Methods for detecting the presence and/or quantity of an immunoreactant, such as an antibody or antigen, present in a human physiological fluid or tissue test sample often employ labelled reagents in a specific complexing process for the detection of these immunoreactants. Commonly used labels include radioactive, enzymatic, fluorescent and chemiluminescent labels. The label chosen for a particular detection method often depends upon the balancing of the advantages and disadvantages of the labels under consideration. Factors such as label size, label stability, available instrumentation, practicability and sensitivity influence the choice of label. See R. M. Dauphinais, Solving and preventing problems in ligand assay, in N. R. Rose et al., Manual of Clinical Laboratory Immunology, Third Edition, Ch. 16, pp. 88-98 (1986).
The avidin-biotin labelling technique has been shown to provide a high level of sensitivity. E. A. Bayer et al., The use of avidin-biotin complex as a tool in molecular biology. Methods Biochem. Anal. 26:1-43 (1980). Avidin is a basic glycoprotein derived from egg albumin which has a molecular weight of 68,000 daltons. It has a high affinity for the vitamin biotin which has a molecular weight of approximately 244 daltons. Each avidin molecule has four binding sites for biotin. The avidin-biotin system employs an immunoreactant which is usually an antibody linked to biotin and a label which is usually an enzyme linked to avidin. The strong interaction between avidin and biotin is used to link the biotinylated antibody to the enzyme-labelled avidin, thus serving as a means of amplifying the sensitivity of the immunoassay in which it is so used. The avidin-biotin labelling system thus has been employed to enhance sensitivity over other labelling systems, including the direct enzyme conjugate labelling system. U.S. Pat. No. 4,228,237 describes a method to determine the presence of a ligand, such as, a labelled antibody, in a liquid medium which utilizes enzyme-labelled avidin and a biotin labelled reagent. Also, U.S. Pat. No. 4,684,609 teaches a biotinavidin label and its use for labelling tissue sections.
Although the avidin-biotin labelling system can provide an enhanced signal over previous labelling systems, problems are encountered with its use. Since the biotin and avidin usually are coupled with a ligand and a label, an additional incubation step and additional washing steps are required to be performed. Furthermore, the hydrophobic nature of the biotin molecule gives rise to higher background "noise" and false positive reactions in the performance of an assay method which utilizes avidin and biotin. Background noise is herein defined as the binding of the labelled species or fragments thereof to reagents in the assay system which causes non-specific precipitation of interference that is unrelated to the specific protein-bound fraction.
Direct antibody-enzyme conjugates are well known in the immunoassay art and have been described in the literature. See, for example, E. Ishikawa et al., J. Immunochemistry 4(3):209-327 (1983). The degree of sensitivity obtained with these direct antibody-enzyme conjugates, however, often is much lower than the degree of sensitivity obtained with the avidin-biotin system. This lower degree of sensitivity is due to the low enzyme-to-antibody ratio obtained for these conjugates in these preparations. In fact, the majority of these direct antibody-enzyme conjugate preparations give enzyme-to-antibody rations of 0.3 to 1.0 (E. Ishikawa, ibid, page 221). Higher enzyme-to-antibody ratios usually are not desirable, if obtainable, because of the potential loss in antibody activity and higher background "noise".
It would be advantageous to provide a labelling system wherein direct antibody-enzyme conjugates could achieve high enzyme-to-antibody ratios and therefore provide a higher degree of sensitivity approaching or equal to the avidinbiotin labelling system. Such an improved direct antibody-enzyme conjugate would not require the additional incubation step and washing steps as the biotin-avidin labelling system requires. Also, such a system would avoid the problems of using a hydrophobic molecule such as biotin, and therefore, lessen the chance of higher background "noise" and false positive reactions.
The invention herein provides for such a improved direct antibody-enzyme conjugate and a labelling system for direct antibody-enzyme conjugates which achieves a high enzyme-to-antibody ratio of approximately three (3) without any significant loss of antibody activity or higher back ground "noise". This conjugate was prepared by using long chains between the antibody and enzyme molecules. In the procedure, sulfhydryl groups are introduced to the antibody. Additionally, maleimide groups are introduced into an enzyme, for example, Horseradish Peroxidase. This modified antibody and modified enzyme are combined and allowed to react. A mean enzyme-to-antibody ratio of approximately three (3) is achieved. A higher level of sensitivity is provided by the improved method without any increase in background "noise". Thus, one incubation and several washing steps required by the biotin-avidin labelling system are eliminated by the labelling system of this invention. It also eliminates the false positive reactions obtained by the use of hydrophobic biotin molecule in the biotin-avidin enzyme indirect method described hereinabove.