WO1988010430A1 - Determination of hdl2 and hdl3 concentration by use of extract from bacteria - Google Patents

Determination of hdl2 and hdl3 concentration by use of extract from bacteria Download PDF

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Publication number
WO1988010430A1
WO1988010430A1 PCT/NO1987/000052 NO8700052W WO8810430A1 WO 1988010430 A1 WO1988010430 A1 WO 1988010430A1 NO 8700052 W NO8700052 W NO 8700052W WO 8810430 A1 WO8810430 A1 WO 8810430A1
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WO
WIPO (PCT)
Prior art keywords
hdl
extract
concentration
bacteria
serum
Prior art date
Application number
PCT/NO1987/000052
Other languages
French (fr)
Inventor
Arve Osland
Arne T. HØSTMARK
Curt Endresen
Original Assignee
Arve Osland
Hoestmark Arne T
Curt Endresen
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
Priority claimed from NO855291A external-priority patent/NO159821C/en
Application filed by Arve Osland, Hoestmark Arne T, Curt Endresen filed Critical Arve Osland
Priority to PCT/NO1987/000052 priority Critical patent/WO1988010430A1/en
Publication of WO1988010430A1 publication Critical patent/WO1988010430A1/en

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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
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • 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
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G01N33/559—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody through a gel, e.g. Ouchterlony technique

Definitions

  • the present invention utilizes extract from bacteria (e.g. from Staphylococcus capitis CCS 82) for measuring the concentration "high density lipoprotein 2" (HDL 2 ) and “high density lipoprote 3" (HDL3) in whole serum, plasma and in other solutions contain ing these lipoproteins by a simple radial diffusion in agar.
  • extract from bacteria e.g. from Staphylococcus capitis CCS 82
  • HDL3 high density lipoprotein 2
  • HDL3 high density lipoprote 3
  • the novel invention makes it possible to estimate the concentra tion of HDL 2 and HDI. 3 in e.g. whole serum (plasma): 1) of a ver large amount of samples, 2) by using small volumes of serum (30 ul), 3) without expensive equipment as an ultracentrifuge, during a relatively short time (incubation over night) and 5) b simple operations (pipetting and visual reading) .
  • the principle of the invention is to prepare an HDL 2 -precipitat ing extract from bacteria (e.g. S.capitis) , which are grown on solid medium containing the amino acid cysteine in concentratio exceeding 10 mg/1, or in an atmosphere with an increased concen tration of C0 2 .
  • HDL 2 -precipitating extract can also be made by growing the bacteria in liquid medium supplied with cysteine. After growth the bacteria are removed, the nutrient medium is concentrated and used as HDL 2 -precipitating extract.
  • This extract is added to melted agar and th mixture is poured into a petri dish. After the mixture has solidified, wells are made in the agar. Samples that are to be analyzed for HDL 2 are added into these wells. After an incu- bation time of 15-20 hours, precipitation rings will appear around each well. The diameter of the precipitation rings are measured with a measuring magnifier. The HDL 2 concentration is function of the diameter. The HDL3 concentration is estimated indirectly by calculating the difference between the concen ⁇ tration of total HDL (estimated by standard methods) and the concentration of HDL .
  • the invention can be utilized industrially by producing reagent kits containing bacterial extract together with agar and petri dishes, all in suitable amounts for the estimation of HDL and HDL 3 levels.
  • the principle for the measurement of HDL 2 is shown in fig. 1.
  • the figure shows a petri dish (1) with agar containing bacteria extract from S. capitis (2) .
  • a well (3) is made and filled wit the serum sample.
  • After a time of diffusion (15-20 h) a pre ⁇ cipitation ring is formed around the well (4).
  • Bacteria e.g. S. capitis
  • C0 C02-concen ⁇ trations higher than in air
  • the active compound which is precipitating HDL is produced by the bacteria under these conditions (Reference 1) .
  • the extract can also be made by growing bacteria in liquid medi containing cysteine. After growth of bacteria, the bacteria are removed. The medium is concentrated, dialysed against PBS and used as HDL 2 -precipitating extract.
  • the extract and melted agar are mixed at a certain ratio (about 0,3 ml extract to 4 ml 1% agar in physiological sa water (0.85% NaCl)) and poured into a petri dish with diameter cm. After the agar has solidified, wells are made in the agar mm in diameter) . These wells are then filled with serum, plasm or other HDL solutions that are to be measured. After incubati at roomte perature for 15-20 hours for the diffusion of serum- proteins into the gel to take place, precipitation rings are formed. The diameter of these rings are read by using a measur ing magnifier.
  • the evidence that this method measures the HDL 2 concentration is shown in refe ⁇ rence 1 (fig. 4). This figure shows results of cumulative densi floatation of HDL showing that the precipitating activity is present in the lower density part of the total HDL (HDL can be separated in two main fractions: one low density fraction, HDL and one high density fraction, HDL 3 ) (Reference 2).

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
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  • Molecular Biology (AREA)
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  • Urology & Nephrology (AREA)
  • Hematology (AREA)
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  • Physics & Mathematics (AREA)
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  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Dispersion Chemistry (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Use of extract from bacteria for determination of HDL2 and HDL3 concentration in serum, plasma or other solutions containing HDL. The extract is produced from bacteria grown on solid nutrient medium containing more than 10 mg/l of cystein, or on solid nutrient medium in an atmosphere with increased content of CO2. Extract may also be produced by growing the bacteria in liquid nutrient medium containing cystein. After growth, bacteria are removed, and the medium is concentrated, dialyzed and used as extract. The extract is added to melted agar that is poured into Petri dish (1). After solidification of agar with extract, wells (3) are made in the agar. The wells are filled with the HDL samples. After a diffusion time of 15-20 hours, a precipitation ring will be formed around the wells (4). Diameter of the ring is a function of HDL2 concentration in the sample. HDL3 concentration is calculated as the difference between total HDL (determined with known methods) and HDL2. Extract may also be added to dilutions of serum and the precipitation reaction in the solution may be read by use of nephelometry or turbidimetry.

Description

DETERMINATION OF HDL2 AND HDL3 CONCENTRATION BY USE OF EXTRACT FROM BACTERIA.
The present invention utilizes extract from bacteria (e.g. from Staphylococcus capitis CCS 82) for measuring the concentration "high density lipoprotein 2" (HDL2) and "high density lipoprote 3" (HDL3) in whole serum, plasma and in other solutions contain ing these lipoproteins by a simple radial diffusion in agar.
Today HDL2 and HDL3 are analyzed using ultracentrifugation techniques (sequential density-flotation or by the use of zonal rotor) . These are tedious proceedures which may take 60 to 80 hours to perform. Relatively large volumes of serum samples ar neccesary, the equipment is expensive and the capacity small.
The novel invention makes it possible to estimate the concentra tion of HDL2 and HDI.3 in e.g. whole serum (plasma): 1) of a ver large amount of samples, 2) by using small volumes of serum (30 ul), 3) without expensive equipment as an ultracentrifuge, during a relatively short time (incubation over night) and 5) b simple operations (pipetting and visual reading) .
The principle of the invention is to prepare an HDL2-precipitat ing extract from bacteria (e.g. S.capitis) , which are grown on solid medium containing the amino acid cysteine in concentratio exceeding 10 mg/1, or in an atmosphere with an increased concen tration of C02. HDL2-precipitating extract can also be made by growing the bacteria in liquid medium supplied with cysteine. After growth the bacteria are removed, the nutrient medium is concentrated and used as HDL2-precipitating extract.
A certain volume of this extract is added to melted agar and th mixture is poured into a petri dish. After the mixture has solidified, wells are made in the agar. Samples that are to be analyzed for HDL2 are added into these wells. After an incu- bation time of 15-20 hours, precipitation rings will appear around each well. The diameter of the precipitation rings are measured with a measuring magnifier. The HDL2 concentration is function of the diameter. The HDL3 concentration is estimated indirectly by calculating the difference between the concen¬ tration of total HDL (estimated by standard methods) and the concentration of HDL .
It is also observed that addition of defined amounts of bacteri extract, containing HDL2-reacting component, to dilutions of serumsa ples , will cause precipitation in the solutions. This effect can be measured by nephelometer (lightscattering) or turbidimeter (adsorption) . This effect is only observed within certain concentrationranges , and can be explained after the sam principles as those used for explanation of the reaction betwee a ultivalent antigen and antibodies.
By adding special components (e.g. polyethylenglycoll) , this precipitationreaction can be observed within one minute instead of several hours as by the use radial diffusion.
The invention can be utilized industrially by producing reagent kits containing bacterial extract together with agar and petri dishes, all in suitable amounts for the estimation of HDL and HDL3 levels.
The principle for the measurement of HDL2 is shown in fig. 1. The figure shows a petri dish (1) with agar containing bacteria extract from S. capitis (2) . A well (3) is made and filled wit the serum sample. After a time of diffusion (15-20 h) a pre¬ cipitation ring is formed around the well (4). Bacteria (e.g. S. capitis) are grown on medium containing cysteine at concentrations above 10 mg/1 or on media in an atmosphere with increased concentration of C0 (C02-concen¬ trations higher than in air) . The active compound which is precipitating HDL is produced by the bacteria under these conditions (Reference 1) .
An autolysate of the bacteria is produced: The bacteria is incubated in phosphate buffered saline, pH 7.2 (PBS) (wet weigh bacteria in g = volume PBS in ml) . After 20 hours at room temperature, the bacteria are sedimented by centrifugation. T supernatant is concentrated approx. 4 times and dialysed agains PBS. This preparation is called HDL2 precipitating extract. The extract can also be made by growing bacteria in liquid medi containing cysteine. After growth of bacteria, the bacteria are removed. The medium is concentrated, dialysed against PBS and used as HDL2-precipitating extract.
The extract and melted agar (55-60°C) are mixed at a certain ratio (about 0,3 ml extract to 4 ml 1% agar in physiological sa water (0.85% NaCl)) and poured into a petri dish with diameter cm. After the agar has solidified, wells are made in the agar mm in diameter) . These wells are then filled with serum, plasm or other HDL solutions that are to be measured. After incubati at roomte perature for 15-20 hours for the diffusion of serum- proteins into the gel to take place, precipitation rings are formed. The diameter of these rings are read by using a measur ing magnifier. The precipitating activity is defined as r2 = r2 - r where rx is the radius of the precipitatio ring formed by the serum sample, r0 is an estimated radius (found by linear extrapolation of standard concentrationcurve with zero amount of serum added) (Reference 1) . The evidence that this method measures the HDL2 concentration (the HDL fraction having the lowest density) is shown in refe¬ rence 1 (fig. 4). This figure shows results of cumulative densi floatation of HDL showing that the precipitating activity is present in the lower density part of the total HDL (HDL can be separated in two main fractions: one low density fraction, HDL and one high density fraction, HDL3) (Reference 2). Estimation of serum samples have shown that the precipitating activity, ^r2, correlates positivity with the amount of HDL2, measured after separation by ultracentrifugation (r=0,59, p<0,01, n=21) but not with HDL3 (Reference 1). The concentration of HDL3 can then be calculated as the difference between total HDL (deter¬ mined by known methods) and the concentration of HDL2, measured by this new method. It is shown that other bacteria than S. capitis, such as S.warneri and S. xylosus, also produce the HDL2 precipitating factor, under the same growth conditions as described for S. capitis (Reference 2).

Claims

PATENT CLAIMS
1. A method for estimation of HDL and HDL3 concentrations i serum, plasma or other solutions containing HDL, c h a r c t e r i z e d b y use of bacterial extract containi HDL2-reacting component.
2. A method according to claim 1, c h a r a c t e r i z e d b y single radial diffusion in agar containing HDL precipitaring bacterial extract (e.g. from Staphylococcus capitis CCS 82) .
3. A method according to claim 1-2, c h a r a c t e r i z b y the use of purified factions of HDL2-reacting compo nent.
4. A method according to claim 1-2, c h a r a c t e r i z e b y agar containing HDL2-precipitating extract from bacteria grown on solid nutrition medium with a concentra tion of cysteine of more than 10 mg/ml.
5. A method according to claim 1-2, c h a r a c t e r i z e b y agar containing HDL2-precipitating extract from bacteria grown on solid nutrition medium in an atmosphere with increased concentration of C02.
6. A method according to claim 1-2, c h a r a c t e r i z b y agar containing HDL2-precipitating extract, prepare by a concentration of liquid nutrition medium where bac¬ teria, producing HDL2-reacting component, have been grown.
7. A method according to claim 1-6, c h a r a c t e r i z e b y the estimation of HDL2-concentration as a function of the diameter of the precipitation ring.
8. A method according to claim 1-7, c h a r a c t e r i z e b y the estimation of HDL3-concentration as the differen between the concentration of HDL and the concentration of HDL2.
9. A method according to claim 1, c h a r a c t e r i z e d b y nephelometric of turbidiometric measurement of preci¬ pitates in serum or dilutions of serum, after adding bacteria extract containing HDL reacting component to the samples.
10. A method according to claim 1 and 9, c h a r a c t e r i z e d b y adding components (e.g. polyethylenglycoll) th increase the speed of the precipitation.
11. A method according to claim 1 and 9-10, c h a r a c t e i z e d b y the use of purified fractions of HDL2- reacting component.
REFERENCES;
1. Osland, A. and Høstmark, A.T.: "Evidence that an extract Staphylococcus capitis can precipitate human serum HDL2". Acta path, icrobiol. scand. Sect. B, 94: 387-392 (1986).
2. Osland, A.: "Staphylococci producing a non-immune precipi tion line with human serum". Acta path, microbiol. scand. Sect.B. 89: 133-135 (1981).
PCT/NO1987/000052 1985-12-27 1987-06-24 Determination of hdl2 and hdl3 concentration by use of extract from bacteria WO1988010430A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/NO1987/000052 WO1988010430A1 (en) 1985-12-27 1987-06-24 Determination of hdl2 and hdl3 concentration by use of extract from bacteria

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
NO855291A NO159821C (en) 1985-12-27 1985-12-27 USE OF BACTERY EXTRACTS FOR MEASURING HDL2 AND HDL3 CONCENTRATION IN SERUM, PLASMA OR OTHER SOLUTIONS.
PCT/NO1987/000052 WO1988010430A1 (en) 1985-12-27 1987-06-24 Determination of hdl2 and hdl3 concentration by use of extract from bacteria

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4188188A (en) * 1978-09-27 1980-02-12 Bio-Rad Laboratories, Inc. High density lipoprotein cholesterol assay
SE450861B (en) * 1979-05-02 1987-08-03 Fis Fournier Innovat Synerg PROCEDURE AND AGENTS FOR DETERMINATION OF APOLIPOPROTEINES IN SERUM

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4188188A (en) * 1978-09-27 1980-02-12 Bio-Rad Laboratories, Inc. High density lipoprotein cholesterol assay
SE450861B (en) * 1979-05-02 1987-08-03 Fis Fournier Innovat Synerg PROCEDURE AND AGENTS FOR DETERMINATION OF APOLIPOPROTEINES IN SERUM

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ACTA PATH MICROBIOL IMMUNOL SCAN SECT B, 93: 371-376, published 1985, (ARVE OSLAND), "Binding of human serum high density lipoprotein to staphylococcus capitis". *
ACTA PATH MICROBIOL IMMUNOL SCAND SECT B, 94: 387-392, published 1986, (A OSLAND & T HOSTMARK), "Evidence that an extract of staphylococcus capitis can precipitate human serum HDL2". *
ACTA PATH MICROBIOL SCAND SECT B 89: 133-135, published 1981, (ARVE OSLAND), "Staphylococci producing a non-immune precipitation line with human serum". *
CHEMICAL ABSTRACT, Vol. 103, (1985), abstract no. 176418b, "Control of lipopolysaccharide-high-density lipo protein interactions by an acute-phase reactant in human serum", (TOBIAS, PETER S et al.), Infect Immun 1985, 50(1). *

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