Process and test kit for non-radioactive enzymatic detection...

Details Process and test kit for non-radioactive enzymatic detection... Process and test kit for non-radioactive enzymatic detection...

1998-02-10

2001-01-16

Cochrane Carlson, Karen (Department: 1653)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S007400, C435S091200, C435S091210, C536S024330, C536S025400

Reexamination Certificate

active

06174672

ABSTRACT:

PRIOR ART
Currently, the routine detection of retroviruses is performed using the detection of virus-specific antibodies and/or viral components (antigens, RNA, proviral DNA), e.g. anti HIV antibody tests, HIV p24 antigen tests, HIV PCR detection test (Holodniy M. et al., J. Infect. Dis. 163, 862-866, 1991; Henrard DR et al., AIDS Res. Hum. Retrovir. 8, 47-52, 1992). However, this type of analysis is based on a non-functional or a structure-specific molecular interaction between antibody and antigen or PCR primer and proviral DNA, respectively, where no conclusions may be drawn with respect to the presence of a complete and a functional and intact virus. Furthermore, this type of analysis fails to cover all of the stages of a viral infection, e.g. the phase in which an infection has taken place but no antibodies have yet been formed. Thus, these factors sometimes lead to false-positive or negative results. For these reasons, it is urgently required to provide for direct and functional determination of retroviruses in biological samples. The medical and sociopolitical importance of retroviruses contributes further to this need, in particular in view of AIDS and a growing tendency and increasing role of retroviruses in animal and human diseases (leukemia, autoimmune diseases, cancer, etc.). Moreover, the transmission of retroviruses by infections causes severe problems in transfusion and transplantation medicine or otherwise requires qualitative examinations regarding viral contaminations in samples of lymph, saliva, sperm or blood of donors as well as in organs, skin, bone marrow, etc. destined for transplantation. The same problems of viral contamination or infection, respectively, apply to the use of biopharmaceuticals and other biotechnological or genetechnological preparations of biological origin used for therapeutical purposes in humans and animals as well as in medical basic reasearch. A prerequisite to solve the problems mentioned above is the direct and functional detection of retroviruses by means of methods which are reliable, sensitive, and as simple as possible.
Up to now, the direct and biologically functional detection of retroviruses was carried out successfully only in single cases and has been associated with a very high amount of work and time involved by infecting cells and has been possible only in purified viral preparations or in cell culture supernatants. So far, no quantitative, simple and reliable routine methods for the detection of retroviruses exist. A reason for this may be the complex composition of biological samples (blood, organic extracts, etc.) containing proteins, enzymes, vitamins, lipids, sugars, and various inhibitors. These complicate or prevent the direct and functional detection of retroviruses.
Reverse transcriptase is the key enzyme of all retroviruses carrying out the reverse transcription (translation) of viral RNA into complementary DNA (cDNA) and enabling the subsequent integration thereof into the genome of the host cell. The enzyme is retrovirus-specific and its enzyme activity indicates the existence of viral particles as well as their functional integrity since the enzyme is very unstable as a free molecule and is rapidly inactivated.
Although up to now a variety of tests to detect reverse transcriptase have been developed, these technologies failed to establish themselves as a routine method in virological and clinical diagnostics because of the following shortcomings:
a) use of radiolabeled components (substrates labeled by tritium, phosporus) and of health hazardous or enviromentally harmful organic solvents (trichloroacetic acid, toluene);
b) insufficient sensitivity of the non-radioactive methods as compared to ELISA (enzyme-linked immunoadsorbent assay) and PCR (polymerase chain reaction) technology because of the use for labeling and detection of the reaction products (cDNA) of antigen/antibody systems (i.e. digoxigenin/anti-digoxigenin; fluorescein/anti-fluorescein) having relatively low binding constants (Eberle, J. et al., J. Virol. Meth. 20, 347-356, 1992; K. Suzuki et al., J. Virol. Meth. 41, 21-28, 1993);
c) complicated routine handling and performance of kinetic measurements because of insufficient methods for the separation of reaction educts from products caused by double labeling of the newly synthesized cDNAs (membrane adsorption, filtration or capturing on a solid phase, respectively) (Eberle J. et al., J. Virol. Meth. 20, 347-356, 1992; K. Suzuki et al., J. Virol. Meth. 41, 21-28, 1993);
d) inefficient immobilization of the primers on solid phases (plastic) (T. Urabe et al., J. Virol, Meth. 40, 154-154, 1992).
The present method applying for grant of patent describes the direct and enzymatic routine detection of retroviruses using reverse transcriptase.
Solution of the Problem
The method to be patented and the corresponding test kit are based on the use in the reverse transcriptase reaction of a primer-and-template immobilized on magnetic particles in the form of a particle-primer/template complex (PPT complex) and labeling of the cDNA which is newly synthesized on the PPT complex by biotinylated deoxynuclectide triphosphates in the form of a PPT-cDNA complex and the detection thereof by streptavidin-coupled marker enzymes and appropriate substrates in the form of a PPT-cDNA-conjugate complex (see FIG.
2
).
Magnetic particles and ligand complexes immobilized on those particles have the advantage that they may be separated from solutions as a solid phase in the presence of a magnetic field and may again be dispersed in solution in the absense of a magnetic field or by agitation, respectively (see FIG.
1
). This simple, very quick, efficient, and reproducible physical process may upon immobilisation of molecules on magnetic particles be used in biological tests for the processing of these molecules and, if necessary, for their successive separation (immobilisation) and dispersion (solubilisation). This alternating magnetic treatment (
FIG. 1
) is particularly advantageous in enzymatic or biochemical reactions where reaction educts (substrates) have to be partially separated from products. The magnetic treatment of particle-ligand complexes in solution is effected by successive introduction and removal of magnets into and out of the proximity of the reaction vessel or the solution, respectively, whereby alternating separation and dispersion of the complexes or solution changes, washing, reaction stop etc. may be performed (FIG.
1
). In microtiter plates, a so-called microtiter magnet separator (
FIG. 3
) consisting of an optically transparent carrier plate is employed for magnetic treatment which has 24 bar magnets introduced into the external space of the microtiter wells and, thus, separates the particle-ligand complexes present in solution onto the side walls of the wells (FIG.
1
), thereby enabling solution changes, washing, and photometric measurements in the presence and without loss of the magnetic particle-ligand complexes to be conducted without any problems.
The method or test kit, respectively, to be patented uses magnetic particles having an immobilized primer-and-template which is necessary for the reverse transcriptase reaction. As primers, there may be employed a homo- or heterooligodeoxynucleotide triphosphate (e.g. oligo dT, oligo dG) while as a template (matrix RNA molecule) a homo- or heteropolymeric RNA such as poly rA (polyadenosine triphosphate) may be used. Particles, primer, and template together form the so-called particle-primer/template-complex reagent (PPT reagent). In the presence of reverse transcriptase and biotin-labeled as well as unlabeled deoxynucleotide triphosphates dNTP's the corresponding cDNA is synthesized complementary to the template RNA which is also immobilized on the magnetic particles by means of the template-primer and forms the so-called PPT-cDNA complex.
Excess non-incorporated nucleotides are washed off, and afterwards the biotinylated PPT-cDNA complex is incubated with streptavidin-conjugated marker enzymes (e.g. peroxidase, alkaline phosphatase),

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