Immunoassay for hapten

The non-competitive FI immunoassay for haptens recently reported by Gunaratna and Wilson [219] offers rather an interesting means for developing flow-through sensors for this type of compound. 

Fig. 4. Classification of reported noncompetitive immunoassays for haptens based on the assay principle. (A) Assays that include a chemical modification of hapten to allow sandwich-type detection. (B1) Improved single-antibody immunometric assays that separate immune complex and excess labeled antibody, either by using a hapten-immobilized affinity column or based on differences in their physical properties. (B2) A variation of single-antibody immunometric assays based on masking of unoccupied antibody by an immunoreactive macromolecule followed by selective capture and detection of the hapten-occupied antibody. (C) Assays employing a probe molecule specific to a hapten-antibody complex.

Ishikawa, E., Tanaka, K., and Hashida, S., Novel and sensitive noncompetitive (two-site) immunoassay for haptens with emphasis on peptides. Clin. Biochem. 23, 445 53 (1990). 

Grant, S., A. Porter, and W. Harris (1999). Comparative sensitivity of immunoassays for haptens using monomeric and dimeric antibody fragments. J. Agric. Food Chem., 47 340-345. 

A Jacklitsch. Separation-free enzyme immunoassay for haptens. In TT Ngo and HM Lenhoff, eds. Enzyme-mediated Immunoassay. New York Plenum Press, 1985, p 33. 

The remarkable selectivity that is inherent in the reaction of an antibody with the antigen or hapten against which it was raised is the basis for the extensive use of immunoassay for the rapid analysis of samples in clinical chemistry. Immunochemical reactions offer a means by which the applicability of potentiometric techniques can be broadened. A number of strategies for incorporating immunoassay into the methodology of potentiometry have been explored... 

Immunoassays for diuron (Figure 13) are another example of improved assay performance using heterologous assay conditions. One antibody was derived from a hapten that extended the dimethylamine side chain of diuron with methylene groups. 

Figure 13 Structures of haptens used for immunizing and coating antigens in a monoclonal antibody-based immunoassay for diuron. A sensitive assay was developed using coating hapten I that had the handle in a position different from the immunogen hapten. When the oxygen in the urea moiety of hapten I was replaced with a sulfur (hapten 11), increasing the heterology, even greater sensitivity was achieved...

Bocher, M., Giersch, T., and Schmid, R.D. (1992) Dextran, a hapten carrier in immunoassays for s-triazines. A comparison with ELISAs based on hapten-protein conjugates./. Immunol. Meth. 151, 1-8. 

L2. Lovgren, U., Kronkvist, K., Backstrom, B., Edholm, L.-E., and Johansson, G., Design of noncompetitive fiow injection enzyme immunoassays for determination of haptens Application to digoxigenin. J. Immunol. Methods 208, 159-168 (1997). 

T4. Tanaka, K., Kohno, T, Hashida, S., and Ishikawa, E., Novel and sensitive noncompetitive (two-site) enzyme immunoassay for h tens with amino groups. J. Clin. Lab. Anal. 4,208—212(1990). T5. Towbin, H., Motz, J., Oroszlan, R, and Zingel, O., Sandwich immunoassay for the hapten angiotensin II. A novel assay principle based on antibodies against immune complexes. J. Immunol. Methods 181, 167-176 (1995). 

M. Franek, M.V. Pouzar and V. Kolar, Enzyme-immunoassays for polychlorinated biphenyls. Structural aspects of hapten-antibody binding, Anal. Chim. Acta, 347 (1997) 163-176. 

Lee, N., D.P. McAdam, and J.H. Skerritt. 1998. Development of immunoassays for type II synthetic pyrethroids. 1. Hapten design and application to heterologous and homologous assays. J. Agric. Food Chem. 46 520-534. 

Pastor-Navarro, N., S. Morais, A. Maquieira, et al. 2007. Synthesis of haptens and development of a sensitive immunoassay for tetracycline residues Application to honey samples. Anal. Chim. Acta 594 211-218. 

Schneider, P. and B.D. Hammock. 1992. Influence of the ELISA format and the hapten enzyme conjugate on the sensitivity of an immunoassay for S-triazine herbicides using monoclonal-antibodies. J. Agric. Food Chem. 40 525-530. 

Rn 6). Similar approaches could be applied to the development of an immunoassay for tetrachlorodibenzo-p-dioxin using the recently synthesized l-amino-2,3,7,8-tetrachlorodibenzo- -dioxin as a hapten (28). 

F2. Freytag, J. W., and Litchfield, W. J., Liposome-mediated immunoassays for small haptens (digoxin) independent of complement. J. Immunol. Methods 70, 133-140 (1984). 

Monroe, D. (1989). Novel Liposome Immunoassays for Detecting Antigens, Antibodies, and Haptens. J. Liposome Res. 1 339 - 377. 

Blecka, L.J. Shaffar, M. Dworschack, R. Inhibitor enzyme immunoassays for quantitation of various haptens a review. In Immunoenzymatic Techniques Avrameas, S., Dmet, P., Mosseyeff, R., Feldman, G., Eds. Elsevier Amsterdam, 1983 207. 

Table 9.3 shows the effect of increase in molar ratio of an atrazine hapten coupled to the enzjune p-galactosidase (p-GAL) in a flow immunoassay for the determination of atrazine. A high hapten density had a negative impact on the assay sensitivity, i.e., the lowest MDC and IC50 values were obtained for the lowest molar ratio of hapten to enzyme. 

The low detection limits of immunoassays depend on the typically high affinities of antibodies for haptens and antigens, as well as the detection limits of the labels used. The detection limit is usually defined as the concentration that yields a signal that is equal to the mean of the blank signal plus two or three standard deviations. This establishes the confidence range for the zero response. For this calculation, the bound/free versus concentration plot is used. While detection limits allow comparisons of different immunoassay methods at the lower concentration end, they say nothing about assay reliability for this reason, both detection limits and precision profiles should be compared. 

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