Specific binding assays using methyl orange

Details Specific binding assays using methyl orange Specific binding assays using methyl orange

1999-06-08

2001-06-26

Wortman, Donna C. (Department: 1645)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving virus or bacteriophage

C435S007100, C435S007900, C435S007920, C435S960000, C436S518000, C436S544000, C436S820000

Reexamination Certificate

active

06251584

ABSTRACT:

BACKGROUND OF THE INVENTION
Specific binding assays, for example immunoassays, which take advantage of natural binding reactions, have found wide-spread use as analytical techniques in clinical chemistry. Because of the specificity of the reactions, they are particularly advantageous in quantifying biological analytes that are present in very low concentration in biological fluids. Such analytes include, for example, antigens, antibodies, therapeutic drugs, narcotics, enzymes, hormones, proteins, etc.
Many dyes or colorants (hereinafter “dyes”) are used in commercial immunoassay coating procedures to aid monitoring of the dispensing of a reagent or spectrophotometric monitoring of the dispensing of reagent. Many of the dyes are commercially available. Basically, the dye allows for visual detection. This can be particularly useful in monitoring the dispensing of solutions into vessels, e.g., microwells during manufacturing processes for coating biological materials onto the surface of the microwells. The coated vessels are subsequently used as solid phase in immunoassays. It is desirable that the dyes that are used do not interfere with the biological nature of proteins, which are coated onto the solid phase support. However, some proteins can interact with dyes and, as a result, this interaction reduces assay performance. For example, this has been a recognized problem in the manufacturing of microwells for assays for detecting antibodies to hepatitis C virus (HCV).
Therefore, one object of the present invention is to provide a dye that can be used in a solid phase coating comprising HCV antigens and to aid the monitoring of microwell fill volume and yet, does not have a detrimental effect on proteins, specifically HCV antigens, present in an anti-HCV assay. Another object of the present invention is to provide a dye that will actually improve assay performance.
SUMMARY OF THE INVENTION
The invention relates to adding dyes, particularly methyl orange, to a coating solution used in a procedure to prepare a solid phase for use in a specific binding assay and for monitoring the volume of coating solution dispensed into a vessel. Many dyes were investigated for use in anti-hepatitis C virus immunoassay coating formulations to enable a colorimetric monitoring and process control of coating volumes dispensed in the solid phase. Many of the dyes reduced the activity of the HCV recombinant coating proteins. Unexpectedly the applicant found that upon the addition of methyl orange not only could volumes of coating solution be monitored, but also, the use of methyl orange as a dye positively interacts with proteins, especially HCV antigens, resulting in improved specificity of results in an assay. Therefore, as shown by the comparison of
FIGS. 1
a
and
1
b
, a solution for coating a solid phase comprising methyl orange allows for greater discrimination of negative samples from positive samples.
Therefore one embodiment of the present invention provides a solid phase to be used for a specific binding assay comprising at least one immobilized HCV antigen that has been treated with methyl orange. A preferred embodiment of the invention is where the HCV antigen is expressed from the NS3 and/or NS4 regions of the viral genome. A more preferred embodiment is where the HCV antigen is c200.
It is a further object of the present invention to provide a method for improving the specificity of an anti-HCV immunoassay by adding methyl orange to a coating solution in an amount sufficient for improving specificity of an immunoassay conducted using said coating.
In a preferred embodiment of this aspect of the invention, a method for the detection of antibodies to hepatitis C virus is performed by, i) providing a solid phase comprising a coating solution comprising methyl orange and at least one first binding ligand for antibodies to hepatitis C virus; ii) contacting the solid phase with a sample that may contain antibodies to hepatitis C virus; iii) contacting the solid phase with at least one second binding ligand for antibodies to hepatitis C virus, said second ligand labelled directly or indirectly with a detectable group and iv) measuring the amount of the detectable group bound to the solid phase. Alternatively, the amount of detectable group not bound to the solid phase can be measured as an indication of the presence of antibodies to HCV. The detectable group can be, for example, an enzyme, a radioactive atom, a fluorescent molecule or a luminescent molecule.
A preferred detectable group is an enzyme. The assay can be carried out using any enzyme label, which can be attached to the ligand to form a labelled ligand. Enzymes such as oxidases, e.g., glucose oxidase, peroxidases, e.g., horseradish peroxidase (HRP), alkaline phosphatase and galactosidases are preferred labels. It is within the skill of the ordinary worker in clinical chemistry to determine a suitable substrate for a given label. The substrate can be a material that is directly acted upon by the enzyme label or a material that is involved in a series of reactions, which involve enzymatic reaction of the label.
It will be understood by one of ordinary skill in the art that steps i) through iv) above can be done sequentially or simultaneously. It will also be understood that the first and second binding ligands can be the same or different from themselves or each other. Furthermore, the amount of detectable group measured can be correlated to the amount of anti-HCV present in the sample.
Finally, another embodiment of the present invention provides a method for coating a solid phase for an immunoassay, the improvement comprising: adding methyl orange to a coating solution in an amount sufficient for improving specificity of the immunoassay.
Other advantages of the present invention will become clear from the following more detailed description and the following examples.


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van der Poel, Cees L., et al., “Hepatitis C virus six years on”,The Lancet, vol. 344, Nov. 26, 1994 pp. 1475-1479.
Uyttendaele, S., et al., “Evaluation of the Third-Generation Screening and Confirmatory Assays for HCV Antibodies”,Vox Sang1994;66:122-1290.

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