Classifications

• • 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
  • C12Q1/10—Enterobacteria
• • 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
  • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
• • 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
  • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
  • G01N33/553—Metal or metal coated
• • 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
  • G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
• • 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
  • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
  • G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
  • G01N2333/255—Salmonella (G)

Definitions

• the present invention relates to an antisera capable of reacting with the flagellae of substantially all Salmonella serotypes and the use of this antisera preparation in practical immunoassay systems for the rapid and specific detection of salmonellae in specimens.
• the present invention further relates to a method for immobilization of bacteria on a titanous hydroxyoxide (hereinafter referred to as titanous hydroxide) solid phase.
• the Kauffmann- hite schema classifies Salmonella serotypes on the basis of the serological properties of their 0 and H antigens.
• This approach has been very successful as a classification principle, and has resulted, so far, in the recognition of more than 1800 Salmonella serotypes and bioserotypes.
• This immunological diversity has complicated the process of salmonellae detection using immunoassays.
• the use of 0 antigens as a basis for the specific detection of salmonellae by immunoassay is not possible because of the existence of close antigenic relationships of 0 identity between Salmonella, Arizona, Escherichia, Citrobacter, and the yeast, Candida.
• Immunoassays based upon the use of either radioactive isotopes or enzymes as labels such as radioimmunometric assays and enzyme immunometric assays, are characteristically highly specific and sensitive and have consequently been used extensively.
• solid-phase immunoassays have increased because of the simplicity of its separation technique.
• other substances for the immobilization of antibodies and antigens have been introduced such as commercial isothiocyanate-substituted plastic discs, polystyrene balls, nylon, activated thiol-Sepharose and microparticulate cellulose.
• the immobilization of whole microbial cells on solid supports is used commercially to exploit their enzymatic activity for the production or degradation of substances.
• Techniques for immobilizing microbial cells include (i) entrapment in alginate gels, polyacrylamide gels, collagen membranes, metal hydroxide precipitates, agar pellets and liquid membranes, (ii) absorption onto chromatographic separation or ion exchange materials, (iii) selective binding to plant lectins and (iv) covalent binding of whole cells to a support, using bifunctional reagents, e.g. carbodiimide.
• bifunctional reagents e.g. carbodiimide
• the first aspect of the present invention consists in an antisera preparation capable of reacting with substantially all Salmonella serotypes in which said preparation comprises a mixture of individual antisera raised against the flagellae of at least eight of the Salmonella serotypes selected from the group comprising Salmonella oranienburg.
• Salmonella enteritidis Salmonella kentucky. Salmonella waycross. Salmonella abortus-e ui. Salmonella tennessee. Salmonella 4, 12 : d;-. Salmonella 1, 4, 5,12;-;1, 2, Salmonella worthington and Salmonella lille. It was perceived that Salmonella flagella possess common H antigenic determinants (which are not shared by other microorganisms) at sufficiently high concentrations to enable the specific detection of salmonellae. Close examination of the H antigenic composition of Salmonella isolates which have been isolated and serotyped in
• the individual antisera comprising the antisera preparation were raised against flagella isolated from the following strains of Salmonella; S. oranienburg (S.R.L.* No. 1254), S. enteritidis (S.R.L. No. 1267), S. kentucky (S.R.L. No. 1285) S. waycross (S.R.L. No. 1312) S. abortus-equi (S.R.L. No. 1451) S. tennessee (S.R.L. No.
• the flagella are isolated by culturing the bacteria in a defined medium, subjecting the bacteria to a pH of less than 3, and preferably 2 or less, for a time period of no more than 1 hour, and preferably from 20 to 40 minutes, removing the bacterial cells, and recovering the depolymerized flagellae.
• the individual antisera components of the antisera preparation are produced by immunization of suitable antibody producing animals with flagellin emulsions in an adjuvant by multiple intrader al injection.
• the second aspect of the present invention consists in a method for the immobilisation of bacteria on a solid phase comprising shaking or agitating a culture of bacteria with titanous hydroxide.
• the titanous hydroxide was prepared by neutralization of a diluted solution of titanous chloride with an ammonia solution and then washing the suspension with saline to remove ammonium ions.
• the immobilization of microorganisms with titanous hydroxide is a simple, one step method, involving shaking for approximately 10 min.
• the degree of immobilization of microorganisms is usually improved by increasing the duration and/or the intensity of agitation, because of increasing the probability of contact between the cells and the titanous hydroxide particles. Nevertheless, a duration of only 10 min was quite sufficient for almost complete immobilization. It is also possible that agitating too violently or for too long a time can cause dissociation of immobilized mircoorganisms. This, however, did not occur with titanous hydroxide in this investigation, indicating a high degree of stability.
• the third aspect of the present invention consists in a method for the detection of Salmonella serotypes in a specimen comprising immobilizing the bacteria in the specimen on a solid phase, adding a quenching agent to mask unutilized binding sites on the solid phase, adding a Salmonella H antisera preparation according to the first aspect of the invention, and detecting the presence of bound antibodies.
• the specimen is cultured before being immobilized on a solid phase.
• the solid phase is either titanous hydroxide or microtitre trays.
• the presence of bound antibodies are detected by either radioimmunometric or enzyme immunometric assay.
• Salmonella serotypes were cultured in brain heart infusion broth (BHI) , diluted with saline and after removing aliquots for standard plate count determinations, the minimum detectable populations were determined with radioimmunometric assay (RIMA) after the immobilization of salmonella cells on titanous hydroxide.
• the antisera used in these assays were (i) each of the ten individual antisera (ii) a mixture of four antisera raised against flagellins from the Salmonella serotypes S_. abortus-equi, £>. kentucky, S_. tennessee and S_. waycross and (iii) a mixture of the 10 antisera.
• the dilution of each individual antisera or each component in an antisera mixture was 1:1000.
• Radioimmunometric assay for the detection of salmonellae was carried out as follows. Sixteen species of Enterobacteriaceae, other than salmonella, namely, Citrobacter freundii, Edwardsiella tarda, Enterobacter aerogenes, Erwinia herbicola, Hafnia alvei, Proteus rettqeri, Proteus vulgaris, Serratia marcescens, Shigella dysenteriae, Proteus mirabilis, Proteus morganii, Shigella flexneri, Yersinia enterocolitica, Klebsiella pneu oniae, Yersinia pseudotuberculosis, and Escherichia coli, were grown in BHI at their optimum temperatures (30 to 37°C) .
• 125 I-labelled protein A and a specific radiolabelled antibody, against rabbit antibody 125I-labelled donkey anti-rabbit F(ab) 2 fragment, 12*_I-F(ab) 2 ) , purchased from the Radiochemical Centre, Amersham, England. The incubation times during assays, using these reagents, were
• MSCB mannitol selenite cystine broth
• Oxoid either alone or in association with a mixture of the sixteen enterobacterial species.
• the inoculation volumes in MSCB of salmonellae and the enterobacterial mixture were 1:100, respectively. After incubation of MSCB cultures for 18h at 42°C, they were assayed for salmonellae with RIMA, using radio ⁇ labelled protein A.
• the antisera used in these assays consisted of the ten antisera mixture (PFA) (at 1:400) as well as the Spicer-Edwards polyvalent antisera (SEA) (at 1:200) .
• PFA ten antisera mixture
• SEA Spicer-Edwards polyvalent antisera
• Radioimmunometric assay was conducted using varying levels of immobilized Salmonella cells and homologous antisera.
• the log minimum detectable populations of Salmonella serotypes using individual antisera in RIMA are listed in Table 2.
• H antisera H antigens of Salmonella serotypes m,t g,m z 6 z 4' z 23 e,n,x z 29 d 1,2 l,w z 38
• RIMA was ⁇ sonduicted usi:ng each antigen (i.e. Salmonella serotype) over a range of cell concentration. Each antigen was tested with its homologous antisera as well as each of the ni heterologous antisera.
• H antigen of H antisera mixture consisting of
• Ente obacteriaceae speicies using antisera mixture 3 in radioimmuno etric ' ⁇ assay.
• the antisera mixture consisted of ten antisera (Table 3), at a final concentration of 1:1000 of each component.
• This antisera mixture was also capable of detecting other Salmonella serotypes (Table 5) which possessed serotype- specific H antigens (according to the Kauffmann-White schema) differing from the Salmonella antigens used to raise the ten antisera (e.g. S_. infantis, S_. give and S_. birkenhead) .
• the log of minimum detectable populations ranged from 3.48 to 5.63.
• this antisera mixture was highly specific for salmonellae detection using RIMA and did not detect populations of up to 10 colony forming units / lOOul of the species E. coli, £. vulgaris, E. aerogenes and C.
• Salmonella H antisera (1:200) and 3 I-labelled protein A was possible. Most of these salmonellae possessed serotype-specific H antigens which differed from the Salmonella H antigens used to raise the ten antisera utilized in the assay. All serotypes were detectable after growing alone or in association with the enterobacterial species mixture (Table 8) . The results also show that the performance of the ten antisera mixture was superior to that Table 6. Detectability of different species of Enterobacteriacea grown in tetrathionate broth as determined by % bound of 125 'i,-labelled protein A and 125 '.I-labelled F(ab) 2' in radioimmunometric assay.
• Shigella dysenteriae -1.9 -0.6
• the inocula used for the tetrathionate broth consisted of salmonellae and the enterobacterial species at the ratio of 1:100, respectively.
• Spicer-Edwards polyvalent antisera This is indicated by the significantly higher values of specific binding of radiolabelled protein A by the former (p ⁇ 0.001, using paired t test).
• a further aspect of this invention was the development of a method for the immobilization of bacteria on a non-soluble support, thereby facilitating the detection of these bacteria, by solid-phase immunoassay systems. The criteria for such a method were that it should be simple, rapid, efficient and that it should not interfere with the measurement of antigen/antibody reactions by immunoassays.
• Immobilization experiments were conducted using titanous hydroxide, titanic hydroxide and zirconium hydroxide, and Salmonella cells as well as other microorganisms. These microorganisms were grown overnight in BHI broth at 37°C. The cultures were diluted to the desired cell concentrations with sterilized saline (pH 6.8) . The metal hydroxide suspensions were dispensed in polystyrene tubes at the rate of 50jul/tube. Diluted culture suspensions were added (lOO ⁇ l) and the tubes were agitated for 10 min at room temperature, in a reciprocal agitator, at a speed to maintain the metal hydroxides suspended in the tubes, except when otherwise specified.
• a A bbyy mmeettaall hydroxides in the presence of quenching materials A A bbyy mmeettaall hydroxides in the presence of quenching materials.
• Titanous hydroxide Titanic hydroxide Zirconium hydroxide
• Bovine serum albumin 17.5 8.6 24. . 3 28.4 14.4 15.9 (Calbiochem)
• Vigorous shaking applied 200 r.p. ⁇ u.
• Salmonellae were first grown in brain heart infusion broth then dilua
• Salmonellae were first grown in each broth then diluted in fresh brot
• Immobilization of microorganisms other than salmonellae by titanous hydroxide This was carried out using cultures grown in BHI which were diluted with saline. The immobilization efficiency was determined using lOOj ⁇ l diluted culture, 50 ⁇ l of titanous hydroxide suspension and 10 min shaking time. The results (Tables 17 and 18) showed the ability of titanous hydroxide to immobilize a wide range of Gram- negative and Gram-positive bacteria. The immobilization %, ranged from 82.0 to 99.9 and 88.1 to 99.6, respectively. The immobilization of salmonellae and other Gram- negative and Gram-positive bacteria by titanous hydroxide was shown in this investigation to be far superior to other conventional methods. Detection of Salmonellae by Immunoassays
• Salmonellae were cultured in brain heart infusion broth (BHI), Baltimore Biological Laboratory, for 18h at 37°C then inoculated into Oxoid mannitol selenite cystine (MSCB) and tetrathionate (TTB) broths. The selective media were incubated for 18h at 42 and 37°C, respectively.
• MSCB Oxoid mannitol selenite cystine
• TTB tetrathionate
• Salmonellae were also inoculated into MSCB in association with a mixture of 16 cultures of other enterobacterial species, at an inoculation volume ratio of 1:100, respectively. The mixed cultures were incubated at 42°C for 18h. The growth of organisms in pure culture was determined using the standard plate count method.
• food homogenates were prepared (25 g food sample and 225ml of buffered peptone water) , using a Sorvall Omni-Mixer with solid foods.
• the homogenates were inoculated with salmonella cultures in BHI, using a straight inoculation wire, then pre-enriched for 18-24h at 37°C.
• the pre-enrichment cultures were inoculated (1.0ml) into MSCB and TTB, for selective enrichment, and incubated for 18-24h at 42°C and 37°C, respectively.
• 125 I-labelled protein A diluted in PSAG to give approximately 25,000 c.p.m./lOQul were added.
• the tubes were placed in the shaker incubator for lh at 30°C.
• the unbound radioactive reagent was removed by dilution, centrifugation and aspiration and the residual radioactivities were counted using a Packard Selectronic Autogamma Spectrometer. Control tests were conducted as above for each sample, whereby quantities of lOQ ⁇ l PSAG were added in place of salmonella H antisera.
• the net radioactivities bound were determined by deducting the residual radioactivities of the tubes containing no H antisera from those containing the antisera.
• the enzyme immunometric assays were also performed using titanous hydroxide suspension in polystyrene tubes, as the solid-phase using the same method used in the radioimmunometric assay except that the use of the antibacterial agent NaN., was omitted and in place of 125 I-labelled protein A, 200jul of anti- rabbit IgG (whole molecule) - alkaline phosphatase conjugate antibody raised in goat, was used.
• the optimum concentration of the enzyme conjugate was found to be 1:250 as determined by chequerboard titration. After incubation for lh at 37 C and the elimination of the unbound enzyme conjugate fraction, aliquots of 200,ul of alkaline phosphatase substrate were added.
• the enzyme substrate was prepared on the same day of use by adding one tablet (5 mg) of
• a total of 235 food samples were investigated for the presence of salmonellae. These comprised 20 raw milk, 28 pasteurized milk, 30 soft cheese (mozzarella, fetta, ricotta, cottage and cacciotta) , 26 cheddar cheese, 70 milk powder, 20 chicken carcasses, 13 chicken livers, 10 egg products (egg powder and egg noodles) and 18 minced beef samples.
• the samples were tested for the presence of salmonellae using the cultural method of the Standards Association of Australia (1983) . Also after pre-enrichment and selective enrichment of food samples, only MSCB cultures were tested (in duplicate) for salmonella with immunoassays, using PFA and SEA antisera diluted to 1:400 and 1:200, respectively.
• the immunoassays were performed using titanous hydroxide as solid-phase.
• Table 19 ( cont ' d)

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• 1986
  • 1986-01-14 WO PCT/AU1986/000010 patent/WO1986004352A1/en not_active Application Discontinuation
  • 1986-01-14 JP JP50065086A patent/JPS62501726A/ja active Pending
  • 1986-01-14 EP EP19860900716 patent/EP0209554A4/en not_active Withdrawn

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