CA1109790A - Automated assay for bacterial or bacterial antibody - Google Patents
Automated assay for bacterial or bacterial antibodyInfo
- Publication number
- CA1109790A CA1109790A CA302,714A CA302714A CA1109790A CA 1109790 A CA1109790 A CA 1109790A CA 302714 A CA302714 A CA 302714A CA 1109790 A CA1109790 A CA 1109790A
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- gram negative
- complement
- negative bacterium
- incubating
- bacterial
- Prior art date
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- 230000001580 bacterial effect Effects 0.000 title claims abstract description 28
- 238000003556 assay Methods 0.000 title abstract description 12
- 241000894006 Bacteria Species 0.000 claims abstract description 33
- 230000000295 complement effect Effects 0.000 claims abstract description 20
- 239000000203 mixture Substances 0.000 claims abstract description 15
- 230000005764 inhibitory process Effects 0.000 claims abstract description 5
- 238000000034 method Methods 0.000 claims description 16
- 210000002966 serum Anatomy 0.000 claims description 14
- 241000588724 Escherichia coli Species 0.000 claims description 10
- 241000293871 Salmonella enterica subsp. enterica serovar Typhi Species 0.000 claims description 8
- 241000588652 Neisseria gonorrhoeae Species 0.000 claims description 7
- 241000606768 Haemophilus influenzae Species 0.000 claims description 6
- 229920006395 saturated elastomer Polymers 0.000 claims description 4
- 238000011534 incubation Methods 0.000 claims description 3
- 238000007865 diluting Methods 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims 6
- 201000009906 Meningitis Diseases 0.000 claims 3
- 229920001817 Agar Polymers 0.000 claims 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 claims 2
- 239000008272 agar Substances 0.000 claims 2
- 230000000415 inactivating effect Effects 0.000 claims 2
- 238000009877 rendering Methods 0.000 claims 2
- 239000007787 solid Substances 0.000 claims 2
- 102000007562 Serum Albumin Human genes 0.000 claims 1
- 108010071390 Serum Albumin Proteins 0.000 claims 1
- 229910002092 carbon dioxide Inorganic materials 0.000 claims 1
- 239000001569 carbon dioxide Substances 0.000 claims 1
- 230000003467 diminishing effect Effects 0.000 claims 1
- 238000002405 diagnostic procedure Methods 0.000 abstract description 3
- 239000000523 sample Substances 0.000 description 12
- 241000283973 Oryctolagus cuniculus Species 0.000 description 7
- 241000588650 Neisseria meningitidis Species 0.000 description 6
- 239000003855 balanced salt solution Substances 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- 229940098773 bovine serum albumin Drugs 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 239000012472 biological sample Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 206010022000 influenza Diseases 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 102100030550 Menin Human genes 0.000 description 1
- 101710169972 Menin Proteins 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 241001467018 Typhis Species 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 108010074605 gamma-Globulins Proteins 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
Classifications
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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
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
-
- 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
- G01N33/56911—Bacteria
-
- 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
- G01N33/571—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses for venereal disease, e.g. syphilis, gonorrhoea
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Food Science & Technology (AREA)
- Virology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Biophysics (AREA)
- Toxicology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
BACTERIAL OR BACTERIAL ANTIBODY ASSAY
ABSTRACT OF THE DISCLOSURE
Automated tests to evaluate the antibody titer of a sample by incubating a test sample, bacteria, and complement, and detecting bacterial growth in the incubated mixture, and to identify bacteria and its titer in a sample by incubating a test sample, bacterial antiserum, and complement, and detecting inhibition of bacterial growth in the incubated mixture. The automated tests lend themselves to diagnostic use and may be provided in the form of diagnostic test kits.
ABSTRACT OF THE DISCLOSURE
Automated tests to evaluate the antibody titer of a sample by incubating a test sample, bacteria, and complement, and detecting bacterial growth in the incubated mixture, and to identify bacteria and its titer in a sample by incubating a test sample, bacterial antiserum, and complement, and detecting inhibition of bacterial growth in the incubated mixture. The automated tests lend themselves to diagnostic use and may be provided in the form of diagnostic test kits.
Description
~ "
~:`
BACTERIAL OR BACTERIAL ANTIBODY ASSAY
SUMMARY OF THE INVENTIO~
The present invention provides 1) an automated test to determine the antibody titer of a sample by incubating in a microtiter plate a mixture containing a test sample, the bacteria whose antibodv is being evalua-;ed in the sample, and complement, and measuring bacterial growth - in the incubated mixture, and :') an automated test for determining the identity and initial concentration of bacteria in a sample by incubating in a micxotiter plate a mixture containing a test sample, bacterial antiserum, complement, and growth medium and measuring inhibition of bacterial growth.
DETAILED DESCRIPTION
The present invention provides an auto-; mated method to measure antibody titer in a biological sample such as, for example, serum, plasma, blood fractions, gamma globulin, and the like. The sample is mixed with rabbit complement, preferably complement from baby rabbits up to about 4 weeks old, and with the bacteria whose antibody level in the sample is being measured. After in-cubating the mixture for a brief period of time, f~
~:`
BACTERIAL OR BACTERIAL ANTIBODY ASSAY
SUMMARY OF THE INVENTIO~
The present invention provides 1) an automated test to determine the antibody titer of a sample by incubating in a microtiter plate a mixture containing a test sample, the bacteria whose antibodv is being evalua-;ed in the sample, and complement, and measuring bacterial growth - in the incubated mixture, and :') an automated test for determining the identity and initial concentration of bacteria in a sample by incubating in a micxotiter plate a mixture containing a test sample, bacterial antiserum, complement, and growth medium and measuring inhibition of bacterial growth.
DETAILED DESCRIPTION
The present invention provides an auto-; mated method to measure antibody titer in a biological sample such as, for example, serum, plasma, blood fractions, gamma globulin, and the like. The sample is mixed with rabbit complement, preferably complement from baby rabbits up to about 4 weeks old, and with the bacteria whose antibody level in the sample is being measured. After in-cubating the mixture for a brief period of time, f~
-2- 16015 e.g. for from about 30 to about 50 minutes, broth is added and the mixture incubated for from about 15 to about 30 hours, preferably for from about 20 to about 24 hours, at about - 5 37C.
The present invention also provides an automated method to determine the identity and initial concentration of bacteria in a biological sample such as mentioned previously.
The sample is mixed with antisera for the bacteria whose identity is ~eing determined, growth medium and complement. After incubating the mixture for a short period of time, e.g. for from about 30 to about 50 minutes, broth is added and the mixture is incubated for from about 15 to about 30 hours, pre~erably for ~rom about 20 to about 24 hours, at about 37C.
The automated assay method of the present invention is applicable to antibody or bacterial determinations for all gram negative bacteria.
~` It has particular utility in determining antibody to such pathogenic organisms as N. meningitidis, Salmonella, Neisseria gonorrhea, Hemophilus influenza, E. coli and the like, or such patho-genic organisms themselves.
The assay is carried out using standard 96-well assay plates, automatic pipetting equipment and automatic diluting equipment. In this way the assay is automated, uses lesser amounts of ingre-dients than conventional methods, and is faster and mo e accurate. The cost of the assay is re-duced considerably compared to the manual method used heretofore while the precision of the assay is improved.
The method of the present invention may conveniently be carried out in a diagnostic test kit which includes, for bacterial determination, ~ 9~3~
- ~3~ 16015 kit which includes, for bacterial determination, lyophilized samples of antibody to gram negati~e bacteria such as, for example, antibody to E. coli, S. typhi, N. meningitidis strains A
5 and C, H. influenza and N. gonorrhea. Complement may be supplied either as part of the kit or may be provided by the user of the ki~. Such a kit is used to determine the identity of any of the foregoing bacteria in clinical samples.
This test has the advantage of excluding non- -~specific e~fects.
The method of the present invention may conYeniently be carried out in a diagnostic test kit which includes, for antibody determination, lyophilized samples of gram negative bacteria such as, for example, E. coli, S. typhi, N. menin~itidis strains A and C, H. influenza and N. ~onorrhea.
Complement may be supplied either as part of the kit or may be provided by the user of the kit.
This test also has the advantage of excluding nonspecific effects.
~1~90 The following examples illustrate the present invention without, however, limiting the same thereto.
Frozen sera samples are thawed rapidly in cold tap water~ Sera are heat inactivated at 56C for 30 minutes and kept in ice water until removed for assay. 25 microliters of the heat inactivated sexa being tested are added with sterile pipette tips to each of the 12 wells in row 1 of MIC~OTITER plates ~Cooke Laboratory Products). These are disposable, rigid polystyrene plates, sterile and with U wells. From 8 to 96 wells/samples are used depending upon the desired accuracy. Each plate is positioned in the automatic pipetter (Cooke Laboratory Products) and 0.025 - ml of sterile balanced salt solution ~Grand Island Biological Cc.) and 0.1~ bovine serum albumin is added to each of the 96 wells. The plate is then positioned in the automatic diluter (Cooke Laboratory Products) which is operated according to the manufacturer's instructions. This peforms two fold dilutions if 0.025 ml diluters are being used. Using a fresh disposable tray, 0.025 ml of a fresh bacterial culture of Neisseria meningitidis are added to each well. This preparation must contain between 1530 fresh viable organisms/0.025 ml. Each plate is shaken for 2030 seconds after addition of bacteria. Next, 0.025 ml of baby rabbit complement ~PelFreeze) are added to each well with an automatic pipetter. The plates are ` shaken again for 20-30 seconds, and then incubated for 40 minutes in a water saturated 37C incubator.
At the end of this incubation period, 0.125 ml of Mueller Hinton broth ~Difco) are added to each well with the automatic pipetter. This step must be carried with a new sterile head and a new disposable plastic reservoir. The plates are incubated overnight (2024 hours) in a 37C
humidified 5% CO2 incubator. Wells with no bacterial growth are scored as negative. Wells with bact~rial growth are scored as positive. The use of a test reading mirror ~Cooke) makes the plate reading easier. With each test the following controls are included: number of bacteria in 0.025 ml challenge; serum control;
baby rabbit complement control; inactivated baby rabbit complement control; organism control and reference sera.
Manual Petr'-Dish Automated Titer Titer SamplePre Post Pre Post 1 8 64 16 1~8 2 64 204~ 64 1024
The present invention also provides an automated method to determine the identity and initial concentration of bacteria in a biological sample such as mentioned previously.
The sample is mixed with antisera for the bacteria whose identity is ~eing determined, growth medium and complement. After incubating the mixture for a short period of time, e.g. for from about 30 to about 50 minutes, broth is added and the mixture is incubated for from about 15 to about 30 hours, pre~erably for ~rom about 20 to about 24 hours, at about 37C.
The automated assay method of the present invention is applicable to antibody or bacterial determinations for all gram negative bacteria.
~` It has particular utility in determining antibody to such pathogenic organisms as N. meningitidis, Salmonella, Neisseria gonorrhea, Hemophilus influenza, E. coli and the like, or such patho-genic organisms themselves.
The assay is carried out using standard 96-well assay plates, automatic pipetting equipment and automatic diluting equipment. In this way the assay is automated, uses lesser amounts of ingre-dients than conventional methods, and is faster and mo e accurate. The cost of the assay is re-duced considerably compared to the manual method used heretofore while the precision of the assay is improved.
The method of the present invention may conveniently be carried out in a diagnostic test kit which includes, for bacterial determination, ~ 9~3~
- ~3~ 16015 kit which includes, for bacterial determination, lyophilized samples of antibody to gram negati~e bacteria such as, for example, antibody to E. coli, S. typhi, N. meningitidis strains A
5 and C, H. influenza and N. gonorrhea. Complement may be supplied either as part of the kit or may be provided by the user of the ki~. Such a kit is used to determine the identity of any of the foregoing bacteria in clinical samples.
This test has the advantage of excluding non- -~specific e~fects.
The method of the present invention may conYeniently be carried out in a diagnostic test kit which includes, for antibody determination, lyophilized samples of gram negative bacteria such as, for example, E. coli, S. typhi, N. menin~itidis strains A and C, H. influenza and N. ~onorrhea.
Complement may be supplied either as part of the kit or may be provided by the user of the kit.
This test also has the advantage of excluding nonspecific effects.
~1~90 The following examples illustrate the present invention without, however, limiting the same thereto.
Frozen sera samples are thawed rapidly in cold tap water~ Sera are heat inactivated at 56C for 30 minutes and kept in ice water until removed for assay. 25 microliters of the heat inactivated sexa being tested are added with sterile pipette tips to each of the 12 wells in row 1 of MIC~OTITER plates ~Cooke Laboratory Products). These are disposable, rigid polystyrene plates, sterile and with U wells. From 8 to 96 wells/samples are used depending upon the desired accuracy. Each plate is positioned in the automatic pipetter (Cooke Laboratory Products) and 0.025 - ml of sterile balanced salt solution ~Grand Island Biological Cc.) and 0.1~ bovine serum albumin is added to each of the 96 wells. The plate is then positioned in the automatic diluter (Cooke Laboratory Products) which is operated according to the manufacturer's instructions. This peforms two fold dilutions if 0.025 ml diluters are being used. Using a fresh disposable tray, 0.025 ml of a fresh bacterial culture of Neisseria meningitidis are added to each well. This preparation must contain between 1530 fresh viable organisms/0.025 ml. Each plate is shaken for 2030 seconds after addition of bacteria. Next, 0.025 ml of baby rabbit complement ~PelFreeze) are added to each well with an automatic pipetter. The plates are ` shaken again for 20-30 seconds, and then incubated for 40 minutes in a water saturated 37C incubator.
At the end of this incubation period, 0.125 ml of Mueller Hinton broth ~Difco) are added to each well with the automatic pipetter. This step must be carried with a new sterile head and a new disposable plastic reservoir. The plates are incubated overnight (2024 hours) in a 37C
humidified 5% CO2 incubator. Wells with no bacterial growth are scored as negative. Wells with bact~rial growth are scored as positive. The use of a test reading mirror ~Cooke) makes the plate reading easier. With each test the following controls are included: number of bacteria in 0.025 ml challenge; serum control;
baby rabbit complement control; inactivated baby rabbit complement control; organism control and reference sera.
Manual Petr'-Dish Automated Titer Titer SamplePre Post Pre Post 1 8 64 16 1~8 2 64 204~ 64 1024
3 32 2048 32 512
4 2 256 2 256 ExAMæLE 2 The following table summarizes the assay variability as'a function of the number of assays per sample.
11`~97~0 95% Confidence limits for Number of Samples average log potency 1+ 0.692 2+ 0.489 3+ 0.399 4+ 0.346
11`~97~0 95% Confidence limits for Number of Samples average log potency 1+ 0.692 2+ 0.489 3+ 0.399 4+ 0.346
5+ 0.309
6+ 0.282 - '; 0.262 ~+ 0.245 9+ 0.231 10+ 0.219 TEST KIT FOR ANTIBODIES
A clinical serum sample to be tested for the presence of antibodies to E. coli~ S. typhi, N. meningitidis strains A and C, H. influenza and N. gonorrhea is heat inactivated at 56C for 20 30 minutes. 25 Microliters of the inactivated serum being tested is added with sterile pipette tips to each second well of row A of MICROTITERR
~ plates ~Cooka Laboratory Products). The rest of ; the wells of row A receive 25 microliters of 25 sterile balanced salt solution (Grand Island Biologicals). Each plate is positioned in the ~ automatic pipetter and 25 microliters of sterile s balanced salt solution with 0.1~ bovine serum albumin is added to each of the 96 wells. The plate 30 is then positioned in the automatic diluter which is operated according to the manufacturer's , instructions. Two-fold dilutions are used. 25 Microliters of a reconstituted lyophilized bacterial culture of E. coli are added with sterile pipette 1~90
A clinical serum sample to be tested for the presence of antibodies to E. coli~ S. typhi, N. meningitidis strains A and C, H. influenza and N. gonorrhea is heat inactivated at 56C for 20 30 minutes. 25 Microliters of the inactivated serum being tested is added with sterile pipette tips to each second well of row A of MICROTITERR
~ plates ~Cooka Laboratory Products). The rest of ; the wells of row A receive 25 microliters of 25 sterile balanced salt solution (Grand Island Biologicals). Each plate is positioned in the ~ automatic pipetter and 25 microliters of sterile s balanced salt solution with 0.1~ bovine serum albumin is added to each of the 96 wells. The plate 30 is then positioned in the automatic diluter which is operated according to the manufacturer's , instructions. Two-fold dilutions are used. 25 Microliters of a reconstituted lyophilized bacterial culture of E. coli are added with sterile pipette 1~90
- 7 - 16015 tips to all wells of row 1 and 2. Row 1 contains the serum and row 2 ia a bacterial growth control. Rows 3 and 4 receive S. typhi, and rows 5 and 6 receive N. meningitidis A.
The bacterial preparations contain between 15-30 fresh viable organisms/25 microliters. They are supplied in the lyophilized state as part of the test kit and contain after reconstitution the required amount of bacteria. Next 25 micro-liters of baby rabbit complement are added toeach well with an automatic pipetter. Plates are then shaken for 20-30 seconds and incu~ated for 40 minutes in a water saturated 37~C incubator.
Then 125 microliters of Mueller Hinton broth are added to each well with the automatic pipetter.
This step must be carried out with a new sterile head and a new disposable plastic reservoir. The plates are incubated overnight ~20-24 hours in a 37C humidified 5% C02 incubator). If a sample of serum inhibits bacterial growth, the identity as well as the titer are determined.
T~ST KIT FOR BACTERIA
25 Microliters of a clinical serum sample to be tested for the presence of the following bacteria: E. coli, S. typhi, N. meningitidis A and C, E. influenza and N. gonorrhea is added with _.
sterile pipette tips to each well of row A of MICROTITER plates. Then 25 microliters of reference antisera for the tested bacteria are added to each second well of row A. Wall A2 re-ceives antiserum to E. coli, well A4 to S~ typhi, etc. These antisera are part of the kit and are supplied in the lyophilized state.
3L~9790
The bacterial preparations contain between 15-30 fresh viable organisms/25 microliters. They are supplied in the lyophilized state as part of the test kit and contain after reconstitution the required amount of bacteria. Next 25 micro-liters of baby rabbit complement are added toeach well with an automatic pipetter. Plates are then shaken for 20-30 seconds and incu~ated for 40 minutes in a water saturated 37~C incubator.
Then 125 microliters of Mueller Hinton broth are added to each well with the automatic pipetter.
This step must be carried out with a new sterile head and a new disposable plastic reservoir. The plates are incubated overnight ~20-24 hours in a 37C humidified 5% C02 incubator). If a sample of serum inhibits bacterial growth, the identity as well as the titer are determined.
T~ST KIT FOR BACTERIA
25 Microliters of a clinical serum sample to be tested for the presence of the following bacteria: E. coli, S. typhi, N. meningitidis A and C, E. influenza and N. gonorrhea is added with _.
sterile pipette tips to each well of row A of MICROTITER plates. Then 25 microliters of reference antisera for the tested bacteria are added to each second well of row A. Wall A2 re-ceives antiserum to E. coli, well A4 to S~ typhi, etc. These antisera are part of the kit and are supplied in the lyophilized state.
3L~9790
-8- 16015 Each plate is positioned in the automatic pipetter and receives 25 microliters/ well of sterile balanced salt solution with 0.1% bovine serum albumin. The plate is then positioned in the automatic diluter and two-fold dilutions are performed. Next 25 microliters of baby rabbit complement are added to each well with an automatic pipetter. The plates are then shaken for 20-30 seconas and incubated for 40 minutes in a water saturated 37C incubator. Then 125 microliters of Mueller-Hinton broth are added to each well with the automatic pipetter. The plates are incubated overnight ~20-24 hours in a 37C humidified 5% CC2 incubator). If samples of reference sera inhibit bacterial growth this serves to establish the identity and initial con-centration of the bacteria in the tested sera.
.
.
Claims (10)
1. A method for determining the identity of a gram negative bacterium present in a serum sample comprising:
(a) incubating the serum sample together with a refer-ence antiserum to a known gram negative bacterium, a serum albumin, and a complement at a physiologically acceptable temperature;
(b) adding a liquid medium to the mixture resulting from step (a);
(c) incubating the mixture resulting from step (b) at a physiologically acceptable temperature; and (d) measuring and scoring the inhibition of bacterial growth in the liquid medium.
(a) incubating the serum sample together with a refer-ence antiserum to a known gram negative bacterium, a serum albumin, and a complement at a physiologically acceptable temperature;
(b) adding a liquid medium to the mixture resulting from step (a);
(c) incubating the mixture resulting from step (b) at a physiologically acceptable temperature; and (d) measuring and scoring the inhibition of bacterial growth in the liquid medium.
2. A method for determining the identity of an antibody to a gram negative bacterium present in a serum sample of the type having the steps (a) inactivating the serum sample;
(b) incubating the inactivated serum sample together with a complement and culture of a gram negative bacterium of known concentration at a physiologi-cally acceptable temperature;
wherein the improvement comprising (c) adding a liquid medium to the mixture resulting from step (b) instead of transfering the mixture to a solid, agar plate and thereby rendering the method automationable;
(d) incubating the mixture resulting from step (c) at a physiologically acceptable temperature, and (e) measuring and scoring the inhibition of the bacterial growth in the liquid medium.
(b) incubating the inactivated serum sample together with a complement and culture of a gram negative bacterium of known concentration at a physiologi-cally acceptable temperature;
wherein the improvement comprising (c) adding a liquid medium to the mixture resulting from step (b) instead of transfering the mixture to a solid, agar plate and thereby rendering the method automationable;
(d) incubating the mixture resulting from step (c) at a physiologically acceptable temperature, and (e) measuring and scoring the inhibition of the bacterial growth in the liquid medium.
3. A method of determining the antibody level to a gram negative bacterium in a serum sample of the type having steps (a) inactivating the serum sample;
(b) diluting the sample serially to obtain aliquots of diminishing concentrations;
(c) incubating simultaneously each of the aliquots together with the culture of a gram negative bacterium of known concentration, and a complement at a physiologically acceptable temperature;
wherein the improvement comprising (d) adding a liquid medium to each of the aliquots from step (c) instead of transferring each aliquot to a solid agar plate and thereby rendering the method automationable;
(e) incubating each of the mixtures resulting from step (d) at a physiologically acceptable temperature; and (f) measuring and scoring the inhibition of bacterial growth in the liquid medium.
(b) diluting the sample serially to obtain aliquots of diminishing concentrations;
(c) incubating simultaneously each of the aliquots together with the culture of a gram negative bacterium of known concentration, and a complement at a physiologically acceptable temperature;
wherein the improvement comprising (d) adding a liquid medium to each of the aliquots from step (c) instead of transferring each aliquot to a solid agar plate and thereby rendering the method automationable;
(e) incubating each of the mixtures resulting from step (d) at a physiologically acceptable temperature; and (f) measuring and scoring the inhibition of bacterial growth in the liquid medium.
4. The method according to Claims 1, 2 or 3, wherein the first incubation is carried out under a water-saturated atmosphere at about 37°C and the second incubation is carried out under a humidified atmosphere containing about 5% carbon dioxide.
5. The method according to Claim 1, wherein the steps (a) to (d) are carried out in a multiwell-plate for a series of gram negative bacteria selected from a group eon-sisting of E. coli, S. typhi, N. meningitis, H. influenzae and N. gonorrhea.
6. The method according to Claim 2, wherein the steps (a) to (e) are carried out in a multiwell-plate for a series of reference antisera to gram negative bacteria selected from a group consisting of E. coli, S. typhi, N. meningitis, H. influenzae and N. gonorrhea.
7. A test kit for carrying out the method of Claim 1 comprising (a) a lyophilized antiserum to a known gram negative bacterium to be used in step (a) as the reference antiserum;
(b) a medium for the growth of the bacterium; and (c) a complement to promote the bacterial growth.
(b) a medium for the growth of the bacterium; and (c) a complement to promote the bacterial growth.
8. A test kit for carrying out the method of Claim 2 comprising (a) a lyophilized gram negative bacterium for preparing the culture required in step (b);
(b) a medium for the growth of the bacterium; and (c) a complement to promote the bacterial growth.
(b) a medium for the growth of the bacterium; and (c) a complement to promote the bacterial growth.
9. A test kit for carrying out the method of Claim 3 comprising (a) a lyophilized gram negative bacterium for preparing the culture required in step (c);
(b) a medium for the growth of the bacterium; and (c) a complement to promote the bacterial growth.
(b) a medium for the growth of the bacterium; and (c) a complement to promote the bacterial growth.
10. The test kit according to Claim 8 or 9, wherein the lyophilized gram negative bacterium is E. coli, S. typhi, N. meningitis, H. influenzae or N. gonorrhea.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US79477277A | 1977-05-09 | 1977-05-09 | |
| US794,772 | 1977-05-09 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1109790A true CA1109790A (en) | 1981-09-29 |
Family
ID=25163629
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA302,714A Expired CA1109790A (en) | 1977-05-09 | 1978-05-05 | Automated assay for bacterial or bacterial antibody |
Country Status (11)
| Country | Link |
|---|---|
| JP (1) | JPS542318A (en) |
| BE (1) | BE866664A (en) |
| BR (1) | BR7802880A (en) |
| CA (1) | CA1109790A (en) |
| DE (1) | DE2820071A1 (en) |
| ES (1) | ES469586A1 (en) |
| FR (1) | FR2390502A1 (en) |
| GB (1) | GB1604884A (en) |
| IT (1) | IT1117351B (en) |
| NL (1) | NL7804410A (en) |
| PT (1) | PT67981B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2537725B1 (en) * | 1982-12-09 | 1985-07-12 | Pasteur Institut | METHOD FOR THE IMMUNOBACTERIOLOGICAL DETECTION OF PATHOGENIC GERM IN CONTAMINATED BIOLOGICAL MEDIA |
| WO1986005592A1 (en) * | 1985-03-18 | 1986-09-25 | National Research Development Corporation | Antigenically active protein and its use in the diagnosis of gonorrhoea |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3864624A (en) * | 1972-05-31 | 1975-02-04 | Yokogawa Electric Works Ltd | Standard voltage generating circuit |
| GB1378103A (en) * | 1972-08-31 | 1974-12-18 | Research Corp | Serological reagent and test for neisseria gonorrhoeae antibodies |
-
1978
- 1978-04-25 NL NL7804410A patent/NL7804410A/en not_active Application Discontinuation
- 1978-05-02 PT PT67981A patent/PT67981B/en unknown
- 1978-05-03 BE BE187354A patent/BE866664A/en unknown
- 1978-05-05 IT IT49223/78A patent/IT1117351B/en active
- 1978-05-05 FR FR7813376A patent/FR2390502A1/en not_active Withdrawn
- 1978-05-05 CA CA302,714A patent/CA1109790A/en not_active Expired
- 1978-05-05 GB GB18018/78A patent/GB1604884A/en not_active Expired
- 1978-05-08 ES ES469586A patent/ES469586A1/en not_active Expired
- 1978-05-08 BR BR7802880A patent/BR7802880A/en unknown
- 1978-05-08 DE DE19782820071 patent/DE2820071A1/en not_active Withdrawn
- 1978-05-09 JP JP5414378A patent/JPS542318A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| PT67981B (en) | 1980-05-05 |
| IT1117351B (en) | 1986-02-17 |
| PT67981A (en) | 1978-06-01 |
| JPS542318A (en) | 1979-01-09 |
| FR2390502A1 (en) | 1978-12-08 |
| GB1604884A (en) | 1981-12-16 |
| NL7804410A (en) | 1978-11-13 |
| BE866664A (en) | 1978-11-03 |
| IT7849223A0 (en) | 1978-05-05 |
| ES469586A1 (en) | 1979-09-16 |
| DE2820071A1 (en) | 1978-11-23 |
| BR7802880A (en) | 1979-01-02 |
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