WO1979000256A1 - Methode de production d'une preparation stable ayant des proprietes d'agglutination d'immunoglobulines - Google Patents

Methode de production d'une preparation stable ayant des proprietes d'agglutination d'immunoglobulines Download PDF

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Publication number
WO1979000256A1
WO1979000256A1 PCT/SE1978/000066 SE7800066W WO7900256A1 WO 1979000256 A1 WO1979000256 A1 WO 1979000256A1 SE 7800066 W SE7800066 W SE 7800066W WO 7900256 A1 WO7900256 A1 WO 7900256A1
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Prior art keywords
bacteria
killed
immunoglobulin
binding
substance
Prior art date
Application number
PCT/SE1978/000066
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English (en)
Inventor
U Jonsson
Original Assignee
U Jonsson
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by U Jonsson filed Critical U Jonsson
Publication of WO1979000256A1 publication Critical patent/WO1979000256A1/fr

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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
    • G01N33/531—Production of immunochemical test materials

Definitions

  • protein A in part as a sub ⁇ stance bound to the ' exterior of the cell " wall, and in part as a soluble substance excreted into the growth medium of the bacteria.
  • protein A was procured in soluble form after initial heat 0 extraction from washed bacteria. By heating suspended staphylococci close to the boiling point, cell-wall protein A was solubilized.
  • Radioim unoassay is the designation of a nowadays classical radioimmunological method of identification by which it is possible to make quantitative determinations of a given substance
  • 25 labelled antigen is separated into a free and an antibody-bound fraction, respectively.
  • protein A-containing staphylococci added as a finely dispersed suspension, have been demonstrated to reach the end-point in their reaction with immunoglobulin within one minute. Then, centri ⁇ fugation is performed, the supernatant fluid is decanted, and the tube is brought to a gamma radiation counter for the determination of the anti ⁇ body-bound fraction of labelled antigen, associated with the bacteria on the tube bottom.
  • the preparations of staphylococci first applied for the purposes mentioned usually originated from a broth culture, the bacteria being treated by repeated washing in a centrifuge, resuspension in a 0.5 % (v/v) formalin solution in neutral buffer, where the staphylococci were incubated for 3 hours under constant mixing, and final repeated washing in a centrifuge.
  • the resulting suspension was stored in neutral phosphate-buffered saline with some pre ⁇ servative added such as sodium azide (NaN , 1 gram per liter.
  • Kronvall The use of protein A-containing Staphylococcus aureus as a solid phase anti-IgG reagent in radio- immunoassays as exemplified in the quantitation of alpha-foetoprotein in normal human serum, Eur. J.. Immunology 4:29, 1974.
  • the object of the present invention is to simplify the production of preparations having immunoglobulin-binding properties in a drastic manner and to provide such a preparation which is stable and is easy to transport and distribute and also may be stored for a practically unlimited period without significant destruction.
  • the object referred to is achieved by the method according to the invention which is characterized in that bacteria of species having immunoglobulin-binding capacity beyond that based on a reaction between anti ⁇ gens of the bacteria and specific antibodies directed against such antigens, e.g. Staphylococcus aureus or Streptococcus pyogenes, are grown in a substrate wherein the bacteria form animmunoglobulin-binding substance, that the bacteria are killed, and that the killed bacteria are lyophilized for producing a bacteria suspension that can be reconstituted with liquid.
  • a preparation produced in this way can be reconstituted at any time during a period of probably several years to form a suspension of killed bacteria which may be used directly without further washing, e.g.
  • Staphylococci to be used for the purposes referred to herein are best grown in a fluid sub ⁇ strate, also designated nutrient medium or broth, since it often comprises an extract obtained from meat by boiling.
  • a fluid sub ⁇ strate also designated nutrient medium or broth
  • fermentors of about 10 litre volume, i.e. vessels where it is possible by effective mixing, aeration, neutralisation with pH-control etc to optimize the growing conditions.
  • the experiments of the inventor have demonstrated that the chemical composition of the substrate may have effects during the subsequent treatment of the material.
  • the so-called CCY-medium a particularly rich substrate for growing staphylococci
  • the substrate contains substance (s) which tend(s) to precipitate and/or sustain aggregation of the bacteria themselves at 80 C, e.g. proteins which denature at this tempera ⁇ ture.
  • TSB medium used at advantage by the inventor, just because it allows heat-killing of bacteria suspended in it at 80 C without significant precipitation or aggregation.
  • heat-killing of Staphylococcus aureus may be performed also after growth in CCY-medium avoiding the phenomena of precipitation and/or aggregation observed at heat-killing at 80 C as mentioned above, i.e. if heat-killing is performed as a non-limiting example, at 60 C during a correspondingly longer incubation time, about 30 minutes according to the inventor's experience.
  • the transition to the killing is performed also after growth in CCY-medium avoiding the phenomena of precipitation and/or aggregation observed at heat-killing at 80 C as mentioned above, i.e. if heat-killing is performed as a non-limiting example, at 60 C during a correspondingly longer incubation time, about 30 minutes according to the inventor's experience. The transition to the killing
  • temperature should preferably take place rapidly, e.g. in a heat exchanger located in a waterbath of the desired temperature, and should not be calculated as part of the so-called keeping time (incubation time) of about 30 minutes necessary for complete killing. It has also_ ⁇ >een demonstrated tfrat it is possible to heat-kill Staphylococcus aureus at 56 C without precipitation or aggregation phenomena, but total killing requires about 3 hours' incubation at this temperature, and apparently there are no additional advantages of killing bacteria for the present purposes at this lower temperature.
  • the inventor has also cultured various immuno ⁇ globulin-binding strains of Streptococcus pyogenes in so-called Todd-Hewitt broth, an established fluid medium for good yields of streptococci, and performed heat-killing at 80°C according to the same principles as for Staphylococcus aureus according to the above cited paper by Jonsson and Kronvall. Also in the case of streptococci the bulk of immunoglobulin-binding capacity present on living strains remains after direct heat-killing, i.e. without prior formalin treatment. Furthermore, also similar to staphylococci, streptococci grown and left in the rich Todd-Hewitt medium during heat-killing at 80°C form aggregates.
  • Such effects may be minimized, also in the case of streptococci, either by exchanging the Todd-Hewitt medium for fresh buffer before-heat-killing at 80°C or by performing the heat-killing in the used growth medium at about 60 C.
  • the latter procedure is pre ⁇ ferred since it implies less processing of the bac ⁇ teria.
  • Bacteria heat-killed in one of the manners mentioned above without prior formalin treatment have been washed and stored as suspensions in neutral buf- fer at refrigerator temperatures.
  • heat-killing may be performed at significant- ly lower temperatures, e.g. at 37°C by adding formaldehyde to a final concentration well below the formaldehyde concentration that would be required for killing with formaldehyde at room temperature.
  • the step of the inventor-to modify heat treatment by the addition of a small amount of formaldehyde - unable to kill per se at so-called room temperature - constitutes a significant step of progress in the processing of immunoglobulin-binding bacteria, a step to be followed by lyophilization for the exploitation of the additive stabilizing effect of such treatment.
  • Preparations produced according to the invention has been tested as a reagent in various radioimmuno- assays, e.g. for hepatitis B-antigen, antibody to this antigen and for aminoglycoside antibiotics such as gentamicin.
  • a suspension of staphylo ⁇ cocci was used which had been derived from lyophilized staphylococci reconstituted in a neutral phosphate- -buffered saline solution with addition of a so-called
  • the reagent of reconstituted staphylococci has been successfully tested in an established method for the classification of specific antibodies directed to infectious agents such as rubella virus or toxoplasma parasites. Infections with one of these agents during pregnancy involves a substantial risk for infection and malformation of the fetus. Therefore, various routine procedures have been developed for the study of the immune response towards these antigens and many others. It is an established fact that the primary antibody response of mammals towards foreign substances (antigens) starts with the production of specific antibodies of the IgM class followed by specific antibodies (i.e. antibodies directed to " the same antigen in question) belonging to the IgA class and later antibodies of the IgG class.
  • IgG antibodies constitute the long term protective antibody immunity against infectious agents, which is often boostered by several injections of vaccines where this is possible.
  • IgM and also IgA antibodies tend to disappear within a few weeks after their first appearance.
  • the presence of specific IgM antibodies - and to some extent also the presence of specific IgA antibodies - is generally regarded as an indication of recent infection caused by the infectious agent forming the antigen in question.
  • formalin- and heat-treated protein A-containing staphylococci may advantageously be used for the characterization of specific anti ⁇ bodies in this regard.

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  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Methode de production d'une preparation bacterienne stable ayant des proprietes agglutinantes d'immunoglobulines non specifiques. Les bacteries, par exemple staphylococcus aureus ou strepotococcus pyogenes, avec un composant agglutinant d'immunoglobulines non specifique, tel que la proteine A du staphylococcus aureus, a la surface des bacteries sont cultivees sur un substrat ou les bacteries forment le composant agglutinant d'immunoglobulines. Ensuite, les bacteries sont tuees et lyophilisees, la lyophilisation etant effectuee peu apres la mort des bacteries. Apres reconstitution, les bacteries lyophilisees sont utilisees comme reactif dans des reactions immunologiques.
PCT/SE1978/000066 1977-10-31 1978-10-31 Methode de production d'une preparation stable ayant des proprietes d'agglutination d'immunoglobulines WO1979000256A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SE7712244A SE7712244L (sv) 1977-10-31 1977-10-31 Forfarande for framstellning av preparationer av avdodade bakterier
SE7712244 1977-10-31

Publications (1)

Publication Number Publication Date
WO1979000256A1 true WO1979000256A1 (fr) 1979-05-17

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ID=20332722

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/SE1978/000066 WO1979000256A1 (fr) 1977-10-31 1978-10-31 Methode de production d'une preparation stable ayant des proprietes d'agglutination d'immunoglobulines

Country Status (7)

Country Link
EP (1) EP0007953A1 (fr)
CH (1) CH649385A5 (fr)
DE (1) DE2857506A1 (fr)
FR (1) FR2472612A1 (fr)
GB (1) GB2050386B (fr)
SE (1) SE7712244L (fr)
WO (1) WO1979000256A1 (fr)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2604259A1 (fr) * 1986-09-19 1988-03-25 Yissum Res Dev Co Reactifs, trousses et procedes d'essais immunologiques
US4948725A (en) * 1987-12-10 1990-08-14 University Of Florida Research Foundation, Inc. Novel type VI bacterial Fc receptors
US4977082A (en) * 1987-12-10 1990-12-11 University Of Florida Research Foundation, Inc. Type VI bacterial FC receptors
US5085984A (en) * 1987-12-10 1992-02-04 University Of Florida Research Foundation, Inc. Novel type VI bacterial Fc receptors
US5102788A (en) * 1988-11-21 1992-04-07 Hygeia Sciences, Inc. Immunoassay including lyophilized reactant mixture
EP0613005A2 (fr) * 1993-02-25 1994-08-31 Quidel Corporation Dosages utilisant des microorganismes teintés comme label
US6391634B1 (en) * 1986-07-29 2002-05-21 G. D. Searle & Co. Monoclonal antibodies and their production and use
US7691608B2 (en) 2006-12-06 2010-04-06 Repligen Corporation Nucleic acids encoding recombinant protein A

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Am J Clin Path, Vol 55, 1971, April, p 452-p 452, LEAVELLE D E et al, Staphylococcal Clumping on Microtiter Plates:..., see p 453, column 1. *
Eur J Immunol Vol 4, 1977, p 29-33, JONSSON S KRONVALL G, The use of protein-A-containing staphylococcus aureus as a solid phase anti-IgG reagent... *
Klinisk Mikrobiologi, STU-seminarium 1976, STU-information Nr 54, 1977, p 116-119, JONSSON S KRONVALL G, Protein A-barande stafylokocker som reagens i serologi. *
Scand J Immunol Vol 3, 1974, p 397-98, GHETIE V et al, Identification of cell surface Immunoglobulin markers by protein A-containing fluorescent Staphylococci. *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6391634B1 (en) * 1986-07-29 2002-05-21 G. D. Searle & Co. Monoclonal antibodies and their production and use
FR2604259A1 (fr) * 1986-09-19 1988-03-25 Yissum Res Dev Co Reactifs, trousses et procedes d'essais immunologiques
GB2197468B (en) * 1986-09-19 1991-03-13 Yissum Res Dev Co Immunoassay methods and kits
US4948725A (en) * 1987-12-10 1990-08-14 University Of Florida Research Foundation, Inc. Novel type VI bacterial Fc receptors
US4977082A (en) * 1987-12-10 1990-12-11 University Of Florida Research Foundation, Inc. Type VI bacterial FC receptors
US5085984A (en) * 1987-12-10 1992-02-04 University Of Florida Research Foundation, Inc. Novel type VI bacterial Fc receptors
US5102788A (en) * 1988-11-21 1992-04-07 Hygeia Sciences, Inc. Immunoassay including lyophilized reactant mixture
EP0613005A2 (fr) * 1993-02-25 1994-08-31 Quidel Corporation Dosages utilisant des microorganismes teintés comme label
EP0613005A3 (fr) * 1993-02-25 1995-07-05 Quidel Corp Dosages utilisant des microorganismes teintés comme label.
US7691608B2 (en) 2006-12-06 2010-04-06 Repligen Corporation Nucleic acids encoding recombinant protein A

Also Published As

Publication number Publication date
GB2050386A (en) 1981-01-07
CH649385A5 (de) 1985-05-15
EP0007953A1 (fr) 1980-02-20
FR2472612A1 (fr) 1981-07-03
SE7712244L (sv) 1979-05-01
GB2050386B (en) 1983-01-26
DE2857506A1 (de) 1981-04-16

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