US3864467A - Method for diagnosis of infectious mononucleosis - Google Patents

Method for diagnosis of infectious mononucleosis Download PDF

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Publication number
US3864467A
US3864467A US243421A US24342172A US3864467A US 3864467 A US3864467 A US 3864467A US 243421 A US243421 A US 243421A US 24342172 A US24342172 A US 24342172A US 3864467 A US3864467 A US 3864467A
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US
United States
Prior art keywords
absorbent
mononucleosis
diagnosis
blood cells
suspension
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Expired - Lifetime
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US243421A
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English (en)
Inventor
E Juhani Leikola
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Orion Oyj
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Orion Yhtyma Oy
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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
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)
    • 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

Definitions

  • ABSTRACT A method for preparing a storageable absorbent for diagnosis of infectious mononucleosis from blood serum is disclosed.
  • the absorbent is prepared by treating mammal red blood cells with a ptoteolytic enzyme or neuraminidase to destroy the mononucleosis receptors and the red blood cells are hemolyzed to make a cell-free stroma suspension which does not interfere with the agglutination step of the diagnosis, when mammal blood is added to the mixture of blood serum to be tested and the absorbent on a slide.
  • the invention relates to the field of preparing a stable absorbent for diagnosis of infectious mononucleosis from blood serum and a method for carrying out the diagnosis.
  • Infectious mononucleosis is a rather common viral disease occuring mainly in adolescents. The diagnosis of this disease is based mainly on the demonstration of an antibody in the blood serum of the patient which agglutinates sheep or horse red blood cells (RBC). In certain circumstances the antibody can also agglutinate beef RBC. However, since normal personssometimes have antibodies with similar properties, it is necessary to absorb these natural antibodies before it can be concluded that the agglutination reaction is due only to the specific mononucleosis antibodies.
  • the presence of the specific mononucleosis antibody can be demonstrated by making serial dilutions of the patients serum in test tubes, or by simple slide tests.
  • serial dilutions In the original method of serial dilutions (Davidsohn, I., Stern, K., and Kashiwagi, C., Amer. J. Clin. Path. 21. l 101 1951) ground guinea pig kidney tissue was added to the serum to be tested to absorb the unspecific, natural antibodies.
  • Guinea pig kidney tissue is not ideal for this purpose, since it is able to absorb only part of the natural antibodies, and, on the other hand, it sometimes removes some part of the specific mononucleosis antibody.
  • this method includes another, simultaneous absorption. This confirming absorption is based on the fact that beef RBC have a reversed effect on the patients serum as compared with guinea pig kidney tissue, i.e., they absorb the specific mononucleosis antibody but leave the natural antibodies intact.
  • two serial dilutions are made of the serum in test tubes, and a certain concentration of sheep RBC suspension is added into the tubes (horse RBC can be used as well).
  • the reaction is concluded to be due to the specific mononucleosis antibody, and, hence, the diagnosis of infectious mononucleosis can be established.
  • the reversed case i.e., there is no agglutination after absorption with guinea pig kidney but some degree of agglutination remains after the confirming absorption, this is due to the natural, unspecific antibodies.
  • the present method is based on the same principle as described in the Description of the Prior Art above. However, a more advantageous slide modification has been developed for the test and the problem of stability has been solved. In addition to the greater specificity and sensitivity, it is also simplier than the present slide tests which are based on the principle of dual absorption.
  • this method a large amount of sheep RBC are treated with enzyme. After the treatment, the RBC are carefully washed with physiological saline (0.9 weightpercent NaCl), and then hemolyzed. The hemoglobin is removed anda fine suspension is made of the resulting stromata.
  • the cells are first treated with enzyme and only thereafter hemol'yzed, since the cell membrane is stret ched in the intact RBC and thus readily ac; cessible to the enzyme.
  • the fine stro niasiispension is stable for at least several months especially if an appropriate preservative is added, and the suspension does not intefere with the final agglutination reaction, since the suspension does not contain any cells.
  • an absorbent which is storageable and suitable for slide tests by homolyzing the red blood cells before or after these have been treated with a proteolytic enzyme or neuraminidase in order todestroy the cells and prepare a fine stroma suspension which does not interfere with the slide test diagnosis.
  • the following reagents are required for the method: (a) the blood sereum to be tested, (b) the absorbent, (c) sheep blood.
  • the serum is removed from the whole blood by allowing the blood to coagulate. After separation of the serum from the clot, the serum is heated at 56C for 15 min to destroy the complement.
  • the absorbent is prepared in the following way, Sheep blood is taken into an anticoagulant medium to prevent the clotting of the blood.
  • the blood cells are washed four times with 0.9 percent NaCl solution, centrifuging the cells between each washing.
  • One part of washed sheep blood cells is mixed with four parts of 0.9 percent NaCl solution, and one part of papain solution is added (this is prepared by dissolving 1 gram of dry papain into 100 ml of0.l5M phosphate buffer, pH 5.4, and into this solution, 0.5M cystein is added to activate the enzyme).
  • the mixture is incubated in 37C water bath until the mononucleosis receptors are destroyed (usually 60-120 min).
  • the blood cells are washed several times with 0.9 mg digitonin in 1 ml of water).
  • the RBC hemolyze, and the resulting stromata are washed at least 5 times by centrifuging the suspension at 12,000 rpm for min each time.
  • the washed stromata are made into a suspension (usually l0- pecent) in 0.9 percent NaCl solution that is buffered to pH 7.2 with 0.02M phosphate buffer.
  • sodium azine is added in a ratio of l to 2,000.
  • the sheep blood is taken into an anticoagulant medium (e.g. Alsevers solution).
  • an anticoagulant medium e.g. Alsevers solution
  • test is performed as follows.
  • One drop of the serum to be tested is put on a suitable slide (e.g.,
  • a method for diagnosis of infectious mononucleosis comprising forming an absorbent by treating sheep or horse red blood cells with a proteolytic enzyme or neuraminidaze in order to destroy the mononucleosis receptors, hemolyzing the red blood cells by adding a hemolyzing agent, separating the resulting stromata from the hemoglobin, and preparing a stroma suspension from the stromata and diluted saline which suspension is free from blood cells interfering with the diagnosis; and mixing blood serum to be tested, said absorbent and sheep or horse blood on a slide whereby if mononucleosis antibodies are present agglutination takes place.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
US243421A 1971-04-14 1972-04-12 Method for diagnosis of infectious mononucleosis Expired - Lifetime US3864467A (en)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
FI711032A FI46893C (fi) 1971-04-14 1971-04-14 Menetelmä mononukleoositaudin toteamiseksi veriseerumista käyttämällä entsyymikäsiteltyjä verisolustroomia epäspesifien vasta-aineiden absor boimiseksi

Publications (1)

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US3864467A true US3864467A (en) 1975-02-04

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ID=8504994

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US243421A Expired - Lifetime US3864467A (en) 1971-04-14 1972-04-12 Method for diagnosis of infectious mononucleosis

Country Status (5)

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US (1) US3864467A (OSRAM)
DE (1) DE2217801A1 (OSRAM)
FI (1) FI46893C (OSRAM)
FR (1) FR2133748B1 (OSRAM)
GB (1) GB1364180A (OSRAM)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4139606A (en) * 1976-05-24 1979-02-13 Zichis Joseph Preparation of antigen and test for heterophil antibody
US4460694A (en) * 1981-03-26 1984-07-17 University Of Miami Bovine glycoproteins and use in diagnosing infectious mononucleosis
RU2172956C1 (ru) * 2000-01-26 2001-08-27 Научно-исследовательский институт детских инфекций Способ диагностики тяжелых форм инфекционного мононуклеоза, вызванного вирусом эпштейна-барр, у детей

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
Chem. Abstr. Vol. 65, 1966, pp. 7,799-7,800. *
Davidsohn, AJCP, Vpl. 41, 1964, pp. 115-125. *
Springer, J. Lab. & Clin. Med., Vol. 65, 1965, pp. 617-627. *
Tomcsik, Path. and Microbio. Vol. 23, 1960, pp. 172-183. *
Tomcsik, PSEBM, Vol. 69, 1948, pp. 562-565. *
Williams and Chase, Methods in Immuno, and Immunochem. Academic Press, NY. Vol. III, 1971, pp. 370-374. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4139606A (en) * 1976-05-24 1979-02-13 Zichis Joseph Preparation of antigen and test for heterophil antibody
US4460694A (en) * 1981-03-26 1984-07-17 University Of Miami Bovine glycoproteins and use in diagnosing infectious mononucleosis
RU2172956C1 (ru) * 2000-01-26 2001-08-27 Научно-исследовательский институт детских инфекций Способ диагностики тяжелых форм инфекционного мононуклеоза, вызванного вирусом эпштейна-барр, у детей

Also Published As

Publication number Publication date
DE2217801A1 (de) 1972-10-19
GB1364180A (en) 1974-08-21
FR2133748A1 (OSRAM) 1972-12-01
FI46893C (fi) 1973-07-10
FR2133748B1 (OSRAM) 1977-11-25
FI46893B (OSRAM) 1973-04-02

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