A METHOD FOR AMPLIFYING AND DETECTING A TARGET NUCLEIC ACID SEQUENCE OF HIV-1

Abstract: A direct method for detecting viral nucleic acid of HIV-1 in a biological sample suspected of containing the target nucleic acid by adsorption of the biological sample onto a solid support coupled to an anti-HIV-1 envelope antibody, followed by amplification of the target nucleic acid sequence and detection of the amplified product. Amplification methods which can be used include the polymerase chain reaction and the ligase chain reaction.

Description: A METHOD FOR AMPLIFYING AND DETECTING A TARGET NUCLEIC ACID SEQUENCE OF HIV-1 Background of the Invention The invention relates to a method for detecting a target nucleic acid sequence in a biological sample, and more particularly, relates to an improved method for amplifying and detecting a target nucleic acid sequence in a biological sample. Humans respond to viral infections by generating antibodies to various viral antigens. The number of antibodies and the amount of each different antibody produced depends on the viral antigen which initiates the antibody response. One category of viral infection which is of concern is hepatitis infection. There are at least four different viruses which have been identified as causative agents for hepatitis: the hepatitis B virus (HBV), the hepatitis A virus (HAV), the hepatitis D virus (HDV), and the non-A, non-B (NANB) also known as hepatitis C virus (HCV). Symptoms associated with any of the four hepatitis viruses are often so similar that it is not possible to make a definitive diagnosis without the use of serological markers. Both HAV and HBV produce unique antigens and antibodies in human blood and other biological fluids. These antigens and antibodies follow distinct and individual serological patterns during the course of the infection, and can be used as serological markers. By detecting these antigens and antibodies, it is possible not only to diagnose the type of hepatitis, but also to determine the stage of the infection and probable prognosis as well. Antigens associated with HBV infection include hepatitis B surface antigen (HBsAg), hepatitis B core antigen (HBc) and hepatitis B e antigen (HBeAg). These antigens are present at different times and in varying combinations throughout the course of the infection. For example, HBsAg usually appears in the serum after an incubation period of one to six months; peaks shortly after onset of symptoms; and it typically disappears within one to three months. In most cases a "window period" will then occur in which both HBsAg and anti-HBs, (antibody to the surface antigen), are not detectable. The duration, of the window period is typically two to six weeks. Subsequent appearance of anti-HBs signals recovery from and probable immunity to hepatitis B infection. At this time, the individual is considered immune. In contrast, antibody to HBcAg (anti-HBc) appears concurrently with symptoms and rapidly rises in titer. It is present during the window period and is therefore the most reliable indication of infection in the absence of other markers. Hepatitis e antigen (HBeAg) appears concurrently with HBsAg prior to the onset of symptoms, and, in acute cases, disappears prior to the loss of HBsAg. HBeAg indicates active viral replication and high DNA polymerase activity representing a highly infectious state. Immunoassays for detecting HBsAg and HBeAg are unable to detect viral associated antigens at concentrations at the lower limit of infectivity which can be only a few viruses per ml. Filter hybridization assays for HBV DNA can only detect 0.1 to 1 pg (2500 to 25,000 viruses). While some HBsAg positive (HBsAg+) patients may not be actively producing virus and are not infectious, currently available assays are unable to distinguish these Individuals from patients who have low levels of viral replication and are infectious. Prudent medical management must consider all HBsAg positive patients as infectious. A number of diagnostic assays have been developed which rely on detection of the presence of a particular DNA or RNA sequence as an indication of the presence of an analyte, e.g., a bacterium, virus or genetic defect, in a sample. In some instances the DNA or RNA sequence is present in sufficient quantities to be detected directly, whether by hybridization, reaction with a specific antibody, or by some other method. However, if the sequence of interest is present only in a small amount, or the background caused by similar sequences present in the sample is sufficiently high, reliable and sensitive detection of the particular DNA or RNA sequence is difficult. Recently, the polymerase chain reaction (PCR) for detecting DNA and RNA has been utilized to analyze extremely small quantities of nucleic acid for the presence of DNA or RNA sequences of interest. The PCR technique amplifies the target nucleic acid sequence by using specific oligonucleotide primers in repeated cycles of target DNA denaturation, primer annealing, and extension with a DNA polymerase. Saiki R. et al., Science. 239:487-491 (1988); Ou C- Y et al.. Science. 239:295-297 (1988); U.S.Patent Nos. 4,683,202 and 4,683,195. A difficulty with employing PCR methodology to detect HBV DNA sequences is that sera contain inhibitors of DNA polymerase. This has necessitated the extraction and purification of nucleic acid prior to performing PCR. Kaneko S. et al., PNAS. 86:312-316 (1989); Theirs V.^et al., Lancet. ii:1273-1276 (1988). Purification of nucleic acid is not totally efficient, often resulting in loss of nucleic acid sequences of interest. The sensitivity provided by PCR has prompted numerous studies on the detection of HIV-1 proviral DNA contained in infected T-cells. Recent data has even suggested that quantitation of proviral load may allow assessment of anti-viral therapy and identification of a group of patients at lower risk of progression to AIDS. D.D. Ho et al., N. Enαl. J. Med. 321 :1621-1625 (1989); N. V. Chelyapov et al., i Acquir. Immune Defic. Svndr. 4:314 (1990); S. M. Schnittman et al., Ann. Intern. Med. 113:438-443 (1990); and M. T. Scechter et al.. AIDS 5:373- 379 (1991). However, detecting the proviral form of a retrovirus provides no information on its replication status. Therefore, only measuring viruses or viral components can provide a true reflection of viral load. Quantitative plasma culture for HIV has demonstrated that titers of infectious circulating HIV virus correlate with the clinical stage of the disease. D.D. Ho, supra. N.V. Chelyapov et al., supra. S.M. Schnittman et al., supra. M.T. Schechter et al., supra, and R.W. Coombs et al., N. Enα. J. Med. 321 :1626-1631 (1989). However, culture is cumbersome and requires up to five weeks to complete. The presence of HIV antigen is a good prognostic marker of progression, but it is detected in less than 15% of asymptomatic individuals and in about 50% of AIDS patients. The detection of HIV-1 RNA in plasma using extraction of total nucleic acids has been described. I. K. Hewlett et al., J. Clin. Immunoassay 11 :161- 164 (1988); and M. Holodniy et al., J. Infect. Pis. 163:862-866 (1991). These methods however require more test sample than may be available. It is an objective of the present invention to provide a an efficient method for capturing the target nucleic acid sequence which then is amplified and detected. It is a further objective of the present invention to provide an assay such that one can reliably directly detect small quantities of nucleic acid. Summary of the Invention The present invention comprises a method and kit for detecting a target nucleic acid sequence of HIV-1 in a biological sample. The method involves adsorbing the biological sample which may contain a target nucleic acid sequence of HIV-1 onto a solid support coupled to an anti-HIV-1 envelope antibody such as anti-HIV-1 gp120 or anti-HIV-1 gp41 antibody; removing unbound sample, amplifying the target nucleic acid sequence and detecting the resultant amplified product present in the sample. Optionally, passing the biological sample through a column containing chromatographic material prior to the adsorption can be utilized to remove possible interfering substances which may be present in the sample and thus to further increase the sensitivity of the assay method. The kit involves a solid support coupled to an anti-HIV-1 envelope antibody such as anti-HIV gp120 or anti-HIV gp41 or anti-HIV p24 antibody; means for removing unbound biological sample from the solid support, means for amplifying the target nucleic acid sequence and means for detecting the amplified product present in the biological sample. Brief Description of the Drawings Figure 1A the location, length (size in base pairs) and orientation (direction) of the primers used to perform PCR on HBV DNA; and Figure 1 B illustrates the amplification of various segments of serum HBV DNA using various clockwise and counterclockwise primer pairs originating in the s, c, and x regions on a HBV DNA. Figure 2A shows the results of an experiment in which monoclonal anti- biotin coated microparticles detected as few as 30 viruses in 200 μl of serum; and Figure 2B shows the results of an experiment in which monoclonal anti- HBs coated microparticles detected 3 viruses. Figure 3 shows results of serial dilutions of tissue-culture supernatant from HIV lll-B-H9-infected cells assayed for reverse transcriptase activity ("RT", shown as a solid line between solid squares), p24 antigen test without Triton (shown as a solid line between open triangles), p24 antigen test with Triton (shown as a solid line between closed triangles) and the viral capture assay of the invention (shown as a solid line between closed circles). Figure 4 shows the detection of HIV-1 RNA using the method of the invention and HIV p24 antigen using a commercially available antigen assay, during seroconversion. Figure 5 shows the specific detection of HIV-1 RNA in the presence or absence of reverse transcriptase for one seroconversion series. Detailed Description of the Invention The present invention is directed to a method for amplifying and detecting a target nucleic acid sequence in a biological sample. The method involves adsorbing the biological sample onto a solid support; removing unbound sample, amplifying the target nucleic acid sequence and detecting the resultant amplified product present in the sample. The sensitivity of the assay method can be further increased by passing the biological sample through a column containing chromatographic material prior to adsorption onto the solid support to remove possible interfering substances which may be present in the sample. One application of the present invention provides a method for a more sensitive PCR assay enabling the direct detection of nucleic acid by adsorption of the infectious agent containing the target nucleic acid sequence to a solid matrix. While the method is applicable to the diagnosis of a variety of viremias, the following examples describe the detection of serum HBV DNA and HIV RNA by this method. The following examples are illustrative of specific embodiments of this invention and do not limit the scope of the claims. Those skilled in the art will readily determine obvious variants of the following examples that are within the scope of the present invention. DEFINITIONS Nucleic acid sequence: A nucleic acid sequence detectable by the present method is a sequence of nucleotides including ribonucleotides or deoxyribonucleotides. Nucleic acid sequences may be either single-stranded or double-stranded or partially single-stranded and partially base-paired regions. Ribonucleic acid sequences include both messenger and ribosomal RNA. Proteinase: A proteinase is an enzyme capable of breaking a peptide bond and includes both exopeptidases and endopeptidases such as protease, Proteinase K, pronase, trypsin, alkaline protease, subtilisin, and chymotrypsin. Target nucleic acids or target nucleic acid sequences: As used herein, these terms refer to DNA or RNA, whether in single or double-stranded form, including messenger or ribosomal RNA. For example, target nucleic acids can be present in viruses, microorganisms, either prokaryotic or eukaryotic, and any aberrant cell that may be associated with a disease or physiologic state. Target nucleic acids include the nucleic acids of HIV (HIV-1 or HIV-2), hepatitis viruses, herpes viruses, human retroviruses, human papilloma viruses, Epstein-Barr virus, Cytomegalovirus, viral RNA transcripts, and replicative intermediates. Also included are nucleic acids of bacteria such as mycoplasmas, rickettsiae and chlamydiae and eukaryotic pathogens such as fungi, yeast and abnormal or variant host cells, particularly those containing oncogenes or genetic defects or genetic markers. Biological sample: As used herein, a biological sample is any sample which may contain a target nucleic acid of interest. The source of the biological sample includes plants, insects and animals. Among the preferred biological samples are serum, plasma, synovial fluid, biopsy material, tissue culture cells or growth medium from cells in tissue culture, tissue extracts and membrane washes. Specific binding pair: As used herein, a specific binding pair comprises two different molecules, wherein one of the molecules has an area on its surface or in a cavity which specifically binds to a particular spatial and polar organization of another molecule. The members of the specific binding pair are often referred to as a ligand and receptor or ligand and anti-ligand. Examples include antibody-antigen, hormone-receptor pairs, enzyme-substrate pairs, biotin-avidin pairs and glycoprotein-receptor pairs. Also included are fragments or portions of specific binding pairs, including cellular or viral proteins, which are either directly or indirectly associated with the DNA to be amplified, for example, any HBV viral protein which is either directly or indirectly associated with the DNA to be amplified, such as HBeAg or HB pol gene products. Also included are fragments or portions of specific binding pairs which retain binding specificity, such as fragments of immunoglobulins, including Fab fragments and the like. The antibodies can be either monoclonal or polyclonal. Solid support: Suitable solid supports include wells of reaction trays, test tubes, beads, strips, membranes, filters, microparticles or other solid supports which are well known to those skilled in the art. Polymerase Chain Reaction (PCR As used herein, this method uses specific oligonucleotide primers in repeated cycles of target DNA denaturation, primer annealing, and extension with a DNA polymerase. Extension products generated from one primer serve as additional target sequences for the other primer. The degree of amplification of target sequence is controlled by the number of cycles that are performed and is theoretically calculated by the simple formula 2/n where n is the number of cycles. Given that the average efficiency per cycle ranges from about 65% to 85%, 25 cycles are needed to yield from 0.3 to 4.8 million copies of target sequences. Amplification techniques: Any method for multiplying the number of copies of a target polynucleotide sequence such as polymerase chain reaction (PCR) and ligase chain reaction (LCR), as well as other methods which are known to those skilled in the art. The Ligase Chain Reaction (LCR) amplifies sections of DNA by copying the section of DNA, and copying the copies of that section of DNA, many times over. This method is described in European Patent Application No. 0 320 308 published June 14, 1989. In this procedure, two probes (for example, A and B) complementary to immediately adjacent regions of a target sequence are hybridized and ligated. This ligated probe then is denatured away from the target, after which it is hybridized with two additional probes (A' and B') of sense opposite to the initial probes A and B. The secondary probes are themselves then ligated. Subsequent cycles of denaturation/hybridization/ligation create the formation of double-length probes of both sense (+) and antisense (-). In LCR, the nucleic acid of the sample is provided either as single stranded DNA or as double-stranded DNA which is denatured to separate the strands. Four probes are utilized: the first two probes (A and B) are the so- called primary probes, and the second two probes (A' and B') are the so-caiied secondary probes. The first probe (A) is a single strand capable of hybridizing to a first segment of the primary strand of the target nucleotide sequence. The second probe (B) is capable of hybridizing to a second segment of the primary strand of the target nucleotide sequence. The 5' end of the first segment of the primary strand of the target is positioned relative to the 3' end of the second segment of the primary strand of the target to enable joining of the 3' end of the first probe to the 5' end of the second probe, when the probes are hybridized to the primary strand of the target nucleotide sequence. The third probe (A 1 ) is capable of hybridizing to the first probe, and the fourth probe (B 1 ) is capable of hybridizing to the second probe (B). The hybridized probes are ligated to form reorganized fused probe sequences. Then, the DNA in the sample is denatured to separate the ligated probes from sample DNA. Successive cycles wherein the ligated probes and target DNA undergo the above-described process are performed to increase the amount of detectable DNA in the sample. The amount of cycles performed is dependent upon the sequences used and the sensitivity required of the test. Usually, the cycle can be repeated from 15 to 60 times. At least one of the probes can be conjugated to a signal generating compound. Detection of Amplified Target Nucleic Acids: Detection of the amplified target nucleic acid can be performed by any one of the general method for hybridization reactions and probe synthesis disclosed in Molecular Cloning by T. Maniatis, E.F. Fritsh and J. Sambrood, Cold Spring Harbor Laboratory, 1982 and Basic Methods of Molecular Biology bv L. Davis, M. Dibner and J. Battey, Elsevier Science Publishing Co., Inc., 1986. These techniques include EIA, dot blot analysis, agarose gel electrophoresis, Southern Blot, liquid hybridization and other DNA hybridization techniques as well as other methodologies which are known to those skilled in the art. DNA Sequencing of Amplified Nucleic Acids: The amplified target nucleic acids can be sequenced by the procedure outlined in the Methods section as well as other methodologies known to those skilled in the art. Denaturing-Agents: As used herein, denaturing agents include disassociating agents such as detergents, alkaline, SDS, urea, guanidine HCI, Nal and other chaotropic agents known to those skilled in the art. Chromatographic Column Material: Desalting can be accomplished by using a chromatographic column. The column material is porous media allowing passage of biological fluid leading to separation of chemical species. Suitable methods include gel filtration wherein molecules are separated according to size. Other suitable methods include ion exchange, affinity and hydrophobic chromatographic methods. Pharmacia (LKB Biotechnology Products Catalogue [1989]). These methods provide for the removal of interfering substances which may be present in the sample. METHODS Sera and Cells for Examples 1 and 2 HBsAg positive, HBV DNA positive sera were obtained from a variety of patients with chronic HBV infection. Sera were obtained from healthy blood donors who had normal ALT, and no serological markers for HIV, HBV, and syphilis. Plasma from volunteer HBsAg positive blood donors were collected along with serial plasma samples from two plasmaphoresis donors who became HBsAg positive. These patients were frequently plasmaphoresed during the course of their acute hepatitis B infection. AH sera and plasma were stored at -20°C. Serological Assays HBsAg, anti-HBc IgM and HBeAg were tested by commercial enzyme linked immunoassays (Abbott Laboratories, North Chicago, IL). Pre-S2 antigen was assayed by a radioimmunoassay in which 200 μl sera were incubated overnight at room temperature with beads from the Auszyme® assay (Abbott Laboratories, North Chicago, Illinois). After washing, 1 25 l-labeled anti-Pre- S2 monoclonal antibodies were incubated with the beads for two hours at 40°C, washed, then counted. A signal to negative control ratio (S/N) of greater than 3.0 was considered positive. Serum HBV DNA was detected by the Abbott Genostics Hepatitis B Viral DNA assay, Kuhns M.C. et al., Viral Hepatitis and Liver Disease ed. by A.J. Zuckerman, Alan Liss Inc., New York, NY, pp. 258-262 (1988), and by dot blot hybridization, Zeldis J. B. et al., Journal of Virological Methods. 14:152- 166 (1986). The sensitivity of each assay is virtually equivalent. The HBV DNA dot blot analysis detects approximately 0.1 pg per 100 μl of serum after seven day autoradiography. The estimate of viral DNA concentration based on densitometry of autoradiograms of samples using cloned HBV DNA was within one logl 0 order of magnitude of the viral titer determined by electron microscopy, core protein concentration, or chimpanzee infectivity. Faint signals at the limit of detectability (0.1 pg DNA) gave inconsistent results, not verifiable by the liquid hybridization assay; therefore, only spots that gave a signal after 24 hours autoradiography (1 pg signal) were scored positive. Primers Figure 1 lists the location, length and orientation of the primers used to perform PCR. Figure 1 demonstrates the lengths of different amplified DNA segments created by PCR, using various clockwise and counterclockwise primer pairs originating in the s, c and x regions jn a HBV DNA containing serum. Since PCR often generates spurious DNA bands that range from 50 to 250 base pairs in length, primer pairs that create fragments greater than 400 base pairs long were primarily employed. Forty cycles were used to achieve greater than a million-fold amplification. The fragments created were from right to left: 1452 bp (position 1825 to 77); 1417 bp (position 1861 to 77); 927 (position 2451 to 77); 2132 (position 1861 to 692); 2285 (position 1861 to 845); 3015 (position 1861 to 1575); 1441 (position 134 to 1575). Note that the sixth column from the left creates a 3015 nucleotide fragment producing by priming from either side of the ends of the 1 strand of HBV. Some s gene sequences were derived from those supplied by J. Snϊnsky and D. Mack of Cetus Corp., Emeryville, CA. The other primer sequences were derived from published HBV DNA sequences analyzed from GenBank using DNAStar® alignment programs. (DNA-Star, Madison, Wisconsin: 14, Ono Y et al., Nucleic Acid Research. 11 :1747-1757 (1983). The oligonucleotϊde primers were synthesized by phosphoramidite chemistry. Polymerase chain reaction detection of serum HBV DNA In all assays, phosphate buffered saline (PBS), negative control sera from normal blood bank donors, and 1 ng of cloned HBV DNA were analyzed. The samples were handled on a laboratory bench removed from the reagents used in the PCR to avoid contamination. DNA Sequencing of Amplified DNA Sequences After PCR, the reaction mixture was passed through a Sephadex G-50® spin column, C.F. Meares et al., Analytical Biochemistry. 142, 68-78 (1984); H.S. Penefsky, The Journal of Biological Chemistry. 252, No. 9, 2891-2899, (1977). The reaction mixture was then sequenced by the dideoxy method of Sanger using the Taq DNA polymerase according to manufacturers' instruction. Sanger F. et al., PNAS. 74:5463-5467 (1977) (Taquence DNA Sequencing Kit, USB Corporation, Cleveland, Ohio) using 35 S labeled dATP and 7% polyacrylamide sequencing gels. The autoradiograms were analyzed using a SeqEasy® digitizer and DNAStar® software. (DNAStar, Madison, Wisconsin) Example 1 Polymerase Chain Reaction Detection of Serum HBV DNA Bv Nonspecific Adsorption Method This example illustrates the claimed method for detecting a target nucleic acid sequence in a biological sample wherein the biological sample is first adsorbed onto a solid support; unbound sample is removed, and the target nucleic acid in the sample is then amplified and detected. This example further illustrates the claimed method wherein the target nucleic acid, serum HBV DNA, is amplified by the method of PCR and the solid support onto which the virus is first adsorbed is a microcentrifuge tube (Eppendorf tube) as hereafter described. Fifty to 100 μl of serum were incubated overnight at 20°C in an 0.5 ml Eppendorf tube. The serum was then discarded and the tube washed three times with PBS. Sixteen μl of water and 100 μl of mineral oil were added; the tube was heated to 95°C for ten minutes. Three μl of ten times PCR buffer (500 mM KCI, 100 mM, Tris pH 8.3, 15 mM, MgCl2, 0.1% (w/v) gelatin) were added plus 10 microliters of a 250 μg per ml solution of proteinase K (Boehringer- Mannheim). The mixture was incubated at 60°C for 60 minutes. Twenty-one μl of a "master mix" were added consisting of nine μl of water, two μl of ten time PCR buffer, eight μl of dNTP mix (1.5 mM final concentration each of datp, dctp, dgtp, " FTP, pH 7), 1 μl each of a clockwise and counterclockwise primer (final concentration of each primer is approximately 1 μM). The reaction was heated to 95°C for 10 minutes both to denature the proteinase K and to anneal primers to single stranded DNA, then cooled to 50°C for five minutes. Five μl (0.5 units) of Taq DNA polymerase (Perkin Elmer-Cetus, Norwalk, CN) were added. The reaction was cycled forty times between 72°C for 30 to 180 seconds depending on the length of DNA being amplified, 95°C for 1 minute, and 50°C for 2 minutes. After the fortieth cycle, the 72°C elongation step proceeded for an additional ten minutes. The target nucleic acid sequence present in the sample was detected as follows. The mixture was heated to 95°C for 1 minute, then cooled to 4°C until analyzed by dot blot analysis, electrophoresis in 1.8% agarose in 1 x TAE (40 mM Tris-acetate, pH7, 2 mM EDTA) with 0.5 μg/ml ethidium bromide, Southern blot analysis, or liquid hybridization. Dot-blot hybridization was performed following the method of Scotto et al., Hepatology 3:279-284 (1983), summarized as follows. 100 μl of serum sample was pipetted in an Eppendorf tube. 200 μl of 1N NaOH and 100 μl of 2M NaCI were added to the Eppendorf tube. The so-formed mixture was vortexed for 10 seconds and incubated at room temperature for 10 minutes. Then, the tube was centrifuged for 30 seconds and the supernatant was applied in the well of a bio dot apparatus. Also, a neutralization solution comprising 0.5M Tris pH 7.4 and 3M NaCI was applied to the same well. The filter was air-dried on a Whatman 3mm paper, and then baked for 12 to 2 hours at 80°C. The dried filter paper was incubated with a solution of 6X Standard Saline Citrate (SSC), 0.5% SDS, 5X Denhardt, 0.1 μg/ml salmon sperm DNA (denatured) for one to two hours. This solution was removed. DNA probe used for hybridization was labeled by nick translation using the method of Rigby et al., J. Molec. Biol. 113:237-251 (1977). The resultant mixture was hybridized overnight. Following this, the mixture was washed twice with 2X SSC and 0.5% SDS for 15 minutes at room temperature, and then washed twice with 0.1X SSC and 0.1% SDS at 68°C for one hour. Autoradiography was performed using Kodak XAR-5 film. Example 2 Polymerase Chain Reaction Detection of Serum HBV DNA by Antibody Microparticle Method Example two illustrates the claimed method of detecting a target nucleic acid in a biological sample wherein the solid support is a microparticle. Example two further illustrates the claimed method wherein the target nucleic acid, serum HBV DNA, is amplified by PCR. Carboxylated latex microparticles (0.1-0.3 μm diameter) were obtained from Seradyne Inc. (Indianapolis, IN). Monoclonal IgG or IgM molecules were covalently coupled to these microparticles using 1-ethyl-3-3 (dimethylaminopropyl) carbodiimide chemistry. Nathan C. F. et al., J. Exp. Med.. 154:1539-1553 (1981); Quash G. et al., J. Immunolooical Methods. 22:165-174 (1978). Fifteen (15) ml of Seradyne microparticles (approximately 30% solids) were mixed with 30 g of Dowex resin, and incubated for two (2) hours with rocking, at room temperature. Then, this mixture was passed through a glass wool syringe or equivalent such as to separate large Dowex particles from the microparticles. The microparticles then were diluted to approximately 200 ml. Next, the microparticles were centrifuged for 15 minutes at 17,000 x g, and washed. This centrifugation and washing step was repeated twice more. The microparticles were resuspended at 180 ml or approximately 2,5% solids and stored at 2° to 8° C. One (1) ml of buffer A comprising 0.1% Tween-20® in 15 mM MES (pH 4.75, prepared fresh prior to use) was added to a 1 ml Eppendorf tube. Then, 100 ml of microparticles (at 2.5% solids) prepared as described herein was added to the tube to form a mixture. This mixture then was vortexed. Next, 100 μl of 0.4 mg/ml of EDC (1-ethyl-3(dimethylaminopropyl) carbodiimide in distilled water (prepared fresh prior to use) was added to the mixture. Monoclonal IgG or IgM antibody (2 mg/ml in PBS without NH2 containing buffer) was added so that the final concentration of the reaction mixture was 0.3-0.2 mg/ml of monoclonal antibody. The reaction mixture then was vortexed, and rocked for two to four hours at room temperature (approximately 20°C). After incubation, the reaction mixture was centrifuged at 10,000 rpm for five to ten minutes to pellet form. The supernatant was removed and the pelleted microparticles were resuspended with 1 ml PBS/2% Tween 20.® It was noticed that at times the pellet could be resuspended with vortexing, but at other times would require passage through a syringe with 23-25 gauge needle to break up microparticle clumps. This centrifugation and wash procedure was repeated twice more. Then pellet was then resuspended with syringing of 1 ml of resuspension buffer (which comprised 150 mM TRIS, 100 mM NaCI, and 1.5% calf skin gelatin) after the final wash to assure adequate dispersion of microparticles, to a final concentration of 2.5% solids. After coupling microparticles were centrifuged for 15 minutes at 17,000 rpm (JA19 rotor); supernatant decanted; and microparticles resuspended at 1.6% v/v in 150 mM Tris pH8.0, 100 mM NaCI, 0.5% gelatin, 0.1% Tween 20®, 9.5% sucrose and One to two hundred μl of sera or plasma were incubated for 30 minutes at room temperature with a 25 μl suspension of microparticles (total surface area of 24-100 cm 2 ) coated with monoclonal antibodies to HBsAg, pre-S1 region of HBsAg, pre-S2 region of HBsAg, or biotin obtained from Abbott Laboratories, North Chicago, Illinois. The microparticles were pelleted in a microcentrifuge, washed once with PBS, suspended in water, heated, digested with proteinase K, and heated again as described above in Section a. The reaction mixture was centrifuged to pellet the microparticles. The supernatant was placed in another tube and PCR was performed as described above in Section a. In some cases, the sera or plasma were centrifuged through a 0.9 ml spin column of Sephacryl 300 (Pharmacia, Piscataway, NJ) prior to adding the antibody coated microparticles. The target nucleic acid sequence present in the sample was detected as follows. Oligonucleotide primers were end-labeled by T4 polynucleotide kinase in a total volume of 15 μl containing 15 mM Tris pH 7.4, 10 mM MgCI2, 0.1 mM EDTA, 0.1 spermidine, 5 mM DTT and 75 μM of gamma labeled 32 P-dATP at 3,000 mCi per mMole. The mixture was heated to 65°C for 10 minutes, then placed in ice for 2 minutes. One μl (approximately 5 units) of T4 polynucleotide kinase was added; reaction proceeded for 30 minutes at 37°C; heated for 65°C for 10 minutes; and then directly used for the hybridization. HBV DNA was nick translated as previously described. Zeldis J.B. et al., Journal of Virolooical Methods. 14:152-166 (1986). RESULTS The above-described method, as illustrated in Examples 1 and 2, demonstrates the claimed method of detection of a target nucleic acid sequence in a biological sample by adsorbing the biological sample onto a solid support; removing unbound sample, amplifying the target nucleic acid and detecting the amplified target nucleic acid present in the sample. Siliconized Eppendorf tubes are as efficient in adsorbing virus as untreated polypropylene Eppendorf tubes based upon end point titration of the lowest concentration of virus needed for serum HBV DNA detection. Dilution of viral specimens in PBS was more sensitive than specimens diluted into serum. Viral adsorption to microparticles was found to be superior to Eppendorf tubes because of their greater surface area (24 to 100 sq. cm. vs. 1.4 to 2.5 sq. cm.). Additionally, the ability to couple the microparticles with antibodies enables increased antibody specific adsorption of the virus to the microparticles. Figure 2 shows the results of an experiment in which monoclonal anti-biotin coated microparticles were capable of detecting as few as 30 viruses in 200 μl of serum; monoclonal anti-HBs coated microparticles were capable of detecting as few as 3 viruses in 200 μl of serum. FIG. 2 shows a comparison of the sensitivity of anti-biotin coated microparticles, wherein FIG. 2A shows the sensitivity of anti-biotin coated microparticles versus that of anti-HBs coated microparticles, and FIG. 2B shows a comparison of the sensitivity of anti-biotin coated microparticles in detecting serum HBV DNA. 200 μl of HBV DNA positive serum serially diluted in control serum was analyzed for HBV DNA as described in the Methods using two primers in the s gene that were 690 nucleotides apart. As a result of using the method of the claimed invention, the yield of amplified DNA using monoclonal anti-HBs, anti- Pre-S1, and anti-Pre-S2 coated microparticles was so great that ethidium bromide staining of the agarose gels was as sensitive as Southern blot analysis in detecting less than 10 viruses per serum specimen. While most serum specimens did not have to be diluted in PBS to improve the sensitivity of the assay, occasionally the intensity (yield) of the ethidium. bromide amplified DNA fragment would increase if the sample were diluted in PBS. The yield of amplified DNA and the sensitivity of the assay improved if serum or plasma was first passed through a Sephacryl 300 spin column prior to adsorption to microparticles. Additionally, amplified DNA can be directly sequenced by the dideoxy method of Sanger as described herein below. Sanger F. et al., PNAS. 74:5463- 5467 (1987). Using the method of the claimed invention, the nucleic acid and amino acid sequences of the amplified serum HBV DNA fragments obtained after PCR from two HBV DNA positive sera, serum 1 and serum 2, were compared to the published sequences for hepatitis B virus, subtype adw, Ono Y. et al., Nucleic Acid Research. 11 :1747-1757 (1983). The nucleotide sequence of a 191 base sequence from serum 1 , subtype adw HBsAg positive, HBeAg positive, anti-HBc positive serum, was identical to that of adw. The nucleotide sequence of a 172 base sequence from serum 2, a subtype adw HBsAg÷, anti-HBc serum, was greater than 98% identical to that of adw. The HBV DNA concentration of serum 2 was an approximately 20,000 virus per ml at the lower limit of sensitivity of a dot blot assay. The 57 amino acid sequence translated from the nucleic acid sequence in serum 2 was identical to that of adw, except in position 213 in which a phenylalanine (F) was substituted for a tyrosine (Y), demonstrating that utilization of the claimed method of detection can be used to sequence the amplified DNA sequences with a high degree of accuracy. A further comparison of the claimed method of detection was carried out as described below. More than forty HBsAg positive sera were analyzed for HBV DNA by both a conventional dot blot assay and the claimed method using PCR amplification. In each instance in which sera were positive by the dot blot assay, the PCR method using either the antibody coated microparticle or Eppendorf tubes also produced a positive signal. Conversely, HBsAg positive serum samples were identified that were HBV DNA positive by the claimed method using PCR amplification but negative by the conventional dot blot assay. Table 1 summarizes the assay results for HBV DNA performed by the dot blot, liquid hybridization, and PCR methods of 24 HBsAg positive blood donors. Seven were HBV DNA positive by either liquid hybridization or dot blot assay. In two cases (#8, #22), the results of each assay differed. The claimed method for detecting HBV DNA using PCR for amplification identified all HBV DNA samples that were previously identified as positive by the methods of liquid hybridization or dot blot assay as well as one (#16) that the other assays scored as negative. Table 2 illustrates the results from a study in which twenty-one plasma samples from two patients with acute self-limited hepatitis were analyzed for HBV DNA by either PCR or by a liquid hybridization assay whose sensitivity approached that of the dot blot method. Kuhns M.C. et al. r Viral Hepatitis and Liver Disease ed. by A. J. Zuckerman, Alan Liss Inc., New York, NY, pp. 258- 262 (1988). Three samples were positive by liquid hybridization and six by PCR analysis. There were no false positive samples by either assay. For each patient, HBsAg and Pre-S2 antigenemia preceded serum HBV DNA. In one, viremϊa was found after the patient was no longer HBsAg positive. (1/31/89, Patient E). Example 3 Detection of HIV-1 Plamsa Viremia HIV-1 RNA By Antibody Microparticle Method Patient Samples A total of 51 plasma samples were obtained from six individuals prior to and during seroconversion as part of a prospective seroconversion study carried in two plasma collection centers. For five individuals, the number of donations ranged from two to seven per month, with an average interval of six days between collections. One donor (E) had much longer intervals between collections, averaging 44 days between donations. Eighteen samples were obtained from seropositive asymptomatic individuals who had tested negative for HIV p24 antigen and positive for anti-p24 antibodies. Twelve samples were collected from seropositive patients diagnosed with AIDS for 0.5-2 years, all of whom had received AZT. Three of these patients had discontinued treatment a median of 0.5 years prior to sampling. Monoclonal Antibodies HIV-1 antibodies which bind to the envelope region of the HIV-1 virus may be used. Monoclonal antibodies to HIV-1 gp41 or HIV-1 gp120 were used as described hereinbelow. The HIV-1 gp41 monoclonal antibodies used were as follows: 83-1371-208 (secreted by hybridoma cell line 83-1371-208, A.T.C.C. Deposit No. HB10951), 5-10-2 (secreted by hybridoma cell line 5- 10-2, A.T.C.C. Deposit No. HB 10953); 10-15-64 (secreted by hybridoma cell line 10-15-64, A.T.C.C. Deposit No. HB10952) and 56-1338-193 (secreted by hybridoma cell line 56-1338-193, A.T.C.C. Deposit No. HB 10955). These hybridoma cell lines were deposited at the American Type Culture Collection (A.T.C.C), 12301 Parklawn Drive, Rockville, MD 20852 as of December 20, 1991, under the terms of the Budapest Treaty. The HIV-1 gp120 monoclonal antibodies used were as follows: 52-445-22 (secreted by hybridoma cell line 52-455-22, deposited at the A.T.C.C. under the terms of the Budapest Treaty as of December 20, 1991 , under Deposit No. 10954), 52- 581-290 (secreted by hybridoma cell line 52-581-290, deposited at the A.T.C.C. under the terms of the Budapest Treaty as of March 28, 1990, under Deposit No. HB 10399), and 52-684-290 (secreted by hybridoma cell line 52-684-290, deposited at the A.T.C.C. under the terms of the Budapest Treaty as of March 29, 1990, under A.T.C.C. deposit No. HB 10400). Also, although monoclonal antibodies were used, it is contemplated that polyclonal antibodies specific for HIV-1 antigenic determinants also can be used. It further is contemplated that multiple target nucleic acid sequences could be detected by employing the method of the invention and using a cocktail comprising more than one antibody to more than one antigenic determinant of HIV-1 , or by using solid phases prepared as described below which then are mixed together to form a mixture of solid phases each able to capture the HIV 1 virus. Other types of HIV-1 monoclonal antibodies, other than those described herein as those directed to the HIV envelope region, also may be used to capture an detect HIV-1 target nucleic acid sequences. Assay Procedure Carboxylated latex microparticles (0.1-0.3 μm in diameter) were obtained from Seradyn, Inc. (Indianapolis, IN). Monoclonal anti-gp120 or anti-gp41 as described above was covalently coupled to these microparticles using 1-ethyl-3-3 (dimethylaminopropyl) carbodiimide chemistry following the method of Quash et al., J. Immunol. Methods. 22:165-174 (1978). After coupling, microparticles were centrifuged for 15 minutes at 13,000 rpm in a microcentrifuge, the supernatant was decanted, and the microparticles were washed with 1X PBS and resuspended at 8-10% vol/vol in 150 mM Tris, pH 8.0, 100 mM NaCI, 0.5% pork skin gelatin, 0.1% Tween-20®, 9.5% sucrose, and 0.02% NaN3. Fifty microliters of serum/plasma were diluted with 150 μl of 1X PBS and then incubated for three hours at room temperature with a 50 microliter suspension of microparticles prepared as described above. The microparticles were pelleted in a microcentrifuge, washed once with PBS, and resuspended in 200 μl of 20 mM Tris pH 8.3, 0.5 mg/ml proteinase K, 0.25% SDS, 20 μg yeast tRNA. After a one hour incubation at 55°C, the mixture was phenol/choroform extracted and the RNA precipitated with ethanol. After centrifugation, the RNA pellet was resuspended in 15 μl of sterile water. cDNA and Amplification First strand cDNA synthesis was performed by adding 5 μl of a solution made of 2.5 units of AMV reverse transcriptase in Cetus Taq buffer containing 50 mM KCI, 20 mM DTT, 25 M of each dATP, dTTP, dCTP, dGTP and 25 ng of each PCR primer (SK38/39) (S. Kwok et al., J. Virol. 61 :1690-1696 [1987]) to the 15 μl of sample and incubating at 37°C for 30 minutes. The mixture then was diluted with 30 μl of water and heated at 95° C for ten minutes. Amplification was carried out in a final volume of 100 μl in the presence of 50 ng of each primer, 50 mM of each dATP, dTTP, dXTP, dGPT and 1.0 unit of Taq enzyme (available from Cetus Corporation, Emeryville, CA) in 1X Taq buffer. The reactions were submitted to 35 cycles at 94°C for 90 seconds and at 60°C for three minutes. Liquid hybridization was used for detection as previously described and also discussed in S. Kwok et al., supra, followed by polyacrylamide gel electrophoresis and autoradiographic exposure for 1-5 hours with intensifying screen at -70°C. The relative intensity of amplified fragments detected on the autoradiograms was expressed as arbitrary units determined visually or by scanning densitometry. p24 Antigen and Serological Measurements HIV p24 antigen was measured in a solid-phase sandwich-type enzyme- linked immunosorbent assay (EIA) (available from Abbott Laboratories, Abbott Park, IL, "HIV-1 p24 antigen assay"). This commercially available assay used a viral lysate and a polyclonal capture antibody, and has a sensitivity of 8-10 pg/ml of p24 antigen. The presence of p24 also was tested with an assay which utilized a capture monoclonal p24 antibody which has a sensitivity of 3-5 pg/ml. Anti-HIV IgG antibodies were detected with a commercially available, FDA-licensed HIVAB®-1 EIA (available from Abbott Laboratories, Abbott Park, IL), which used an HIV-1 lysate. Western blots were performed according to the manufacturer's instruction (DuPont, Wilmington, DE). Antibodies to p24 antigen were measured in an indirect enzyme immunoassay in which recombinant p24 antigen was used as the capture antigen (available from Abbott Laboratories, Abbott Park, IL), and as described in H. W. Sheppard et al., J. Acq. Immun. Defic. Svndr. 4:704-712 (1991). Reverse Transcriptase (RT) Assay Reverse transcriptase assay was done by adding 100 μl of RT reaction mix containing 10 mM Tris pH8.2, 0.3M KCI, 0.15 M MgCl2, 0.01 M DTT to template RNA and 3H-TTP to 25 microliters of tissue culture supernatant. Each sample was assayed in duplicate. Disrupted virus from culture supernatant was used as positive control and buffer alone as negative control in a duplicate assay. Samples were incubated for one hour at 34°C, then spotted onto Whatman DE81 filters and allowed to sit for 1-10 minutes. After washing and drying the filters, radioactivity was measured for one minute in the presence of scintillation fluid. Results 1. Viral capture assay of the invention compared to HIV p24 antigen and reverse transcriptase. Serial dilutions of tissue-culture supernatant from H9lll-B-infected cells were assayed for reverse transcriptase activity, p24 antigen and by the viral capture assay of the invention. Briefly, ten-fold dilutions of tissue culture supernatant, corresponding to 1 to 10" 6 tissue culture infective dose (TCID) were tested with each assay. These results are summarized in Figure 3. Reverse transcriptase (RT) activity was not detected in any of the samples, as seen by the graphed line between open squares. Fifty μl of sample were diluted to 200 μl with PBS prior to testing for p24 antigen with the polyclonal HIVAG®-1 test. HIV antigen was measured with (as seen graphed between closed triangles) and without (as seen graphed between open triangles) the addition of Triton X-100®. in this assay, a sample was considered positive if containing 10 pg/ml or more p24 antigen. In the presence of Triton, free p24 as well as antigen released from disrupted virions were measured (i.e., total antigen). In the absence of Triton, only free p24 was detected. Therefore, the difference between the results obtained in the presence or absence of Triton reflected the proportion of p24 associated with intact virions. In the absence of Triton, p24 antigen was detected at 1 (>200 pg/ml), 10" 1 (>200 pg/ml) and 10 "2 (80 pg/ml) TC1D50- In the presence of Triton, a signal equivalent to the cutoff of the assay was still detected at 10" 3 TCID50. For HIV-1 RNA detection, seen in Figure 3 as a graphed line between closed circles, the relative intensity of amplified fragments detected on the autoradiograms was expressed as arbitrary units determined visually or by scanning densitometry. In contrast, the signal obtained from HIV-1 RNA was maximum above 10 "2 TCID50 and decreased in a linear fashion between 10 "2 and 10" 5 TCID50. Therefore, the viral capture assay of the invention was about 100 times more sensitive than p24 antigen assay. 2. HIV-1 RNA Detection During Seroconversion HIV-1 viral capture performed as described hereinabove was used to determine the level of HIV-1 viremia in 51 serial plasma samples collected from six individuals (A-F) during the early phase of infection. These results are shown in Figure 4. These samples were tested by the assay of the invention (viral capture assay) according to the protocol described above and by the commercial EIA test for the presence of HIV antibodies (available from Abbott Laboratories, Abbott Park, IL). A monoclonal p24 antigen test was performed on 200 μl of sample. In this assay, a sample was considered positive if containing 3-5 pg/ml or more p24 antigen. For HIV RNA assay, 50 μl of sample were tested. The arrow on Figure 4 indicates the 110 bp fragment amplified by using SK38/39 primers. Autoradiograms were developed after 6- 12 hours exposure. A non-specific band of higher molecular weight was observed in some experiments. In Figure 4, "HCV" represents an HCV PCR- positive HCV sample. It was found that 29/29 (100%) of seropositive samples were positive for HIV-1 RNA, compared to 13/29 (45%) for HIV antigen. Six samples (donor A, day 31 and 43; donor D, day 1 and 5; donor F, day 8 and 11) immediately preceding seroconversion were positive for both HIV-1 RNA and antigen. Of 15 pre-seroconversion samples negative for HIV antigen, HIV-1 RNA was found in three samples from one individual (donor B, day 1, 29 and 78) eight to five months prior to seroconversion, and in one sample from another person (donor A, day 29) two days before antigen positivity. One sample from the latter individual (donor A, day 1) gave a p24 antigen signal 30 days prior to antigen positivity and 45 days before seroconversion, but was negative for HIV-1 RNA. To ensure that the signal detected was due specifically to HIV-1 RNA, an analysis was performed on some seroconversion series in the presence or absence of the reverse transcription step. Serial samples from donor F were submitted to viral capture according to the method of the invention described above. Extracted nucleic acids were divided in two aliquots: one was treated with 2.5 units of reverse transcriptase (RT) (+) and the other left untreated (-) prior to amplification with Taq. Autoradiographic exposure time was 1.5 hours. As shown in Figure 5, no amplified signal was detected in the absence of reverse transcriptase, indicating that the viral capture method detected exclusively viral RNA. As another control for specificity, a sample was added to panel B from a patient chronically infected with hepatitis C (HCV), which was positive by RNA PCR (Dr. M. Kuhns, personal communication), and found it to be negative by HIV-1 viral capture. Finally, seroconversion series from donor F was obtained from an individual with chronic hepatitis B infection. The results obtained with these samples are similar to those of the other panels, indicating that other viruses do not have detectable interference with the HIV-1 capture assay. 3. HIV-1 RNA Detection in Asymptomatic and AIDS Patients Results from seroconversion studies thus indicated that 16/29 (55%) of seropositive samples negative for p24 antigen had HIV-1 RNA detectable by the viral capture assay of the present invention. In order to determine whether asymptomatic HIV-infected individuals with no detectable HIV-1 p24 antigen had measurable level of circulating HIV-1 particles, a small population of 18 subjects was analyzed. All samples were pre-selected as negative for HIV p24 antigen. These specimens also had detectable levels of antibodies to HIV-1 p24. The method of the invention as described in this example was used to determine the presence of HIV-1 RNA in these samples. After performing the assay as previously described in this Example, 10/18 (60%) of samples were found to be positive for HIV-1 RNA. No correlation was observed between HIV RNA detection and titers of antibody to HIV-1 p24. It has been reported that the majority (85%) of patients have viremia detectable by plasma culture. R. W. Coombs et al., N. Enol. J. Med. 321:1626- 1631 (1989). To determine the proportion of AIDS patients positive by viral capture assay, we analyzed samples collected from twelve subjects diagnosed with AIDS (CDC class IV-c). By following the method of the invention as previously described in this example, 11/12 (92%) of these samples were positive for HIV-1 RNA. CONCLUSION The above experimental results demonstrate that by following the steps of adsorbing virus to a solid support, washing the support, and then removing and purifying the target nucleic acid sequence with proteinase K and other constituents as described herein, PCR could detect serum HBV DNA and HIV RNA at concentrations below the sensitivity of conventional dot blot and liquid hybridization assays. For HBV detection, desalting serum or plasma through a Sephacryl 300 spin column prior to adsorption also increased the sensitivity of the assay. A rough quantitatϊon of viral DNA can be estimated by serially diluting a sample in HBV DNA negative serum or PBS. As few as 3 viruses per 200 μl can be detected using anti-HBs, anti-Pre-Sl, or anti-Pre-S2 coated microparticles. For HIV detection, a variety of highly sensitive serological assays for antigens, antibodies and nucleic acids are available for both the clinician and researcher. Although HBeAg positivity correlates with high titer serum HBV DNA in chronic HBsAg carriers, the absence of HBeAg or antibody to HBeAg does not exclude the presence of serum hepatitis B virus DNA. Zeldis J. B. et al., Journal of Virological Methods. 14:152-166 (1986). It was determined that approximately 20% of chronic HBsAg, anti-HBe positive cirrhotics were serum HBV DNA positive as assayed by conventional spot hybridization assay. With development of an even more sensitive polymerase chain reaction assay, a higher percentage of patients may be found to contain circulating viral DNA and therefore be potentially Infectious. The increased sensitivity of the PCR assay is illustrated by Tables 1 and 2 in which this method identified all sera and plasma identified as HBV DNA positive by liquid hybridization and dot blot analyses and identified some samples not scored positive by either assay. In the survey of 24 HBsAg positive blood donors (Table 1), only HBeAg positive samples were HBV DNA positive. In two cases of acute infection (Table 2), hepatitis B surface and Pre-S2 antigenemia preceded serum HBV DNA. This does not necessarily reflect the greater sensitivity of the immunoassays for HBsAg and Pre-S2 antigen, since a plasma sample obtained over two months into the infecti n (1/31/89, patient E) was viremic and not antigenemia A majority of the PCR studies on HIV have focused on the detection and sometimes semi-quantitation of integrated provirus. There also have been some reports Indicating that the level of proviral DNA may decrease over time in patients undergoing AZT therapy. D. Ho et al., N. Eng. J. Med. 321 :1621-1625 (1989); N. V. Chelyapov et al., J. Acquir. Immune Defic. Syndr. 4:314 (1990). Recent data have suggested that seropositive individuals having a low proviral (DNA) load may have a better prognosis than those with high proviral load. S.M. Schnittman et al., Ann. Inter. Med. 113:438-443 (1990); M. T. Schechter et al., AIDS 5:373-379 (1991). However, measuring the proviral form of a retrovirus provides no information on its replication status, which is determined by the amount of mature virions released by infected cells. Therefore, only methods measuring infectivity (plasma culture) or viral components (p24 antigen, HIV-1 RNA) provide accurate assessment of viral load. The isolation of RNA from HIV-1 seropositive plasma followed by cDNA synthesis and amplification has been described previously. I. K. Hewlett et al., J. Clin. Immunoassav 11:16 1-164 (1988); M. Holodniy et al., J. Infect. Pis. 163:862-866 (1991). However, the methods described so far have involved extracting total RNA from relatively large volumes of plasma. The assay of the invention provides a more specific and sensitive method for detection of HIV-1 RNA in smaller samples based on the capture of viral particles by monoclonal antibodies covalently coupled to a solid support. There are several advantages to using the method of the invention compared to those previously described. First, samples are processed non- destructively and can be analyzed by other methods after reaction with the solid phase. Second, almost any volume of sample can be processed before centrifugation of the microparticles and extraction of HIV-1 RNA, thereby increasing further the sensitivity of the assay. Third, the removal of the sample and washing of the solid phase virtually eliminates the risk of RNA degradation by plasma RNAases during proteinase K digestion. Although it is difficult to estimate the sensitivity of the viral capture cDNA/PCR assay without directly comparing to a standard of known RNA copy number, an approximation can be made by comparing the RNA PCR results to those obtained with p24 antigen and DNA PCR. It has been estimated that about 108 viral particles are produced per ml of culture, corresponding the approximately 10 ng or 2.5 x 10 1 0 molecules of HIV-1 p24 antigen. M. Popovic et al., Science 224:297-500 (1984). Thus, one can estimate that roughly 100 p24 core proteins are present in each viral particle. A. S. Bourinbaiar, Nature 349:111 (1991). Using 200 μl of specimen, the sensitivity of the p24 antigen monoclonal assay is about 1.0 pg/200 μl or 2.5 x 10 6 molecules of p24 antigen, which would represent about 10 4 virions. Therefore, the viral capture assay of the present invention, which was at least 100 more times sensitive than HIV-1 p24 antigen assay, may detect 100 or fewer virions in a 50 μl sample. We have been able to detect consistently reliable data with 10 molecules of target HIV-1 DNA. Since the reverse transcriptase is relatively inefficient, converting 10-50% of RNA into cDNA, the approximation of the viral capture sensitivity appears appropriate. After seroconversion, all seropositive samples were positive for HIV-1 RNA by the method of the invention, while only 13/29 (45%) of these had detectable p24 antigen by the described commercially available p24 antigen assay method. This is similar to the observations of Daar et al. who showed that after seroconversion, three of four patients retained positive plasma cultures for at least two weeks without having detectable p24 antigen. Before seroconversion, HIV-1 RNA was detected concomitantly, or slightly prior to HIV antigen. In one case (donor B), three samples were positive, although weakly, for HIV-1 RNA from eight to five months prior to seroconversion. It is possible that positivity at this time reflects the passive transmission of virus coinciding with infection, and that viral replication started several months later. Alternatively, these samples may contain defective non-infectious particles. In that case, the actual infection inducing seroconversion would have occurred somewhat later. It was observed that the amounts of amplified material were somewhat parallel to the levels of p24 antigen, although no attempt was made to accurately quantitate the intensity of the PCR signals obtained from viral capture. Samples having 200 pg/ml or more of p24 antigen gave saturating PCR signal and, in general, samples positive for HIV antigen gave stronger PCR signals than those negative for HIV antigen. The rise and decrease in the amounts of amplified material paralleled that observed for p24 antigen. These results support previous observations made by using semi-quantitative plasma culture to determine the levels of plasma vϊremia in individuals seroconverting for HIV. E. S. Daar et al., N. Eno. J. Med. 324:961-964 (1991); S. J. Clark et al., N. Eng . J. Med. 324:954-960 (1991). Interestingly, in one of our series (Donor C), the PCR signals decreased sharply concomitantly with the appearance of anti-gp120/anti-gp160 antibodies (day 10). The two samples preceding day 10 in Donor C were positive for anti-p24 only by Western blot analysis. Like Clark et al., supra, and Daar et al., supra, we observed that some seroconverters appear to control viral replication rapidly and effectively while others maintain high levels of circulating virions for much longer periods of time. For example, donors A and C have slightly detectable viremia 12 and 10 days post-seroconversion, respectively. In contrast, donors B and F appeared to have much more circulating virus at 189 and 27 days post-seroconversion, respectively. Although we could not obtain follow-up data from these individuals, or the asymptomatic patients, we can hypothesize that patients with lower viral load have a better prognosis than those high high levels of circulating HIV RNA. The method of the invention thus has several important applications for diagnosis and monitoring of HIV infection. It can provide a rapid means of assessing infection in newborns. Also, achieving a good semi-quantitation may allow prediction of vertical transmission and prognosis of AIDS development based on the levels of viral load. Finally, capturing viral particles may provide those sequences presumably most relevant to infection and progression. Therefore, sequence analysis of the virus itself may lead to new insights into the pathogenicity of HIV. Thus, this invention describes a direct method of detecting viral nucleic acid in a biological sample by adsorption of the biological sample onto a solid support followed by amplification of the target nucleic acid sequence and detection of the resultant amplified product. The target nucleic acids capable of detection by this inventive method include not only viruses but also microorganisms, either prokaryotic or eukaryotic, and any normal, abnormal or aberrant cells that may be associated with a disease or physiologic state. In addition to PCR, any technique which multiplies the number of copies of the target polynucleotide sequence such as ligase chain reaction (LCR) can be used for amplification of the target nucleic acid. This method is particularly useful to provide an extremely sensitive immunoassay for the detection of hepatitis B virus (HBV) DNA and HIV-1 RNA in serum. All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims. Table 1. Analysis of HBsAg+ Blood Donors * AII samples were anti-HBe+ except for samples 21 and 23. ND, Assay not performed. iDot blot performed as described in Methods. Borderline positive spots (<1 pg density) were scored as negative. 2 Results of Abbott Genostics HBV DNA assay. 3 The s gene primers generated a DNA fragment that was 690 bp long. Anti-HBs coated microparticles were employed after passing 100 μl sample through a 0.9-ml Sephacryl 300 spin column as described in Methods. Table II. A Blind Analysis of Plasma from Two Patients with Acute HBV HBV DNA Anti-HBe Date HBsAg Pre-S2 1 IgM L H. 2 PCR 3 + + + + + Table II. A Blind Analysis of Plasma from Two Patients with Acute HBV (Continued) ----- Anti-HBe Date HBsAg Pre-S2 1 IgM L H. 2 PCR 3 The pre-S2 assay was performed as described in Methods. Listed are the S/N ratio. 2 Results of Abbott Genostics HBV DNA assay. 3 The s gene primers generated a DNA fragment that was 690 bp long. Anti-HBs coated microparticles were employed after passing 100 μl sample through a 0.9 ml Sephacryl 300 spin column as described in Methods. ND, Assay not performed.

Claims: CLAIMS 1 . A method for detecting a target nucleic acid sequence of HIV-1 which may be present in a biological sample comprising the steps of: a) adsorbing the biological sample onto a solid support coupled to an anti-HIV-1 envelope antibody specific for HIV-1; b ) removing unbound biological sample from the solid support; c ) amplifying the target nucleic acid sequence; and d) detecting the amplified product present in said biological sample. 2. The method of claim 1 wherein interfering substances which may be present in the sample are subjected to desalting prior to step a. 3. The method of claim 2 wherein desalting of the sample is accomplished by passing the sample through a column containing chromatographic material and removing interfering substances which may be present in the sample. 4. The method of claim 1 wherein the solid support is a microparticle. 5. The method of claim 1 wherein the antibody is a monoclonal antibody. 6. The method of claim 5 wherein the monoclonal antibody is secreted by the hybridoma cell line selected from the group consisting of A.T.C.C. deposit no. HB 10951, A.T.C.C. deposit no. HB 10952, A.T.C.C. deposit no. HB 10953, A.T.C.C. deposit no. HB 10954, A.T.C.C. deposit no. HB 10955, A.T.C.C. deposit no. HB 10399 and A.T.C.C. deposit no. HB 10400. 7. The method of claim 1 wherein the sample is heated prior to step 8. The method of claim 7 wherein the sample is heated at 95°C prior to step d. 9. The method of claim 1 wherein the sample is treated with a proteinase prior to step d. 1 0. The method of claim 9 wherein the proteinase is proteinase K. 1 1 . The method of claim 1 wherein the sample is denatured by a denaturing agent prior to step d. 12. The method of claim 1 wherein the sample is denatured by thermal denaturation prior to step d. 13. The method of claim 1 wherein the target nucleic acid is amplified by polymerase chain reaction. 14. The method of claim 1 wherein the target nucleic acid is amplified by ligase chain reaction. 15. The method of claim 1 wherein the detection of step e is carried out by dot blot analysis, Southern blot analysis, or liquid hybridization. 1 6. The method of claim 1 wherein the target nucleic acid sequence is deoxyribonucleic acid. 17. The method of claim 1 wherein the target nucleic acid sequence is ribonucleic acid. 18. A kit suitable for detecting a target nucleic acid sequence of HIV- 1 which may be present in a biological sample comprising: a) a solid support coupled to an anti-HIV-1 envelope antibody; b ) means for removing unbound biological sample from the solid support; c) means for amplifying the target nucleic acid sequence; and d) means for detecting the amplified product present in said biological sample. 1 9. The kit as in claim 18 wherein the solid substrate is a microparticle. 20. The kit as in claim 18 wherein the antibody is a monoclonal antibody secreted by the hybridoma cell line selected from the group consisting of A.T.C.C. deposit no. HB 10951, A.T.C.C. deposit no. HB 10952, A.T.C.C. deposit no. HB 10953, A.T.C.C. deposit no. HB 10954, A.T.C.C. deposit no. HB 10955, A.T.C.C. deposit no. HB 10399 and A.T.C.C. deposit no. HB 10400.

Inventors: HENRARD DENIS R, NIMMS LARRY T, ZELDIS JEROME B, HENRARD, DENIS, R., NIMMS, LARRY, T., ZELDIS, JEROME, B.
Applicants: ABBOTT LAB, ZELDIS JEROME B, ABBOTT LABORATORIES, ZELDIS, JEROME, B.
Application year: 1993
Main classification: C12Q1
IPC codes: C07H21, C12N15, C12P21, C12Q1, G01N33