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
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56983—Viruses
• • 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
  • G01N33/555—Red blood cell
  • G01N33/556—Fixed or stabilised red blood cell
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S436/00—Chemistry: analytical and immunological testing
  • Y10S436/811—Test for named disease, body condition or organ function
  • Y10S436/812—Infectious mononucleosis
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S436/00—Chemistry: analytical and immunological testing
  • Y10S436/826—Additives, e.g. buffers, diluents, preservatives
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T436/00—Chemistry: analytical and immunological testing
  • Y10T436/10—Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
  • Y10T436/101666—Particle count or volume standard or control [e.g., platelet count standards, etc.]
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T436/00—Chemistry: analytical and immunological testing
  • Y10T436/10—Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
  • Y10T436/107497—Preparation composition [e.g., lysing or precipitation, etc.]
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T436/00—Chemistry: analytical and immunological testing
  • Y10T436/10—Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
  • Y10T436/108331—Preservative, buffer, anticoagulant or diluent

Definitions

• This invention relates to a method for the diagnosis of infectious mononucleosis and to a novel composition useful therein; more particularly, the invention relates to an improved test for diagnosing such disease with improved accuracy, rapidity and simplicity.
• Infectious mononucleosis is an acute disease most commonly found in patients within the 1625 age group, which is characterized by fever, generalized lymph node enlargement and lymphocytosis with atypical Lymphocytes.
• Heterophile antibodies i.e., antibodies having an aflinity for related or identical antigens found in sheep cells, guinea pig tissues and many other unrelated biological substances, are usually present in the blood of mononucleosis patients and, in fact, provide the basis for the commonly employed heterophile antibody test for this disease.
• heterophile antibodies are found in the sera of 90% of those patients infected with mononucleosis, such antibodies may also be present in the blood of individuals not having the disease.
• the antibodies may be produced by the injection of biologicals containing horse serum, particularly in persons who manifest serum sickness following such injections. Alternatively, .they may occur naturally in the blood stream in low titer as so-called 'Forssman antibodies, or native heterophile. It is because of this variety of hetero phile antibodies which may be present in test sera that it has heretofore been necessary to employ relatively complicated multistage serological tests to identify and distinguish the heterophile antibodies characteristic of mononucleosis.
• the common serological tests for heterophile antibodies are based upon the fact that such antibodies can be detected by their ability to cause agglutination of sheep erythrocytes. In order to diagnose mononucleosis, making use of such phenomenon, a 3-stage diagnostic test has evolved.
• a slide test is performed, in which the serum suspected to contain heterophile antibodies is titrated with sheep erythrocytes, antibodies present therein agglutinating the erythrocytes.
• a third or differential heterophile test involves the treatment of two further samples of the original test serum with two antigens, the first of which is capable of adsorbing any Forssman and serum sickness antibodies present in the sample treated, and the second of which is capable of adsorbing any infectious mononucleosis and serum sickness antibodies present in the other sample.
• antigen prepared from horse kidneys will adsorb the Forssman and serum sickness antibodies
• antigen prepared from beef erythrocytes will adsorb the infectious mononucleosis and serum sickness antibodies.
• the type of antibodies present in the test serum may be determined.
• the heterophile antibody test for infectious mononucleosis is positive if, in the differential test, adsorption of the serum with the horse kidney antigen fails to completely remove the anti-sheep cell agglutinins, whereas adsorption with the beef cell antigen completely removes the anti-sheep agglutinins.
• a further object of the invention is to provide such a method which, in view of its improved specificity, is substantially free of false positive indications and does not, therefore, require the high proportion of time-consuming presumptive and differential testing required by heretofore known heterophile antibody serological diagnoses.
• Yet a further object of the invention is to provide a composition useful in the improved diagnostic method of the present invention, which composition is stable and may be employed even after long periods of storage.
• the aldehyde-treated equine erythrocyte compositions employed in the practice of the method hereof are prepared by treating one volume of equine erythrocytes with from 0.2 to one volume of the appropriate aldehyde, preferably formaldehyde.
• the treatment may be conducted at any suitable temperature, it being preferred to react the reagents at temperatures of from about 4 to 40 C.
• the thus aldehyde-treated cells are thereafter incorporated in a saline suspension containing from about 2% to 6% by volume of the cells, and desirably, additionally containing a suitable preservative, e.g., sodium azide, phenol, or Merthiolate (a trade name for sodium ethyl mercurithiosalicylate).
• a suitable preservative e.g., sodium azide, phenol, or Merthiolate (a trade name for sodium ethyl mercurithiosalicylate).
• Reagents thus prepared have been found so
• EXAMPLE 1 A 4% saline suspension of formalin-treated horse erythrocytes containing 0.1% sodium azide preservative was prepared.
• the formalized erythrocytes were initially obtained as follows: Horse blood was obtained by bleeding directly into Alsevers solution so that the final composition was one-half horse blood and one-half Alsevers solutin. To 50 ml. of the aforegoing mixture was added 20 ml. of 50% ormalin and the mixture incubated at a temperature of 37 C.
• saline suspension was added to one drop of each serum to be tested on a fiat glass slide, mixed at room temperature with a wooden applicator stick, rotated for two minutes and read for agglutination within such period, using indirect lighting.
• a saline control was carried out concurrently with each slide test. Clear or fine granular patterns were regularly viewed in the slide tests employing the saline controls. Positively reacting sera, on the other hand, produced coarse agglutination patterns.
• the formalized horse cells thus prepared were utilized in slide tests of 978 different test sera, and the results thus obtained compared with the serological diagnoses obtained employing the slide test-presumptive test-differential test technique described by Davidsohn.
• the results obtained by the use of the simple slide test employing formalized horse erythrocytes differed from those obtained by the differential test procedure in only nine cases, equivalent to a diagnostic accuracy of over 99%.
• the test results are summarized in Table I below, comparing the positive and negative indications as to the presence of infectious mononucleosis (IM) obtained by the respective tests:
• test sera titrated with the formalized horse erythrocytes were also titered against sheep cells to compare the efficacy of slide tests employing the conventional raw sheep cell reagent, with such tests utilizing the formalized horse cell reagent of the present invention.
• 250 sera obtained from blood donors and patients suffering from a variety of diseases were used as controls.
• one serum from a patient with bronchial asthma showed a positive agglutination, while 249 were negative with the formalized horse cells.
• EXAMPLE 2 A 4% saline suspension of glutaraldehyde (C H O treated horse erythrocytes containing 0.1% sodium azide preservative was prepared.
• the glutaraldehyde-treated erythrocytes were initially obtained as follows: Horse blood was obtained by bleeding directly into Alsevers solution so that the final composition was one-half horse blood and one-half Alsevers. solution.
• To 50 ml. of the foregoing mixture was added 80 ml. of 25% glutaraldehyde, 16 ml. sodium carbonate to a pH of 7.0, and 50 ml. of 0.15 M phosphate buffer to a pH of 7.2. The mixture was thereafter incubated at 25 C. for 8 hours.
• the glutaraldehyde-treated cells thus prepared were utilized in slide tests of 20 different test sera, and the results thus obtained compared with the serologic diagnoses obtained employing the presumptive test-differential test technique described by Davidsohn. Of the 20 tests so performed the results differed from the Davidsohn procedure results in only one case.
• EXAMPLE 3 A 4% saline suspension of pymvaldehyde-treated horse erythrocytes containing 0.1% sodium azide preservative was prepared.
• the pyruvaldehyde erythrocytes were initially obtained as follows: Horse blood was obtained by bleeding directly into Alsevers solution so that the final composition was one-half horse blood and onehalf Alsevers solution. To 50 ml. of the foregoing mixture was added 50 m1. of a pyruvaldehyde solution containing 8 ml. of 45% pynu-valdehyde, 17 m1. of a 1% sodium carbonate solution, and ml. of 0.15 M sodium phosphate buffer, the resulting mixture having a pH of 6.5. The mixture was thereafter incubated at a temperature of 25 C.
• the pyruvaldehyde-treated horse cells thus prepared were utilized in slide tests of 20' different test sera, and the results thus obtained compared with the serologic diagnoses obtained employing the presumptive test-differential test described by Davidsohn. Of the 20 tests performed, the results obtained by the simple slide test of this example differed in only one case from those obtained by the Davidsohn procedure.
• an improved method for the diagnosis of infectious mononucleosis and a novel composition useful in the practice of such method See footnote 5, column 4.
• a method for the specific diagnosis of infectious mononucleosis which comprises titrating a test serum with a saline suspension of aldehyde-treated equine erythrocytes, said erythrocytes having been treated with a water-soluble aldehyde compatible therewith, whereby the presence in said serum of heterophile antibodies associated :with infectious mononucleosis effects agglutination of the thus treated erythocytes.
• said erythrocytes consisting essentially of horse erythrocytes and having been treated with formalin in an amount of from 0.2 to 1 volume of formalin per volume of horse erythrocytes, and in which said suspension contains from 2% to 6% by volume of the thus formalized cells.
• said suspension additionally contains, a preservative selected from the group consisting of sodium azide, phenol and merthiolate.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• Immunology (AREA)
• Hematology (AREA)
• Cell Biology (AREA)
• Biomedical Technology (AREA)
• Molecular Biology (AREA)
• Urology & Nephrology (AREA)
• Chemical & Material Sciences (AREA)
• Microbiology (AREA)
• Biochemistry (AREA)
• Food Science & Technology (AREA)
• Medicinal Chemistry (AREA)
• Physics & Mathematics (AREA)
• Analytical Chemistry (AREA)
• Biotechnology (AREA)
• General Health & Medical Sciences (AREA)
• General Physics & Mathematics (AREA)
• Pathology (AREA)
• Virology (AREA)
• Mycology (AREA)
• Tropical Medicine & Parasitology (AREA)
• Investigating Or Analysing Biological Materials (AREA)

Applications Claiming Priority (2)

Publications (1)

Family

ID=27048663

Family Applications (1)

Country Status (6)

Cited By (5)

Families Citing this family (2)

Citations (2)

• 1966
  • 1966-05-13 US US557851A patent/US3426123A/en not_active Expired - Lifetime
  • 1966-08-08 GB GB35398/66A patent/GB1155315A/en not_active Expired
  • 1966-08-25 DE DE19661598928 patent/DE1598928B2/de active Pending
  • 1966-08-30 SE SE11682/66A patent/SE341450B/xx unknown
  • 1966-09-07 NL NL6612579A patent/NL6612579A/xx unknown
  • 1966-09-09 JP JP41059258A patent/JPS5242849B1/ja active Pending

Patent Citations (2)

Also Published As

Similar Documents