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
  • C12Q1/14—Streptococcus; Staphylococcus
• • 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
  • G01N33/56944—Streptococcus
• • 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
  • G01N2333/315—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Streptococcus (G), e.g. Enterococci

Definitions

• This invention relates to a new and novel method of utilizing labeled antibodies especially to detect the presence of Group A Streptococci in, for example, a swab sample.
• Serological grouping of human p-hemolytic Streptococcal infections is important to the medical profession, especially the presence of Group A Streptococci which necessitates therapy. As has been reported, a significant error may result from the assumption that all fi-hemolytic Streptococci from the human nasopharynx belong to Group A.
• a number of known methods are used to identify Group A Streptococcus. One involves resort to a fluorescent antibody that make for a specific identification of infectious organisms. However, this laboratory procedure involves a costly fluorescent microscope. In another method used in detecting Streptococci, a bacterial culture is grown. However, positive identification of Group A Streptococci requires 48 hours, involving in some cases an undesirable time lag of proper diagnosis and treatment.
• the method of the invention here incorporates advantages of sensitivity, rapidity, and use only of an ordinary light microscopy for identification purposes rather than resorting to an expensive fluorescent microscope.
• the diagnostic method here, in brief, involves a method of detecting the presence of Group A Streptococci in the presence of other Streptococci and Staphylococci organisms in an unknown sample.
• First is prepared a series of at least 3 dry slide smears, one of said smears acting as a negative control comprising a suspension of only Group G Streptococci and Staphylococci aureus.
• a second positive control smear comprising a suspension of only Group A Streptococci.
• a third smear is prepared comprising a suspension of unknown organisms to be tested for presence of Group A Streptococci. Separately prepared is an antibody conjugate.
• the first step in the invention involves development of an antibody in a conventional manner by resort to a test animal such as a rabbit.
• a conjugate of the antibody to Group A Stroptococcus and peroxidase is then made by bridging or conjugating the two by resort to a coupling agent, such as glutaldehyde or diisocyanate.
• a coupling agent such as glutaldehyde or diisocyanate.
• typical peroxidase enzymes which can be used are a horse radish or lacto peroxidase.
• each antibody has a counterpart in some antigen such as a Group A Streptococcus.
• An antibody specific to an antigen will become attached to that antigen whenever encountered by the antibody. If an antibody that is known to be specific for a particular antigen within a group of antibodies is isolated from the globulin portion of serum or plasma of a host animal which was stimulated to produce that antibody, then the antigen and antibody specific to that antigen will become attached to each oher.
• the peroxidase acts as a type of amplifier which will later allow the organism to be properly observed under a microscope by resort to a dye.
• a preferred method of preparing the reagent here is to form a conjugate of antibody and peroxidase, and then add the conjugate to the Group A Streptococcus.
• Smear slides of a swab sample and a positive and a negative control sample are prepared in the usual manner.
• the lower control limit organisms (negative control) are prepared from a NCDC-derived panel of Group G Streptococci and Straphylococcus aureus which are coagulase positive, non-viable and have been reconstructed from a lyophilized stock.
• the upper control limit organisms likewise are obtained from a NCDC-derived panel of Group A Streptococci which are non-viable and reconstituted from lyophilized stock.
• the upper control limit organisms act as a positive control.
• the three air-dried smears are usually rinsed in a buffer such as a tris-saline buffer, pH 7.5 (buffer) for a few minutes and thereafter drained, usually with a filter paper.
• a buffer such as a tris-saline buffer, pH 7.5 (buffer) for a few minutes and thereafter drained, usually with a filter paper.
• a buffer such as a tris-saline buffer, pH 7.5 (buffer) for a few minutes and thereafter drained, usually with a filter paper.
• all three smears may be placed on a single slide, if desired, in separate inscribed areas.
• One drop of the conjugated reagent prepared as described above is placed on each dry smear and they are incubated. In a typical situation, incubation may be carried out in a moist chamber for 20 minutes at room temperature. The slides are then rinsed again with buffer solution and drained. In one specific technique the slides are immersed in a buffer solution for say about 5 minutes.
• a detecting dye usually an electron donor dye.
• a typical dye useful here is 3- am1no-9-ethyl carbazole.
• Other suitable dyes which may be used include 3,3'-diaminobenzidine, p chloroaniline and a mixture of u-naphthol and p-phenyldenediamine dihydrochloride.
• an oxidizing agent such as hyrogen peroxide.
• Other oxidizing agents WhlCh may be used are methyl peroxide and ethyl peroxide (CH OOH; C H OOH).
• the peroxidase enzyme breaks down the hydrogen peroxide.
• the hydrogen peroxide breakdown product in turn causes the dye to become colored, which colored material in turn becomes attached to the Streptococci.
• EXAMPLE I Preparation and fixation of organisms from throat swabs
• the pharyngeal mucosal swab is swirled vigorously for approximately 30 seconds in 0.5 ml. saline in a Kahn or similar tube.
• the saline is expressed from the swab.
• the same swab can be used to inoculate a blood agar plate for isolation of fi-hemolytic Streptococci.
• a disposable Pasteur pipette is used to transfer one drop (approximately 25 microliters) of the saline suspension of bacteria onto the inscribed area of a microscope slide.
• Samples (0.01 ml., 0.0001 ml.) of the same saline rinse can be taken into a calibrated loop and spread on blood agar plates to titer the saline wash. The test area, on the microscope slide is then allowed to air dry. The smears and fixed by immersion in absolute methanol for 5 minutes and drained.
• Streptococcus antibody was first conjugated to horseradish peroxidase. Two drops (approximately 50 ,ul.) of the conjugated antibody is added to each dry smear, using a Pasteur pipette. Using separate applicator sticks or toothpicks, carefully the reagent is spread over each test area. The applicator stick can be held horizontally to catch the meniscus of the fluid in a manner that avoids scraping cells from the slide.
• the slide is incubated at room temperature for about 20 minutes in a humid chamber using, for example, a slide box, or an inverted petri dish with moist paper towels fitted into or around the dish.
• the slide is then rinsed with ml. of tris-saline buffer dripped from a pipette and then washed by immersion in a staining dish of tris-saline buffer for 5 minutes.
• the slide is dried by tilting at an angle and placing an absorbent piece of paper (paper towels) at the edge of the inscribed area.
• the slides are examined for staining under a light microscope using a 40X objective and l2.5 oculars (final magnification of 500x).
• the advantage of this procedure lies in its speed without the use of an expensive fluorescent microscope.
• the relative low magnification allows a larger area to be viewed when scanning the 1 cm. diameter circle on the slide, thus allowing greater detection limits.
• the slide areas are scanned under a light microscope at a final magnification of 500x.
• the defined detection limits are not directly applicable when other objectives and/ or magnification ranges are used with this procedure.
• the positive control (Group A Streptococci) is clearly distinguished from the negative control (Group G Streptococci and Staphylococcus aureas) by a bright red color.
• the Group A Streptococci directly from the throat swab stain with the same intensity and possess the same single cell morphology as the positive control. Primary focus must be on intensity of color and on presence and extent of chained organisms.
• EXAMPLE II In a second embodiment of the invention as a variation of the techniques described in Example I, the pharyngeal mucosal swab is placed in 1 ml. of Todd-Hewitt broth and incubated at 37 C. for 2 to 5 hours, the broth is then centrifuged 5 minutes at about 2000 r.p.m. to pack the organisms. The supernate fluid is decanted and then the cells resuspended in 1 ml. of saline and recentrifuged.
• the saline is decanted and the tube placed in a rack for 2 to 3 minutes to allow residual saline and organisms to collect in the bottom of the tube.
• the organisms are mixed thoroughly in the residual saline.
• some of the washed sediment is removed and placed within the inscribed area on a single slide.
• test areas are then allowed to air dry.
• the slide is fixed in absolute methanol for 5 minutes and allowed to drain dry.
• the smears are then processed as when the cells are eluted directly from the swab. All controls and data interpretations are the same.
• a method of detecting the presence of Group A Streptococci in the presence of other Streptococci and Staphyllococci organisms in an unknown sample comprising the steps of conjugating a Streptococcus antibody with a Group A Streptococcus by forming a complex of said antibody and Group A Streptococcus through means of a peroxidase enzyme coupling agent, preparing a series of at least three slide smears; one of such smears acting as a negative control comprising a suspension of Group G Streptococci and Staphylococcus aureus; a second positive control smear comprising a suspension of only Group A Streptococci and a third smear comprising a suspension of unknown organisms, adding the conjugate to each smear, incubating the treated smears, adding hydrogen peroxide and an electron donor dye to the incubated treated smears, and observing the thus treated slide smears under a microscope
• a method of preparing a reagent useful in a method of detecting the presence of Group A Streptococci in the presence of other Streptococci and Staphylococci organisms in an unknown sample which comprises the step of conjugating a Streptococcus antibody with a Group A Streptococcus by forming a complex of said antibody and Group A Streptococcus through means of a peroxidase enzyme coupling agent, said resulting conjugate being the desired reagent.

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Cited By (13)

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• 1972
  • 1972-07-05 US US00268967A patent/US3790447A/en not_active Expired - Lifetime
• 1973
  • 1973-05-24 CA CA172,155A patent/CA996455A/en not_active Expired
  • 1973-06-04 AU AU56506/73A patent/AU470394B2/en not_active Expired
  • 1973-07-03 JP JP48074472A patent/JPS4943479A/ja active Pending
  • 1973-07-04 DE DE2334061A patent/DE2334061B2/de active Pending
  • 1973-07-05 GB GB3217573A patent/GB1420806A/en not_active Expired
  • 1973-07-05 FR FR7324800A patent/FR2190917B1/fr not_active Expired

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