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
• • 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
  • G01N33/563—Immunoassay; Biospecific binding assay; Materials therefor involving antibody fragments
• • 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
  • 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
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6854—Immunoglobulins
  • G01N33/6857—Antibody fragments

Definitions

• Streptococcus Group A One of the major antigenic determinants of Streptococcus Group A (Strep-A) is N-acetyl-D-glucosamine. Most Anti-Strep A antibodies bind to the N-acetyl-D- glucosamine determinant. Anti-Strep A also possesses on its own structure N-acetyl-D-glucosamine, however, and will self-bind. This gives a high background which may mask the presence of Strep-A or give a false positive reading for Strep-A immunoglobulins . Moreover, staphylococcal organisms found normally on throat or other body surfaces can bind to the Fc or tail portion of antibodies, including those specific for Strep A, thus giving a false positive reading. We here describe a claim a method for overcoming this serious limitation on assays for Streptococcus Group A.
• Staphylococcus A is reduced or eliminated.
• monoclonal Anti- Streptococcal Group A antibody is cleaved to remove the Fc portion which includes an N-acetyl-D-glucosamine moiety.
• the present invention may be embodied in a kit for carrying out assays in the laboratory, hospital or in the physician's office.
• the invention may be described as an improved iramunoassay for an organism, using the IgG Anti-organism antibody, the Fc portion of which possesses a moiety to which the IgG Anti-organism antibody can self-bind; the improvement comprising the following steps:
• step (b) binding Ftab' )-, fragment from step (a) to a solid support to provide thereupon sites for binding antigenic determinant of said organism but which will not cross-bind with the Fc portion of the IgG Anti-organism antibody;
• step (c) labelling F(ab' )_ fragment from step (a) or IgG organism antibody with a labelling moiety which can be detected;
• step (d) reacting the products of steps (b)- and (c) with a test medium suspected of containing said organism; and (e) detecting the labelling moiety on the reagent of step (c) as an indication of the presence of said organism in said test medium.
• the labelling moiety is an enzyme
• step (a) includes reacting IgG Anti-organism antibody with the antigenic determinant of the antigen for said antibody followed by a cleavage reaction to remove said antigen from said antibody
• step (b) includes reacting Ftab' ⁇ with antigenic determinant of the antigen for said antibody followed by coupling the resulting F(ab')_- antigen complex to a solid phase support and then by removal of said antigen
• step (c) includes reacting F(ab') 2 with antigenic determinant of the antigen for said antibody followed by reacting the F(ab ' ) 2 -antigen complex with the labelling reagent.
• step (b) binding F(ab' )_ fragment from step (a) to a solid support to provide thereupon sites for binding antigenic determinant of Streptococcus Group A but which will not cross-bind with Streptococcus A antigen or Staphylococcus organism;
• step (c) labelling F( ab' )_ fragment from step (a) or IgG Anti-Streptococcus A antibody with a labelling moiety which can be detected,
• step (e) detecting the labelling moiety on the reagent of step (c) as an indication of the presence of
• step (a) includes reacting IgG Anti-Streptococcus Group A antibody with N- acetyl-D-glucosarnine followed by a cleavage reaction to remove the N-acetyl-D-glucosamine containing moiety of said antibody
• step (b) includes reacting F(ab ' ) 2 with N- acetyl-D-glucosamine followed by coupling the resulting F(ab ' ) _ -N-acetyl-D-glucosamine complex to a solid phase support and then by removal of N-acetyl-D-glucosamine
• step (c) includes reacting F( ab ' )» with N-acetyl-D- glucosamine followed by reacting the F( ab ' ) -, complex with the label
• the F(ab') fragment of IgG Anti-Strep Group A is prepared according to the method described by Nisonoff, et al., Arch. Biochem Biophys 89: 230-240 (1960).
• the IgG at a concentration of 5 mg/ml is dialyzed against 0.054 NaOAc, 0.07M NaCl, pH 4.8.
• Pepsin is added to give a 3 wt/wt percent concentration and the mixture is incubated at 37 °C for 18 hours.
• the mixture is subsequently dialyzed against borate- buffered saline (BBS), pH 8.4, and then passed over a N-acetyl-D-glucosamine - Sepharose column and washed with phosphate buffered saline (PBS).
• BBS borate- buffered saline
• PBS phosphate buffered saline
• the F(ab') ⁇ is eluted from the column with PBS containing 0.2M N-acetyl-D-glucosamine.
• Polystyrene beads (Poly-Sep (trademark) 2% divinyl benzene (DVB), Polysciences, Inc.) are incubated with bovine serum albumin (B ⁇ A), 2 mg/ml in PBS, for 2 hours at room temperature. The beads are washed 3 times with PBS, and then suspended in 2% glutaraldehyde in PBS and mixed for 30 hours at 4°C. The beads are washed 5 times with PBS to remove glutaraldehyde. After the PBS wash, the beads are incubated in PBS solution containing 1 mg/ml anti-Strep A F(ab ' ) ? antibody and 0.1% glutaraldehyde at 4 C overnight. Thereafter, the beads are again washed 5 times with PBS and incubated with IM ethoanolamine, pH 8.0 for 2 hours at 4°C. Finally, the beads are then washed 5 times with PBS.
• B ⁇ A bovine serum albumin
• labelled second antibody employs Anti-Strep A or the F(ab * ) fragment thereof labelled with the enzyme ⁇ -galactosidase, although the label per se is not critical and the method is fully suitable for radioimmunoassay or other detection procedures .
• the present inventive procedure reduced self-binding of Anti-Strep A by a factor of more than ten.
• Equal volumes of F( a ' ) ⁇ or IgG coupled beads (50 ⁇ l) containing equal number of antibody combining sites were incubated with IgG Anti-Strep A-3-galactosidase conjugate for 20 minutes. The beads were then washed 3 times with PBS-0.2% NP-40. The beads were then incubated in the .substrate for B-galactosidase, o-nitrophenyl- galactopyranoside (ONPG), for 10 minutes.
• the B-galacto ⁇ sidase produces a yellow color when mixed with its substrate. The color formation may be visually detected or quantitatively determined spectrophotometrically.
• One of the features of this invention is a particularly advantageous method for preparing F( ab ' ) , Anti-Strep A - polystyrene beads, and for preparing labelled F(ab') 2 Anti-Strep A and for binding an antibody or fragment to a solid phase support, exemplary of which are the polystyrene beads used to exemplify this invention.
• Polystyrene beads are mixed with BSA, 2.0 mg/ml, in PBS-0.02% NaN, at room temperature for 2 hours, the beads are washed 4 times with PBS and then mixed with PBS containing 2% glutaraldeyhde and 0.02% NaN for 10 hours at room temperature. The beads are then washed 6 times with PBS and mixed in a 1:1 suspension with the affinity purified Anti-Strep Group A antibody in PBS- . 0.02 " i NaN3 with 0. IM N-acetyl-D-glucosamine for 24 hours at 4 °C.
• Strep Group A carbohydrate is prepared according to the method described by Salton, M.R.J. and Home, R.W. Biochim et
• kits for diagnostic determinations and for other uses and in research as well, which includes the major reagents and, of course, would typically include vessels, instructions, etc .
• a kit according to this invention would include labelled F( ab' ) , F(ab' ) 2 - bound to styrene beads or other convenient solid phase support and such vessels, containers and instructions as may be conveniently included. Most conveniently the kit would also contain a developer.
• B-galactosidase is used as the labelling indicator moiety
• the principle of the invention involves the combined steps of providing F( ab ' ).,Anti-Strep A, coupling the same to a solid phase support such as polystyrene beads, and also coupling the F(aB' ) 2 Anti-Strep A antibody to a detectable label.
• a solid phase support such as polystyrene beads
• F(aB' ) 2 Anti-Strep A antibody to a detectable label.
• -galactoside is a preferred label, because it gives a very sensitive and pronounced indication and is easily handled.
• Other labels may, of course, be used.
• radioimmunoassay (RIA) procedures may be used but present a number of disadvantages, not the least of which is the necessity for handling radioactive reagents and wastes.
• Other substrates may be used, but polystyrene beads are particularly advantageous being readily available, easily handled, provide a very sensitive assay and bind effectively with antibody.
• Pepsin cleavage is described, but other cleavage reagents and methods may be used within the concept of the invention.
• cleavage reagents and methods may be used within the concept of the invention.
• papain, CNBr, trypsin and other reagents may be used for removing the Fc region or the carbohydrate moiety from the immunoglobuL i n.
• the sandwich enzyme immunossay is a well-established technique, and the present invention permits the use of this general assay technique in ⁇ very effective, reliable and highJy sensitive assay for Streptococcus Group A.
• OMPI the F(ab') with an antigenic determinant therefor, e.g., N-acetyl-D-glucosamine, during binding to beads and the same procedure applied to stabilizing the F(ab' ) 2 during labelling with a detectable moiety, e.g., ⁇ -galactosidase, provide an extremely efficient technique and high yield, with improved reliability and higher sensitivity when incorporated into the overall method of this invention.
• an antigenic determinant therefor e.g., N-acetyl-D-glucosamine
• This invention finds commercial applicability in the clinical and research assay of Streptococcus Group A as a diagnostic method in the treatment of disease and as a research tool in the development of diagnostic and therapeutic methods and reagents.

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• 1983
  • 1983-11-28 EP EP19840900213 patent/EP0141818A1/de not_active Withdrawn
  • 1983-11-28 WO PCT/US1983/001877 patent/WO1984004169A1/en unknown

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