Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1991-04-08
1993-05-11
Kepplinger, Esther L.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435962, 436518, 436531, 436826, G01N 3353
Patent
active
052100200
DESCRIPTION:
BRIEF SUMMARY
DESCRIPTION
1. Technical Field
The present invention relates generally to the field of quantitation of water borne analytes by immunoassays and, specifically, with assay conditions that enhance the binding reactions that occur in these assays.
2. Background Art
One of the most sensitive versions of immunoassays is the sandwich technique. This technique includes two binding reactions. In one, a sample analyte molecule to be quantitated is bound to an antibody immobilized to a solid phase support (first binding), and the excess unbound sample analyte molecule is removed by washing. In the second, an excess amount of a labeled antibody is added thereto to bind it to the sample analyte molecule on the support (second binding). Thus, the analyte molecule is sandwiched between the immobilized antibody and the labeled antibody and the excess unbound labeled antibody is removed by washing. Finally, a signal is developed from a label substance (e.g., enzymes, radioactive isotopes, etc.) of the labeled antibody by suitable means. The amount of the analyte molecule is determined by measuring the amount of the signal. This is the normally employed general sandwich technique. In addition to this, there is another technique wherein a sample analyte molecule to be quantitated is firstly reacted with a labeled antibody and then they are added to an immobilized antibody to form a sandwich (reverse sandwich technique), or an immobilized antibody, a sample analyte molecule to be quantitated and labeled antibody are simultaneously admixed to react them (one step sandwich technique).
The above two binding reactions of a sandwich technique are of paramount importance. Specific binding among the immobilized enzyme, the analyte molecule and the labeled antibody that takes place during the first and second binding reactions results from the specific affinity among them (e.g., between the antigen and the antibody, etc.). On the other hand, non specific binding is defined as accidental binding of the labeled antibody to the solid phase directly. This type of binding causes a background signal in the assay that is not proportional to the analyte concentration. These specific binding and non-specific binding reactions significantly influence sensitivity and performance of these assays. Thus, one aim of the art is to minimize non-specific binding of the labeled antibody to the support and maximize specific binding between the labeled antibody and analyte bound to the support.
A technique found to be useful for this aim has been to include particular substances which have the property of increasing the specific binding or decreasing the non-specific binding in an incubation solution (reaction mixture) containing (1) the analyte molecule bound to the immobilized antibody and the labeled antibody, (2) the immobilized antibody and the analyte molecule bound to the label substance, or (3) the immobilized antibody, the label substance and the analyte molecule. For example, it was shown that use of high salt concentration in an ELISA (Enzyme Linked Immunosorbent Assay) incubation solution frequently improves the binding reactions [Hashida et al., Clin. Chim. Acta, 135, 263-273 (1983)]. Others have found that inclusion of gelatin in an incubation solution is helpful to decrease non-specific binding and thereby improve sensitivity of ELISAs [Kato et al., Febs. Lett. 99, 172-174 (1979)]. Proteolytically degraded gelatin was also found to be helpful in this regard. Milk protein (casein) is another additive that was found to improve assay performance by decreasing non-specific binding [Johnson et al., Gene. Anal. Techn., 1, 3-8 (1984)]. Although these substances alleviate the problem, they do not work in every system and also much more needs to be done to lower the non-specific binding of labeled antibody to the solid phase support. Additionally, many of these substances decrease both specific and non-specific binding. This creates a new problem by lowering detection sensitivity. For example, addition of extra protein to an incubation solution co
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patent: 5077198 (1991-12-01), Shih et al.
Yanagisawa et al., Nature, vol. 332, pp. 411-415, (1988).
Sigma Chemical Company, 1988 Price List, p. 317.
Hashida, et al., Clinica Chimica Acta, 135:263-273 (1983).
Kato, et al., FEBS Letters, 99(1):172-174 (1979).
Johnson, et al., Gene Anal. Tech., 1:3-8 (1984).
Chemical Abstracts, vol. 99, No. 9, Aug. 29, 1983, (Columbus, Ohio, US), J. F. Kennedy et al., p. 456, abstract 68597z, & Eur. J. Biochem. 1983, 138 (3), 697-705.
Kondo Koichi
Motsenbocker Marvin A.
Buckley Linda M.
Conlin David G.
Kepplinger Esther L.
Takeda Chemical Industries Ltd.
Wolski Susan C.
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