Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1997-11-18
1999-11-23
Budens, Robert D.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 5, C12Q 168, C12Q 170
Patent
active
059898194
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for quantitatively determining an antibody, contained in a biological sample, having the ability to inhibit the activity of a reverse transcriptase derived from a human immunodeficiency virus (HIV). More particularly, the present invention is concerned with a method for quantitatively determining an antibody, contained in a biological sample, having the ability to inhibit the activity of a reverse transcriptase derived from an HIV, which comprises the steps of: reacting a hybridization product (immobilized on a solid phase) of a primer consisting essentially of oligodeoxythymine nucleotide and an RNA template consisting essentially of adenine ribopolynucleotide with a reverse transcriptase derived from HIV in an aqueous reaction system, to bind the reverse transcriptase to the hybridization product, thereby obtaining a hybridization product-bound reverse transcriptase; conducting a synthesis of a complementary deoxyribonucleic acid to the RNA template from a deoxymononucleotide triphosphate added to the aqueous reaction system under the action of the hybridization product-bound reverse transcriptase in the presence of the antibody having the ability to inhibit the activity of the reverse transcriptase; and measuring the amount of the synthesized complementary deoxyribonucleic acid and quantitatively determining the antibody having the ability to inhibit the activity of the reverse transcriptase, based on the measured amount of the synthesized complementary deoxyribonucleic acid. More particularly, according to the present invention, there is provided a method for quantitatively determining an antibody, contained in a biological sample, having the ability to inhibit the activity of a reverse transcriptase derived from an HIV, in the above-mentioned steps, wherein the reverse transcriptase is reacted with the antibody present in the aqueous reaction system, and wherein the reaction of the reverse transcriptase with the antibody is conducted after the binding of the reverse transcriptase to the hybridization product and before the addition of the deoxymononucleotide triphosphate; or before the binding of the reverse transcriptase to the hybridization product and before the addition of the deoxymononucleotide triphosphate; or after the binding of the reverse transcriptase to the hybridization product and simultaneously with the addition of the deoxymononucleotide triphosphate and antibody.
Conventionally, it has been difficult to classify the antibodies against the viral antigens into groups according to the types of the epitopes which the antibodies recognize, and quantitatively determine each of the groups of antibodies. However, the present invention provides an easy and highly reproducible method for classifying and quantitatively determining the antibodies against the epitopes of a viral reverse transcriptase. Using the method of the present invention, not only the virus which has mutated during a latent period thereof, and the properties which the virus exhibits as a result of the mutation, but also a virus which has acquired drug resistance as a result of the administration of the drugs to the host, can be studied by quantitatively determining the antibodies. Further, by the method of the present invention, it has also become possible to accurately and promptly determine the changes in the infected host, such as the change in the ability to produce antibodies, which occur in accordance with the progression of illness. In addition, the above-mentioned method is advantageous in that the biological samples used are easily available, and that cumbersome procedures, such as culturing of the virus, are not required. Therefore, the method of the present invention is effective for the diagnosis and the like of HIV which necessarily include testings for an extremely large number of test items. Further, the method of the present invention can be advantageously used for the study of HIV infection because an antibody is
REFERENCES:
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Asahi Kasei Kogyo Kabushiki Kaisha
Budens Robert D.
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