AU630117B2 - Diagnostic test for hblv - Google Patents
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- AU630117B2 AU630117B2 AU43364/89A AU4336489A AU630117B2 AU 630117 B2 AU630117 B2 AU 630117B2 AU 43364/89 A AU43364/89 A AU 43364/89A AU 4336489 A AU4336489 A AU 4336489A AU 630117 B2 AU630117 B2 AU 630117B2
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- 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
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Description
OPI DATE 18/04/90 Pr AOJP DATE 24/05/90 INTERNATIONAL APPLICATION PUBLISHED U APPLN. ID L133614 89 PCT NUMBER PCT/US89/0L1190 NDER THE PATENT COOPERATION TREATY (PCI) (51) International Patent Classification 4 International Publication Number-, WNO 90/03447 C12Q 1/70, C12N 7/02 Al(3InentoaPulctoDae5Api1900.49) GO IN 33/53, 33/531(4)nralolPulatoDae5Api1900.09) (21) International Application Number: PCT/US89/04190 (74) Agents: OLIFF, James, A, et al,; OlifC& Berridge, 2775S.
(22) International Filing Date: 27 September 1989 (27.09.89)WahntnSreAxndiV 231(U) (81) Designated States: AT (European patent), AU, BE (Euro- Priority data:- pean patent), CH (European patent), DE (European pa- 250,301 28 September 1988 (28.09.88) US tent), FR (European patent), GB (European patent), IT (European patent), JP, LU (European patent), NL (Eu- (71) Applicant- THE UNITED STATES OF AMERICA, repre- oen aetSE(uoenptn) sented by THE SECRETARY, UNITED STATES DE- PARTMENT OF COMMERCE [US/US]; Washington, Published DC 20231 Wth, interniational searchi report, (72) Inventors: SAXINGER, Carl 6814 Renita Lane, Bethesda, MD 20817 GALLO, Robert, C. 8513 Thornden Terrace, Bethesda, MD 20814 (US).
ABLASHI, Dharam, V. 4117 Barnsley Lane, Olney, MD 20832 SALAHUDDIN, Syed, Zaki ;12600 Newgate Road, Potomac, MD 20854 (US).
(54) Title: DIAGNOSTIC TEST FOR HBLV (57) Abstract A serodiagnostic assay and kit fur detecting exposure to or infection by human B-lymphotropic virus are described.
I
WO 90/03447 PCT/US89/0 4 190 -4- DIAGNOSTIC TEST FOR HBLV The present invention is related to a qualitative and quantitative test for serodiagnosis of human B-lymphotropic virus (HBLV). The tests heretofore employed for the detection of HBLV infection utilize immunofluorescence assay (IFA) using fixed HBLV-infected cells mounted on microscope slides or indirect enzymeimmuno assay (EIA) using a lysate of HBLV-infected cells spotted on a nitrocellulose membrane. Both of these IFA and EIA tests are, however, evaluated by laboratory personnel by observing with the eye and, therefore, imprecise and inaccurate to a certain degree, are prone to errors of subjectivity in the interpretation of the results and the preparation of IFA test slides with uniform quality is quite difficult. An objective serological, spectrophotometric assay for detecting HBLV infection was heretofore not available.
SUMMARY OF THE INVENTION It is, therefore, an object of the present invention to provide an objective, spectrophotometric, serological test for determining the exposure and the level of humoral immunity to HBLV.
It is a further object of the present invention to provide a kit for serodiagnosis of HBLV infection.
Other objects and advantages of the present invention will become evident from the following detailed description of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS These and other objects features and many of the attendant advantages of the invention will be better understood upon a reading of the following detailed description when considered in connection with the accompanying drawings wherein: Figure 1 shows the frequency distribution of HBLV ELISA test results for normal blood donors. Antibody reactivity are expressed as the absorbance for each serum divided by the absorbance for the standard normal control.
WO 90/03447 PCT/US89/04190, 2 Figures 2A and 2B show the lack of immunological cross-reactivity between HBLV and CMV. Figure 2A HBLV ELISA testing was performed as described in methods using 3 different test sera representing high reactivity (HBLV virus positive case), intermediate reactivity (pooled normal human serum of commercial origin) and low reactivity (single donor serum, RV=0.25). Each serum was tested for HBLV antibody by ELISA after preabsorption with HBLV-infected HSB2 cells, uninfected HSB2 cells, and CMV antigen as described in methods. Figure 2B CMV ELISA testing was performed as described in methods using 2 different test sera representing a CMV positive serum and the same normal human serum pool used in Figure 2A. Both sera were tested for CMV antibody by ELISA after preabsorption with HBLV-infected HSB2 cells, uninfepted HSB2 cells, CMV antigen and CMV negative control antigen as described in methods. The normal human pool serum activity against CMV was competed by CMV antigen but not by CMV negative control antigen (data not shown).
DETAILED DESCRIPTION OF THE INVENTION The above and various other objects and advantages of the present invention are achieved by a diagnostic kit and a method for serodiagnosis of infection and progression of infection by human B-lymphotropic virus (human herpes virus type comprising: obtaining a substantially pure, soluble, viral antigen lysate from human B-lymphotropic virus (HBLV) or from cells infected with HBLV such as HSB-2; reacting serum from a subject suspected of exposure to or infected by HBLV with the lysate of step also retesting of said serum over intervals of time to assess progression of infection; reacting labelled anti-(human IgG) or anti-(human IgM) or antibodies against other human antibody isotypes or subtypes, with hunan antibody bound in step then spectrophotometrically comparing absorption at the appropriate wavelength of the reaction product formed either directly in step or after r. WO 90/03447 PCT/US89/04190 -3incubation in the presence of appropriate enzyme substrate, with the HBLV-negative control serum; then determining degree of positivity by comparing the value of the test serum with the distribution of similar values for a normal population and the HBLV-negative control serum, a difference in absorbance of 40% or more, preferably 50% or more, being sufficiently indicative of HBLV infection of the subject from whom the test serum was obtained.
An optional test for absolute positivity (confirmatory test) is performed by retesting of a given test serum after preabsorption of that serum with HBLVinfected cell lysate HBLV-HSB2) using uninfected cells as a negative control HSB2), a difference of more than 40% in the readings being indicative of the presence of HBLV-antibodies in the serum obtained from said subject.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference. Unless mentioned otherwise, the techniques employed herein are standard methodologies well known to one of ordinary skill in the art.
MATERIALS AND METHODS Propagation of virus infected cells and purification of virus.
Virus was grown in HSB2 cells as described by Salahuddin, et al, 1986, Science 234, 596-601; Ablashi, et al, 1987, Nature (London) 329, 207.
Preparation of soluble antigen lysates from cells and virus.
r- r I~u;iiYYryy~h! WO 90/03447 PCT/US89/0419 0 4 HSB-2/HBLV infected cells (108 cells/mi) or HBLV (10 mg/ml) were suspended in a buffer containing 0.05 M Tris (pH8), 1% Triton X 100, 0.6 M NaCI, and additionally 1 mM phenyl,methyl,sulfonylfluoride (PMSF) 1/200 Trasylol for cell lysates. The suspension was incubated for 15 min. at 4 0 C, vortexed vigorously or honogenized briefly, and centrifuged at 40,000 x g for min. Viral antigens and controls from cytomegalovirus (CMV), herpes simplex (HSV), varicella zoster (VZV), and adenovirus were purchased from MA Bioproducts, Inc. and used without further preparation at protein concentrations comparable to the HBLV preparations.
A deposit of substantially pure, antigenic viral lysate of the present invention has been made at the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland, 20851, U.S.A. on September 28, 1988 under the accession number 40500. The deposit shall be viably maintained, replacing if it becomes nonviable, for a period of 30 years from the date of the deposit, or for 5 years from the last date of request for a sample of the deposit, whichever is longer, and made available to the public without restriction in accordance with the provisions of the law. The Commissioner of Patents and Trademarks, upon request, shall have access to the deposit.
HBLV ELISA using polystyrene microtiter plates.
HBLV virions were harvested and concentrated from the medium of infected HSB-2 cell cultures and purified by standard sucrose-density gradient sedimentation. Soluble viral antigen lysate was prepared as above. A dilution of 0.5 ug/100 ul was used to coat the wells of Immulon 1 microtiter plates for about 16 hr at 4°C. Test sera diluted 1/400 in 20% normal goat serum (v/v in PBS, phosphate buffered saline) were incubated in the wells 16 hr at 4 0 C. Bound antibody was detected by reaction with 1/1000 goat anti-human-IgG labelled with alkaline phosphatase (Boehringer) for 1 hr at room tem- WO 90/03447 PCT/US89/04190 5 perature (22-25 followed by 0.05% p-nitrophenyl phosphate (pH 8.5) for 20 min and measurement of A400 in a Dynatech MR600 microtiter plate reader. Sera were tested in duplicate and a normal control serum was tested in four different locations on each plate. Results are expressed as ELISA ratio value or RV, the ratio of the test serum absorbance to that of the normal control serum. These conditions were determined according to the strategy developed for HTLV (Saxinger et al, 1983, Lab.
Invest. 49, 371-377).
Confirmation of HBLV antibody specific reactivity by absorption.
Suspected antibody-positive samples were preincubated with soluble antigen lysate from HSB-2/HBLV or HSB-2 cells, prepared as described above and diluted to 30-fold, for 2 hr at 4 0 C. Duplicate aliquots from each pre-incubation were then assayed for anti-HBLV reactivity as described above. Results were expressed as ratio of ELISA absorbance in the presence of HBLV virus (HSB-2/HBLV) to the ELISA absorbance in the absence of HBLV virus (HSB-2).
Characterization of HBLV immunoreactive proteins by immunoblot.
This procedure was performed essentially as described by Saxinger et al, 1983, supra, for HTLV.
Electrophoresis was modified as required by the use of pre-cast 10 to 20% or 12% polyacrylamide gel slabs purchased from Integrated Separations, Inc. and the Megadalt apparatus. From 10 to 20 slabs were each overlaid with 107 HSB-2/HBLV or HSB-2 cells lysed in SDS sample buffer and electrophoresed under the stacking and running buffer conditions described by Towbin et al, 1979, Proc. Natl.
Acad, Sci. USA 76, 4350-4354. Transfer to nitrocellulose was effected by transverse electrophoresis at 25 V for 16 hr. Dansylated protein molecular weight markers were prepared using dansyl chloride (Pierce Chemicals) and included with the sample and used as internal markers of r_ WO 90/03447 PCT/US89/0 4 190.
6 transfer efficiency and molecular weight positions.
After transfer the sheets were washed briefly with ethylene glycol in saline and stored at -20 0 C. Sera were tested at a 1/1000 dilution by incubation with cut strips in S&S trays containing 5% normal goat serum in Blotto for 16 hr at 4 0 C and reaction in sequence with 1/2000 goat anti-human-IgG labelled with alkaline phosphatase (Boehringer) and NBT-BCIP (Kirkegaard and Perry, nitroblue tetrazolium- chloro,indolyl-phosphate).
The methodology of the present invention was employed in the assessment of HBLV antibody reactivity in U.S. populations and used three types of collections (Table 1, columns A to In one, blood donors were collected on a single day in March, 1987 at two separate blood centers in Minneapolis and Kansas. The second was a randomly selected subset of sera from the NHANES-II population-based health and nutrition survey collected over the years 1976 to 1980 (United States National Center for Health Statistics). In the last, pediatric patients seen during the month of June, 1987 at a hospital in North Carolina were used.
Sera from these collections were screened for antibody reactivity with HBLV antigens by ELISA as described herein supra and the results expressed as ratio value (RV) which is the test result for a given serum indexed to the normal control serum. The mean values and standard deviations for each group are listed in columns E and F of Table 1. The mean ELISA RV for the blood donors and population mini-survey ranged from 0.94 to 0.98, and was 0.77 for the pediatric group. Excepting the pediatric group, there was no significant difference between any of the groups. The mean HBLV ELISA RV for the pediatric group, 0.77, was slightly but significantly lower than the other donor groups. The ELISA test results were normally distributed over a broad range of RV (test of Kolmogorov/Smirnov) with no evidence of bimodality (Fig. 1) which precluded the designation of a WO 90/03447 PCT/US89/04190 7 "cut-off" level between negative and positive sera.
Absorption-competition test results for the various study groups listed in column of Table 1 show that greater than 80% of sera tested are specifically neutralized by HBLV-infected HSB-2 cells and not HSB-2 cells alone. For additional clarification, a -confirmatory step was added consisting of preincubating each serum separately with well-characterized preparations from disrupted HSB-2 cells and virus-infected HSB-2 cells. These neutralizing preparations of HBLV-infected or uninfected HSB-2 cells showed no competitive crossreactivity with antibodies against EBV, CMV, HSV, VZV, HIV, or adenovirus type 2 at levels which reduced IgG binding to HBLV-associated antigen by more than 90%. Nor did the antigens of these other herpesvirus compete in IgG reactions with the HBLV-associated antigen preparations. Data for tests with CMV are shown in Fig. 2 and are typical of results with the other viruses. Furthermore, there as no significant correlation between IgG titers against HBLV-associated antigens and antibody titers against other known herpes viruses. The rankcorrelations (Spearman's Rho) between HBLV and other herpesvirus antibody titers for 50 U.S. donors were 0.15, 0.07, and 0.13 for EBV, CMV, and HSV, respectively (Table Correlations between antibody titer against HBLVinfected HSB-2 cells and either uninfected HSB-2 cells or purified virus were 0.09 or 0.88, respectively, showing a general lack of influence of uninfected HSB-2 cellular antigens on the immunoglobulin binding results.
The method of the present invention was also iployed for the study of the magnitude of anti-HBLV IgG reactivity as a function of age in pediatric clinic population. The results indicated that at birth the quantitative levels (RV) are at approximately adult level (RV=1) or slightly higher. The levels decrease logarithmically over time to reach a minimum at ages between 3 to 6 months and gradually rise again ro reach adult levels WO 90/03447 PCr/US89/04190, 8 between the ages of 2-3 years. This trend, that is the biphasic transition between early decline and later increase of antibody levels was, however, clear and unmistakable.
In summary, the data presented herein clearly demonstrate the practical utility, convenience and feasibility of the serological test described herein.
Of course, the method of the present invention can be fully or partially automated by conventional instrumentation means well known to one of ordinary skill in the art.
A diagnostic kit in accordance with the present invention for detecting HBLV infection comprises containers separately containing the substantially pure, isolated, antigenic soluble lysate of HBLV or, a solid surface coated with said lysate (such as microtiter plate, beads, sticks and the like), HBLV-negative and positive control sera and soluble lysates of HBLVinfected and HBLV-uninfected control cells and instructional material for performing the spectrophotometric assay in accordance with the present invention.
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.
i" i^ WO 90/03447 PCT/US89/04190 9 Table 1 IMMUNOGLOBULIN REACTIVITY WITH HHV-6 (HSB-2) VIRUS-ASSOCIATED ANTIGEN AND ITS NEUTRALIZATION BY HSB-2 CELLS INFECTED WITH HHV-6 COMPARED TO HSB-2 CELLS COMPARED TO HSB-2 CELLS SERUM SOURCE
(A)
Randam U.S.
Kansas City Minneapolis female male Pediatric clinic DATE DONOR COLLECTED AGE
(C)
'76-'80 6-74 1987 adult 1987 35.8 S 36.6 35.3 1987 3.7 No.
(D)
119 200 248 89 159 55 IgG Reactivity ELISA ELISA mean stdev
(F)
.985 1.76 .943 2.21 .956 2.70 1.129 2.53 .894 2.72 .770" 3.60 Neutralization OF SERA
NEUTRALIZED
(G)
97% 81% 77% Sera from Kansas City and Minneapolis blood donors were collected during one day in March 1987. Random U.S.
donors were 119 sera randomly selected from the NHANES-II collection (USNCHS). Pediatric clinic sera were collected during the month of June 1987 in North Carolina.
Median age of pediatric subjects was 0.58 years. ELISA testing was carried out using 0.5 ug/0.1 ml of lysed sucrose-gradient purified virions produced by infected cultures of HSB-2 cells and 1/400 dilution of test serum. ELISA results are expressed as the numerical ratio (RV) of the test sample absorbance to that of a pooled normal human serum (Gibco Inc.). Statistical calculations were performed using log(RV) values. Ab+ samples were neutralized >50% HBLV/HSB-2 relative to HSB- 2 cell extracts. Soluble cell extracts were prepared by gentle homogenization of the cells as described in the text.
WO 90/03447 PCT/US89/0 4 1 90 10 Table 2 Rank-Correlations between HBLV and other Herpes Virus Antibody Titers for 50 Random U.S. Donors (using HBLV/HSB2 infected cells) HBLV/HSB2 Spearman's compared with Rho value EBV 0.15 CMV 0.07 HSV 0.13 HSB2 cells 0.09 HBLV virus 0.88
Claims (4)
1. A method for detecting infection by human B- lymphotropic virus (HBLV), comprising: obtaining a substantially pure, soluble, antigenic lysate of human B- lymphotropic virus and a HBLV negative- control serum; reacting serum from a subject suspected of exposure to or infected by human B-lymphotropic virus with the lysate of step reacting labelled anti-(human IgG) or anti-(human IgM) with antibody bound in step then spectrophotometrically comparing absorption at a specific wavelength of reaction product formed either in step or after incubation in the presence of an enzyme substrate, with HBLV-negative control serum; a difference of more than 40% in spectrophotometric readings being indicative of the presence of HBLV- antibodies in the serum obtained from said subject.
2. A kit for serodiagnosis of HBLV-infection, comprising containers separately containing substantially pure, antigenic, soluble lysate of HBLV or HBLV-infected cells.
3. Substantially pure, isolated, soluble, antigenic lysate of human B-lymphotropic virus (HBLV) or of HBLV-infected cells.
4. The lysate of claim 3 deposited at ATCC under accession number #40500. SUBSTITUTE SHEET 11 SUbTiTUE M El 1PEAMt L
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US25030188A | 1988-09-28 | 1988-09-28 | |
US250301 | 1988-09-28 |
Publications (2)
Publication Number | Publication Date |
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AU4336489A AU4336489A (en) | 1990-04-18 |
AU630117B2 true AU630117B2 (en) | 1992-10-22 |
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Application Number | Title | Priority Date | Filing Date |
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AU43364/89A Ceased AU630117B2 (en) | 1988-09-28 | 1989-09-27 | Diagnostic test for hblv |
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EP (1) | EP0436622A4 (en) |
JP (1) | JPH03504163A (en) |
AU (1) | AU630117B2 (en) |
IL (1) | IL91807A (en) |
WO (1) | WO1990003447A1 (en) |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU7852387A (en) * | 1986-08-11 | 1988-03-08 | United States of America, as represented by the Secretary, U.S. Department of Commerce, The | Testing for the human b lymphotropic virus (hblv) |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4661445A (en) * | 1985-05-24 | 1987-04-28 | Saxinger W Carl | Competitive ELISA for the detection of HTLV-III antibodies |
US4816387A (en) * | 1986-06-06 | 1989-03-28 | Bio-Research Laboratories, Inc. | Method for detection of antibodies to HTLV-III and diagnostic test kit useful therewith |
KR940011593B1 (en) * | 1986-10-31 | 1994-12-22 | 모리 가쓰지 | Overlock swing device for use in sewing machine |
JP2971886B2 (en) * | 1987-06-01 | 1999-11-08 | ベイラー・カレツジ・オブ・メデイシン | Expression of immunologically active proteins of human B-lymphotropic virus. |
US5039604A (en) * | 1987-08-21 | 1991-08-13 | Cellular Products, Inc. | Test device and method of preparing same, assay kit and method for the simultaneous detection of two HTLV or HIV antibodies |
US7130515B2 (en) * | 2004-08-31 | 2006-10-31 | 3M Innovative Properties Company | Triple-band bend tolerant optical waveguide |
-
1989
- 1989-09-27 WO PCT/US1989/004190 patent/WO1990003447A1/en not_active Application Discontinuation
- 1989-09-27 JP JP1510389A patent/JPH03504163A/en active Pending
- 1989-09-27 IL IL91807A patent/IL91807A/en not_active IP Right Cessation
- 1989-09-27 AU AU43364/89A patent/AU630117B2/en not_active Ceased
- 1989-09-27 EP EP19890911133 patent/EP0436622A4/en not_active Withdrawn
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
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AU7852387A (en) * | 1986-08-11 | 1988-03-08 | United States of America, as represented by the Secretary, U.S. Department of Commerce, The | Testing for the human b lymphotropic virus (hblv) |
Also Published As
Publication number | Publication date |
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EP0436622A1 (en) | 1991-07-17 |
IL91807A0 (en) | 1990-06-10 |
JPH03504163A (en) | 1991-09-12 |
IL91807A (en) | 1993-05-13 |
EP0436622A4 (en) | 1993-03-17 |
AU4336489A (en) | 1990-04-18 |
WO1990003447A1 (en) | 1990-04-05 |
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