmed and the material used to generate a calibratron curve to estimate hapten density. 

Figure 1 Schematic sequence of the direct and indirect competitive ELISA. The principle difference is that for direct competitive immunoassay, the well is coated with primary antibody directly, and for indirect competitive immunoassay, the well is coated with antigen. Primary antibody (Y), blocking protein (Y), analyte (T), analyte-tracer ( ), enzyme labeled secondary antibody ), color development ( J)...

Varenne, A., Vessieres, A., Salmain, M., Brassier, P., and Jaouen, G. (1995) Production of specific antibodies and development of a nonisotopic immunoassay for carbamazepine by the carbonylmetallo-immunoassay (CMIA) method./. Immunol. Meth. 186, 195-204. 

Allen RL, Manning W, McKenzie KD, et al. 1992a. Development of a monoclonal antibody immunoassay for the detection of gasoline and diesel fuel in the environment. Assoc Am Railroads Contaminated Soils-Diesel Fuel Contamination Research Triangle Park NC. 

Hoffman et al. [46, 47] found that an LCST polymer remains strongly bound to a substrate, especially to cellulose acetate (CA), at a temperature above its LCST, whereas most of the adsorbed polymer molecules are easily rinsed off below the LCST. For instance, they synthesized a room-temperature-precipit-able terpolymer (LCST = 7-13 °C), consisting of IPAAm, A-butylacrylamide (BAAm) and N-acryloxy succinimide (NASI), which was conjugated to a murine monoclonal antibody. They developed the membrane-affinity concentration immunoassay [48]. 

Marco, M.P. and B.D. Hammock (1995). Immunochemical techniques for environmental analysis. II. Antibody production and immunoassay development. Trends Anal. Chem., 14 415 125. 

Nichkova, M., R. Galve, and M.-P. Marco. 2002. Biological monitoring of 2,4,5-trichlorophenol (I) Preparation of antibodies and development of an immunoassay using theoretical models. Chem. Res. Toxicol. 15 1360-1370. 

Siddle, K., and Soons, M., Monoclonal antibodies for human pituitary glycoprotein hormones and rabbit immunoglobulin. In "Monoclonal Antibodies and Developments in Immunoassay" (A. Albertini and R. Ekins, eds.), pp. 53-66. Elsevier, Amsterdam, 1981. 

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... 

The first immunoassay developed was based on the principle described above, with an insulin competitor, i.e., an analyte derivative labelled with a radioactive isotope ( I) and the anal3d e (insulin) competing for a limited amoimt of immobilized anti-insiilin antibodies. After the equilibrium was reached and the unbmmd competitor was removed, the residual radioactivity was correlated to the concentration of insulin [2], Since then, numerous other variants of competitive immimoassay formats have been described, either in a... 

Although high cross-reactivity is sometimes seen as a problem in immunoassay development, antibodies with a high CR for compounds within the same chemical class can favourably be used either for post-column detection of the cross-reactants separated by HPLC [60-64] or for selective SPE in columns with immobilized antibodies (immunoaffinity SPE), followed by HPLC separation of the trapped cross-reactants (see Fig. 9.12) [65]. 

Besides monoclonal and polyclonal antibodies. Fab or F(ab)2 fragments can be employed for immunoassay development. Their use is of considerable... 

In order to decide the lowest possible working concentrations of competitor and antibody, the development of a competitive immunoassay usually starts by performing a two-dimensional dilution experiment, i.e., the antibody concentration, [Ab], is varied in one dimension and the competitor concentration, [Ag ], is varied in the other. In this way the optimal [Ab] and [Ag ] can be determined experimentally and the lowest possible [Ag ] and [Ab] are usually selected, when 30-70% of the tracer is bound to the antibody ([Ab-Ag ]). However, the contradiction in defining immunoassay sensitivity, as mentioned above, has led to different values of the theoretically predicted... 

Production of Polyclonal Anti-Plcloram Antibody. Picloram antisera was obtained from New Zealand White rabbits following the protocol described by Hall et al. (15). The rabbits were injected subcutaneously with an emulsion consisting of 0.5 to 1.0 mg immunogen dissolved in 0.5 mL of PBS and an equal volume of Freund s complete adjuvant. The injections were repeated 3, 6, and 10 days after the initial injection, substituting Freund s incomplete adjuvant for complete adjuvant. A booster injection was given one month after the initial injection and was repeated at monthly intervals thereafter. The rabbits were bled for antibody titer determinations 10 days after each boost. Antisera for picloram immunoassay development were prepared from a single bleed in each case. 

Generation of Antibodies. Because of their small molecular weight, pesticides such as alachlor are not generally immunogenic (4)- A key step in immunoassay development therefore involves the covalent conjugation of the pesticide or an appropriate analogue to a carrier protein ( ). In considering various... 

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. 

Christofides ND, Sheehan CP. Enhanced chemiluminescence labeled-antibody immunoassay (Amerlite-MAB) for free thyroxine Design, development, and technical evaluation. Clin Chem 1995 41 12-23. 

Micheli, L., Di Stefano, S. Moscone, D., Palleschi, G., Marini, S., Coletta, M., Draisci, R. and delli Quadri, F. Production of antibodies and development of highly sensitive formats of enzyme immunoassay for saxitoxin analysis. Anal. Bioanal. Chem., JiTi, 678-684 (2002). 

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