Classifications

• • 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
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/554—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being a biological cell or cell fragment, e.g. bacteria, yeast cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/0005—Vertebrate antigens
  • A61K39/0008—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/0005—Vertebrate antigens
  • A61K39/0011—Cancer antigens

Definitions

• This invention relates to new and improved methodology for the immunodiagnosis of multiple sclerosis and/or malig ⁇ nant diseases from blood sample analysis.
• Another object of our invention is the provision of methodology as above which provides highly accurate, specific and readily reproducible positive diagnostic re ⁇ sults.
• Another object of our invention is the provision of methodology as above which, in large measure, consists of relatively uncomplicated steps to thus enable the effective and reliable administration thereof by appropriately trained laboratory technicians as opposed to highly trained medical specialists.
• Another object of our invention is the provision of methodology as above which is effective to materially reduce the patient discomfort, time required, and overall cost attendant positive diagnosis of multiple sclerosis and/or malignant diseases.
• Another object of our invention is the provision of methodology as above which is, in not insubstantial measure, particularly adaptable to automation, to even further reduce the time and cost requirements thereof.
• Another object of our invention is the provision of methodology as above which advantageously enables the quantity preparation, well in advance of actual diagnostic testing of patients, of putative antigenic, disease-specific test extracts, and non-specific control extracts which may readily be frozen for effective, long-term storage and/or shipment thereof.
• a further object of our invention is the provision of methodology as above which advantageously enables the determination of diagnostic test results through use of a ' variety of different procedures.
• the new and improved methodology of our invention is based upon the discovery that a multiple sclerosis related material is present in and may be ex ⁇ tracted from the pooled whole bloods of donors known to have multiple sclerosis in accordance with currently accepted diagnostic techniques; and that this material has putative, specific antigenic characteristics when appropriately reacted with the disease-sensitized leukocytes (white blood cells) of whole blood sample from a patient with multiple sclerosis.
• these putative antigenic characteristics of the extracted material effect a specific decrease in the ability of the disease-sensitized leukocytes of the multiple sclerosis patient blood sample, probably as a result of altered cell-mediated immunity, to adhere to a glass surface, thus making possible the prompt and reliable determination of the extent of the putative antigenic extract-sensitized leukocyte reaction by leukocyte adherence inhibition or related assay.
• Control extracts for assay purposes are prepared from the pooled bloods of donors known not to have multiple sclerosis.
• a non-adherence index value of greater than 20 attendant utilization of the new and improved methodology of our in ⁇ vention by way of leukocyte adherence inhibition assay is deemed to warrant a positive diagnosis of multiple sclerosis.
• the specificity and clinical reproducibility of the antigenic extract-sensitized leukocyte reaction are to say, for example, that the putative antigenic extract as prepared from the blood of a donor known to have multiple sclerosis will not appreciably react with, or affect the ability of, the leukocytes from a healthy patient, or the disease- sensitized leukocytes from one suffering, for example, from breast cancer, to adhere to a glass surface.
• the clinical reproducibility of the test procedures render the same particularly adaptable for widespread diagnostic application, especially since the various putative antigenic
• O extracts can be prepared well in advance of actual horrid and stored for long periods by freezing; while the relatively uncomplicated nature of the test procedures render the same readily and effectively administrable by appropriately trained medical technicians. Too, the nature of the test procedures is such that automation thereof to a not insubstantial extent should prove practical.
• Alter ⁇ native preparation of the putative antigenic extract for multiple sclerosis diagnosis from the pooled urines of donors known to have multiple sclerosis, and alternative preparation of the putative antigenic extract for cancer diagnosis from the pooled urines, pleural fluids, ascites fluids, supernates of tumor cell cultures, or tumor cells grown in culture media, respectively, of patients known to have the specific type of cancer of interest, are also disclosed in accordance with the teachings of our invention.
• FIG. 1 is a scattergram illustrating the results pro ⁇ vided by a typical application of the new and improved methodology of our invention
• FIG. 2 is a table setting forth typical non-adherence index values obtained through an application of the method ⁇ ology of our invention to the diagnosis of multiple scler ⁇ osis;
• FIG. 3 is a table setting forth the non-adherence index values obtained through periodic application of the method ⁇ ology of our invention to a group of ten multiple sclerosis patients.
• FIG. 4 is a table setting forth the results of validation testing of the specificity of the putative anti ⁇ genic extracts of our invention. DETAILED DESCRIPTION OF THE INVENTION
• MS multiple sclerosis
• a specific malignant dis ⁇ ease of interest in the nature, for example, of breast cancer, or cancer of the head and neck, from blood sample analysis is typically effected attendant the following steps; it being understood that the nature and/or purpose of these steps may vary somewhat in accordance with the requirements of the specific test procedure to be utilized, all as described in detail hereinbelow:
• test extract a putative antigenic, specific MS related material, or specific malignant disease of interest related material (hereinafter the "test extract") from the pooled bloods of donors known to have MS or the specific malignant disease of interest, and preparation of the con ⁇ trol extract from the pooled bloods of donors known not to have MS or the specific malignant disease of interest:
• NAI non-adherence index
• the leukocyte- rich plasma is then drawn off and centrifuged at 200 X g for 5 minutes at 25°C, whereupon the resultant supernate is discarded and the predominantly white cell pellet resuspended in 2.5 ml of 0.8% ammonium chloride (adjusted, for example, with IN NaOH to pH 7.2) and allowed to stand for 15 minutes at 4°C to lyse the remaining red blood cells.
• the cell pellet is then washed three times in 5 ml of Medium 199 (Gibco preparation containing 25 mm Hepes Buffer Glutamine and Hanks Balanced Salts) , and the final suspension adjusted with additional Medium 199 as required, through use, for example, of a Coulter or like white cell counter, to a concentration of I X 10 leukocytes per ml.
• Test And Control Extract Preparation Twenty ml of venous blood is withdrawn into plastic syringes from each of a group of at least 5 donors, all known to have clinically definite MS in either the relapsing or progressive phase of the disease in accordance, for example, with the Rose et al.
• the resultant supernate is dialyzed for 12-16 hours in a 26 mm cellophane membrane, with a molecular weight cut-off in the range of 12,000 to 14,000 daltons, against two liters of phosphate buffered saline of pH7.4 with three changes of buffer.
• the resultant non-dialysate is centrifuged at 40,000 X g for 15 minutes at 4°C and the resultant supernate concentrated to 30 ml in a sterile 90 mm Millipore Ultra Filtration Unit with a PSAC membrane, with purified nitrogen at 40 psi being used to propel the solution through the membrane.
• the resultant retentate which is never allowed to dry, is diluted with PBS to 50 ml, and clarified by centrifugation at 40,000 X g for 15 minutes at 4°C to com ⁇ plete the preparation of the specific or test extract.
• Storage of the extract is preferably effected in 1 ml ali- quots at -60°C.
• Preparation and storage of the non-specific or control extract is effected in the same manner as preparation and storage of the test extract but from the pooled bloods of each of a group of at least five donors known not to have MS.
• Test And Control Sample Preparation The respective test and control extracts are thawed, if necessary, and diluted to protein concentrations of approximately 5 mg/ml (1:30 dilution) with Medium 199. 0.1 ml aliquots ⁇ ' f- the thusly diluted test and control extracts are then respectively dispensed into disposable glass tubes (for example, Tek Disposable, 16 X 150 mm as marketed by Abbott Laboratories, South Pasadena, California) which have been thoroughly pre- cleaned with ethyl alcohol.
• disposable glass tubes for example, Tek Disposable, 16 X 150 mm as marketed by Abbott Laboratories, South Pasadena, California
• LAI basic leukocyte adherence inhibition
• LAI assay functions to measure the ability of the leukocytes of the respective test and control samples to adhere to a glass surface by counting those of the same which do not so adhere.
• a procedure control sample consisting of 0.1 ml of patient's blood sample, pre ⁇ pared as above, and 0.4 ml of Medium 199 is pipetted into a plastic cuvette containing 20 ml of Isoton or like cell- counting solution, and a Coulter Electronic Cell Counter, of the type manufactured and marketed by Coulter Electronics, Inc.
• test and control extract background cell counts are then obtained with, in each instance, 0.1 ml of the relevant extract being mixed with 0.4 ml of Medium 199 and Isoton, and electronic cell counting accomplished as above.
• 0.1 ml aliquots of the respective test and control sample suspen ⁇ sions are pipetted into plastic cuvettes each containing 20 ml of Isoton II or like cell-counting solution, Counter and the cell counter then utilized to count the non-adherent leukocytes (white blood cells) from each .of the test and control sample suspensions, it being understood that all assays are preferably conducted in triplicate with attendant mean calculation.
• the assay results are expressed in terms of the NAI in accordance with the following equation:
• A is the mean number of non-adherent leukocytes from the test sample minus the test extract background count
• B is the mean number of non-adherent leukocytes from the control sample minus the control extract background count.
• Test And Control Sample Preparation 0.01 ml of loosely packed latex beads or like leukocyte agglutination means of approximately 7 microns in diameter in a 1% NaCl solution is added to each one of three polystyrene tubes (16 X 100 ml) , hereinafter referred to as tube1,," “tube 2,” and “tube 3.” 0.1 ml of the test extract is added to "tube 1,” and 0.1 ml of the control extract is added to "tube 2.” No extract is added to "tube 3" which is to be used as a procedure control, for detection of bead clumping, only. Sufficient Medium 199 is added to each of the tubes to achieve final tube contents volumes of 0.31 ml.
• Test And Control Sample Assay Initially, the three "pro ⁇ cedure control slides" are examined microscopically at, for example, 10 X 45 power, and an agglutination index calcu ⁇ lated as described hereinbelow. If this agglutination index is greater than 30, the procedure is discarded. Thereafter, "slide 3" is microscopically examined as above to determine if clumping of the latex beads and blood sample leukocytes has occurred to indicate insufficient latex bead- solution mixing and require re-preparation of the test and control samples.
• Cell counting may conveniently be effected through use of automatic visual imaging means taking, for example, the form of the Cyto-Tally or similar scanning cell counter. If desired, cell counting may be conducted in duplicate, using the duplicate slides 1 and 2, and an average calculated.
• A is the average number of positive latex beads from slide
• 1, and B is the average number of positive latex beads from slide 2.
• an Al value of greater than 30 has been selected as positive indicia of MS in the patient in question.
• HBSS Hanks Balanced Salt Solution
• LSM Lymphocyte Separation Medium
• lymphocytes which separate at the respective tube inter ⁇ faces during centrifugation are carefully removed with a disposable plastic syringe and long cannula, whereupon the resultant lymphocyte serum suspensions are transferred to and combined in a plastic centrifuge tube, to which is added 3 ml of HBSS, and centrifuged at 200 X g for 10 minutes at 25°C. Centrifugation as last described is re ⁇ peated two additional times with the supernate being dis ⁇ carded in each instance and the pellet re-suspended in 3 ml of HBSS.
• lymphocyte pellet resulting from final centri ⁇ fugation is then re-suspended in 0.5 ml of Medium 199 con ⁇ taining 10% fetal calf serum to adjust the lymphocyte con ⁇ centration to 1 X 10 ⁇ , and 0.2 ml sodium chromate 51 (1 uC/ml) as marketed by New England Nuclear, Boston,
• Test And Control Extract Preparation These preparations are effected in the same manner described in detail hereinabove with regard to utilization of the leukocyte adherence inhibi ⁇ tion procedure.
• Test And Control Sample Preparation Medium 199 with Hepes to which has been added 10% fetal calf sera (hereinafter "Medium”), the test and control extracts, diluted to protein concentrations of approximately 5 mg/ml (1:30 dilution) with Medium 199, and/or the labeled blood sample cells, are re ⁇ spectively disposed in the 96 wells of a plastic microplate, which contains 8 panels (hereinafter “panels 1 through 8") of 12 wells each, as follows:
• the plastic microplate is incubated for 120 minutes at 37°C to complete the test and control extract-labeled cells reactions, if any. After incubation, the plate is turned over on a paper towel and allowed to drain at room temperature for 20 minutes to enable the re ⁇ acted, and thus non-adherent, labeled cells to flow out of the panel wells, whereupon the plate is again placed upright and sprayed with a cell exfoliate spray fixator to fix the adherent, labeled cells in the respective wells.
• test And Control Sample Assay For assay, the plastic micro ⁇ plate is cut into individual wells which are dropped in turn into a plastic counting tube for counting of the adherent, labeled cells in each of the wells by an Abbott gamma counter as manufactured by Abbott Laboratories of Chicago, Illinois.
• the assay results are expressed in terms of the lymphocyte adherence index (LYAI) in accordance with the following equation:
• A is the mean number of gamma counts per minute for all test wells initially containing the test extract and labeled cells
• B is the mean number of gamma counts per minute for all control wells initially containing the control extract and lab ⁇ led cells.
• a value of greater than 30 for the LYAI from Equation 3 has been selected as positive indicia of MS on the part of the patient in question.
• the leukocyte-rich plasma is withdrawn and centri ⁇ fuged in a conical polystyrene tube at 200 X g for 5 minutes, whereupon the supernate is discarded and the pellet re- suspended in 1 ml of tris-buffered ammonium chloride solu ⁇ tion and refrigerated at 4°C for 15 minutes to lyse the red cells. 5 ml of Medium 199 with Hepes is then added to the
• Test And Control Extract Preparation These preparations are effected in the same manner described in detail herein ⁇ above with regard to utilization of the leukocyte adherence inhibition procedure.
• Test And Control Sample Preparation The respective test and control extracts are diluted to protein concentrations of approximately 5 mg/ml (1:30 dilution) with Medium 199.
• Three pairs of polystyrene tubes (16 X 100 mm) are then pre ⁇ pared in the following manner: 0.2 ml of the test extract, and 0.4 ml of the blood sample cell suspension is added to each of the first tube pair; 0.2 ml of the control extract and 0.4 ml of the blood sample cell suspension is added to each of the second tube pair; while 0.4 ml of the blood sample cell suspension is added to the third tube pair for use as a procedure control; it being understood that the extent to which the blood sample leukocytes are broken up, or lysed, in the first and second tube pairs will be propor ⁇ tional in each instance to the extent of the relevant extract-blood sample leukocyte reaction. Thereafter, sufficient Medium 199 is added to each of the tubes to pro ⁇ vide final tube solution volumes of 1 ml.
• Test And Control Sample Assay 0.1 ml of the test and con ⁇ trol sample, and 0.1 ml of the diluted blood sample cell suspension are each added to respective 20 ml quantities of isotonic ayide-free cell counting solution (Diluid or Isoton) in plastic cuvettes, and the leukocytes which have not been lysed counted through use of a Coulter D-2 electronic cell counter to provide an initial count.
• the tubes are then in ⁇ cubated at 37°C for one hour with gentle shaking every 15 minutes, whereupon leukocyte counting is again accomplished as described to provide a middle count for procedure control purposes.
• the tubes are then reincubated for one hour at 37°C with gentle shaking as above, whereupon leukocyte ' counting as described is accomplished for a third and final time to provide a final count.
• the percentage of lysis for each tube pair is then calculated in accordance with the following equation:
• the lysis index (LI) is then calculated in accordance with the following equation:
• an LI value of greater than 20 has been selected as positive indicia of MS in the patient in question.
• test and control extracts for the immunodiagnosis of malignant diseases of interest are effected in the same general manner as that described herein ⁇ above with regard to the preparation of the test and control extracts for the immunodiagnosis of MS. More specifically, and taking, for example, test and control extract prepara ⁇ tion with regard to breast cancer, it may be understood that the test extract would be prepared as described, but from the pooled bloods of a group of at least five donors known to have breast cancer through extraction as described of the breast cancer-related material therefrom; while the control extract would again be prepared as described, but from the pooled bloods of a group of at least five donors known not to have breast cancer. The diagnostic procedures would, in all other respects, remain the same as described hereinabove for the diagnosis of MS.
• O ⁇ leukocytes from the blood samples of patients having the same particular disease as that borne by the donors of the pooled bloods from which the test extract in question was prepared was accomplished by the testing of three different putative antigenic extracts against the sensitized leukocytes from the bloods of MS patients, patients with malignant diseases in the nature of breast cancer, and cancer of the head and neck, respectively, and against the non-sensitized leukocytes of "normal" control patients. The results of this validation testing are presented in the table of FIG.
• FIG. 4 is believed to make clear that non-sensitized leukocytes from the blood samples of normal control donors display a marked non-specificity to all of the putative antigenic extracts in question.
• the respective NAI values of FIG. 5 are believed to indicate the absence of significant cross-reactivity between the respective putative antigenic extracts of interest.
• the respective test and con ⁇ trol extracts may be effectively prepared from body substances other and different from blood which have also been found to contain the MS-related materials and the malignant disease- rela-ted materials of interest; it being understood by those skilled in this art that certain other body substances, as specified hereinbelow, may be more readily available in the necessary quantities to thus facilitate the relatively large scale advance preparation of the extracts.
• the respective test and control extracts may alternatively be respectively prepared from body substances taking the form of the appropriately concentrated, pooled urines of a plural ⁇ ity (again, for example, five) donors known to have MS in accordance with the Rose et al.
• test and control extract preparation would be effected in the manner described in detail hereinabove excepting for the fact that the same would commence, in each instance, with body substances taking the form of the pooled, appropriately concentrated urines, rather than the pooled bloods, of the known MS, and known non-MS, donors. Blood sample preparation, test and control sample preparation, and test and control sample assay would, in each instance, and with regard to each procedure, remain as described.
• test and! control extract preparation may alternatively be respectively effected from the pooled pleural fluids, ascites fluids, supernates of tumor cell cultures, tumor cells grown in culture media, or concentrated urines, or like substances, of a plurality (again, for example, five) donors known to have the specific type of cancer of interest in accordance with traditional diagnostic techniques; and from the pooled pleural fluids, ascites fluids, supernates of tumor cell cultures, tumor cells grown in culture media or concentrated urines of a like plurality of donors known not to have the specific type of cancer of interest in accordance with those same diagnostic techniques.
• blood sample preparation, and test and control sample preparation and assay would, in each instance, and with regard to each procedure, remain as described.
• MS or malignant disease may be readily and positively effected in accordance with the teachings of our invention as described through the simple withdrawal of a blood sample from the patient in question, and without regard for the particular body substance, that is blood, urine, pleural fluid, ascites fluid, supernates of tumor cell cul ⁇ tures, or tumor cells grown in culture media, or like sub ⁇ stance, from which the respective test and control extracts were prepared.

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• 1979
  • 1979-04-30 WO PCT/US1979/000272 patent/WO1980002459A1/en not_active Ceased
• 1980
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