WO1993014225A1 - Procede d'amplification et de detection d'une sequence d'acides nucleiques cible de hiv-1 - Google Patents

Procede d'amplification et de detection d'une sequence d'acides nucleiques cible de hiv-1 Download PDF

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WO1993014225A1
WO1993014225A1 PCT/US1993/000295 US9300295W WO9314225A1 WO 1993014225 A1 WO1993014225 A1 WO 1993014225A1 US 9300295 W US9300295 W US 9300295W WO 9314225 A1 WO9314225 A1 WO 9314225A1
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hiv
nucleic acid
sample
deposit
target nucleic
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PCT/US1993/000295
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Jerome B. Zeldis
Denis R. Henrard
Larry T. Nimms
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Abbott Laboratories
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Priority to JP5512640A priority Critical patent/JPH07503135A/ja
Priority to EP93903459A priority patent/EP0672176A4/fr
Publication of WO1993014225A1 publication Critical patent/WO1993014225A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/702—Specific hybridization probes for retroviruses
    • C12Q1/703—Viruses associated with AIDS
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV

Definitions

  • 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.
  • hepatitis infection One category of viral infection which is of concern is hepatitis infection.
  • 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).
  • HAV hepatitis B virus
  • HAV hepatitis A virus
  • HDV hepatitis D virus
  • NANB non-A, non-B
  • 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.
  • 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
  • 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.
  • anti-HBcAg antibody to HBcAg
  • 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.
  • an analyte e.g., a bacterium, virus or genetic defect
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • an anti-HIV-1 envelope antibody such as anti-HIV gp120 or anti-HIV gp41 or anti-HIV p24 antibody
  • Figure 1A the location, length (size in base pairs) and orientation (direction) of the primers used to perform PCR on HBV DNA
  • 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 ⁇ l 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).
  • RT reverse transcriptase activity
  • 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.
  • 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.
  • 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 refer to DNA or RNA, whether in single or double-stranded form, including messenger or ribosomal RNA.
  • 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.
  • HIV HIV-1 or HIV-2
  • hepatitis viruses HIV (HIV-1 or HIV-2)
  • herpes viruses human retroviruses
  • human papilloma viruses Epstein-Barr virus
  • Cytomegalovirus viral RNA transcripts
  • viral RNA transcripts and replicative intermediates.
  • nucleic acids of bacteria such as mycoplasmas, rickettsiae and chlamydi
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • PCR Polymerase Chain Reaction
  • 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.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • 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 (-).
  • 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
  • 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.
  • 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.
  • 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 ⁇ l 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
  • 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).
  • 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.
  • 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.
  • ⁇ l of a "master mix” consisting of nine ⁇ l of water, two ⁇ l of ten time PCR buffer, eight ⁇ l of dNTP mix (1.5 mM final concentration each of datp, dctp, dgtp, " FTP, pH 7), 1 ⁇ l each of a clockwise and counterclockwise primer (final concentration of each primer is approximately 1 ⁇ M).
  • 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 ⁇ l (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 ⁇ g/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 ⁇ l of serum sample was pipetted in an Eppendorf tube.
  • 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 ⁇ m 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).
  • 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 ⁇ l 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.
  • EDC 1-ethyl-3(dimethylaminopropyl) carbodiimide
  • 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.
  • 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
  • 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.
  • 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 ⁇ l containing 15 mM Tris pH 7.4, 10 mM MgCI2, 0.1 mM EDTA, 0.1 spermidine, 5 mM DTT and 75 ⁇ M of gamma labeled 32 P-dATP at 3,000 mCi per mMole.
  • the above-described method 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.
  • FIG. 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 ⁇ l of serum; monoclonal anti-HBs coated microparticles were capable of detecting as few as 3 viruses in 200 ⁇ l 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • HBsAg positive sera were analyzed for HBV DNA by both a conventional dot blot assay and the claimed method using PCR amplification.
  • the PCR method using either the antibody coated microparticle or Eppendorf tubes also produced a positive signal.
  • 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.
  • Three samples were positive by liquid hybridization and six by PCR analysis. There were no false positive samples by either assay.
  • HBsAg and Pre-S2 antigenemia preceded serum HBV DNA. In one, virem ⁇ a was found after the patient was no longer HBsAg positive. (1/31/89, Patient E).
  • HIV-1 RNA By Antibody Microparticle Method Patient Samples
  • HIV-1 gp41 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).
  • 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 HB 10399
  • 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.
  • 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.
  • Carboxylated latex microparticles (0.1-0.3 ⁇ m 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).
  • 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.
  • RNA pellet Fifty microliters of serum/plasma were diluted with 150 ⁇ l 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 ⁇ l of 20 mM Tris pH 8.3, 0.5 mg/ml proteinase K, 0.25% SDS, 20 ⁇ g 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 ⁇ l of sterile water.
  • First strand cDNA synthesis was performed by adding 5 ⁇ l 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 ⁇ l of sample and incubating at 37°C for 30 minutes. The mixture then was diluted with 30 ⁇ l of water and heated at 95° C for ten minutes.
  • SK38/39 S. Kwok et al., J. Virol. 61 :1690-1696 [1987]
  • Amplification was carried out in a final volume of 100 ⁇ l 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.
  • HIV p24 antigen was measured in a solid-phase sandwich-type enzyme- linked immunosorbent assay (EIA) (available from Abbott Laboratories, Abbott
  • 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).
  • RT Reverse Transcriptase Assay Reverse transcriptase assay was done by adding 100 ⁇ l 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
  • Viral capture assay of the invention compared to HIV p24 antigen and reverse transcriptase.
  • Reverse transcriptase activity was not detected in any of the samples, as seen by the graphed line between open squares.
  • Fifty ⁇ l of sample were diluted to 200 ⁇ l 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).
  • 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.
  • 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.
  • 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.
  • HCV hepatitis C
  • 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.
  • 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.
  • 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.
  • PCR could detect serum HBV DNA and HIV RNA at concentrations below the sensitivity of conventional dot blot and liquid hybridization assays.
  • desalting serum or plasma through a Sephacryl 300 spin column prior to adsorption also increased the sensitivity of the assay.
  • a rough quantitat ⁇ on of viral DNA can be estimated by serially diluting a sample in HBV DNA negative serum or PBS.
  • viruses per 200 ⁇ l can be detected using anti-HBs, anti-Pre-Sl, or anti-Pre-S2 coated microparticles.
  • anti-HBs anti-HBs
  • anti-Pre-Sl anti-Pre-S2 coated microparticles.
  • HIV detection a variety of highly sensitive serological assays for antigens, antibodies and nucleic acids are available for both the clinician and researcher.
  • 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).
  • 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.
  • 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).
  • 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.
  • samples are processed non- destructively and can be analyzed by other methods after reaction with the solid phase.
  • 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.
  • the sensitivity of the p24 antigen monoclonal assay is about 1.0 pg/200 ⁇ l 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 ⁇ l 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.
  • these samples may contain defective non-infectious particles. In that case, the actual infection inducing seroconversion would have occurred somewhat later.
  • 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.
  • 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.
  • 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.
  • LCR ligase chain reaction
  • the s gene primers generated a DNA fragment that was 690 bp long.
  • Anti-HBs coated microparticles were employed after passing 100 ⁇ l 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
  • the pre-S2 assay was performed as described in Methods. Listed are the S/N ratio. 2 Results of Abbott Genostics HBV DNA assay.
  • the s gene primers generated a DNA fragment that was 690 bp long.
  • Anti-HBs coated microparticles were employed after passing 100 ⁇ l sample through a 0.9 ml Sephacryl 300 spin column as described in Methods. ND, Assay not performed.

Abstract

Procédé direct servant à la détection d'acide nucléique viral de HIV-1 dans un échantillon biologique susceptible de contenir l'acide nucléique cible au moyen de l'adsorption dudit échantillon biologique sur un support solide accouplé à un anticorps d'enveloppe anti-HIV-1, suvi par l'amplification de la séquence d'acides nucléiques cible et de la détection du produit amplifié. Les procédés d'amplification pouvant s'utiliser comprennent la réaction en chaîne de polymérase et la réaction en chaîne de ligase.
PCT/US1993/000295 1992-01-13 1993-01-13 Procede d'amplification et de detection d'une sequence d'acides nucleiques cible de hiv-1 WO1993014225A1 (fr)

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JP5512640A JPH07503135A (ja) 1992-01-13 1993-01-13 Ｈｉｖ‐１の標的核酸配列を増幅及び検出する方法
EP93903459A EP0672176A4 (fr) 1992-01-13 1993-01-13 Procede d'amplification et de detection d'une sequence d'acides nucleiques cible de hiv-1.

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JPH07503135A (ja) 1995-04-06

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