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
  • G01N33/563—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
  • G01N33/531—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
  • G01N33/569—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 reliably and with high sensitivity.
• 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.
• 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 important feature of this invention is the method of protecting the antibody during chemical modification of the antibody to be protected, e.g. during cleavage, labelling or binding to a solid phase support thereof, to assure that the ability of the antibody to bind to antigenic determinant of the complement antigen is not impaired.
• 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 is useful in an improved immunoassay 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 selfbind; the improvement comprising the following steps:
• step (a) preparing F(ab') 2 fragment of IgG Anti-organism antibody; (b) binding F(ab') 2 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 ; (c) labelling F(ab') 2 fragment from step (a) or IgG organism antibody with a labelling moiety which can be detected; (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.
• 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
• step (b) includes reacting F(ab') 2 with antigenic determinant of the antigen for said antibody followed by coupling the resulting F(ab') 2 -antigen complex to a solid phase support and then by removal of said antigen; and 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.
• the invention may be used in the immunoassay for Streptococcus Group A, comprising the steps: (a) preparing F(ab') 2 fragment of IgG Anti-Streptococcus Group A antibody, (b) binding F(ab') 2 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; (c) labelling F(ab') 2 fragment from step (a) or IgG Anti-Streptococcus A antibody with a labelling moiety which can be detected, (d) reacting the products of steps (b) and (c) with a test medium suspected of containing Streptococcus Group A , and (e) detecting the labelling moiety on the reagent of step (c) as an indication of the presence of Streptococcus Group A in said
• step (a) includes reacting IgG Anti-Streptococcus Group A antibody with N-acetyl-D-glucosamine 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') 2 -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') 2 with N-acetyl-D-glucosamine followed by reacting the F(ab') 2 complex with the labelling reagent.
• the F(ab') 2 fragment of IgG Anti-Strep Group A is prepared according to the method described by Nisonoff, et al, Arch. Biochem Biophys 89:230-244 (1960). The IgG at a concentration of 5 mg/ml is dialyzed against 0.05M NaOAc, 0.07M NaCl, pH 4.8.
• Pepsin is added to give a 3 wt/wt percent concentration and the mixture is incubated at 37oC 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') 2 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 wi th bov ine serum albumin ( BSA ) , 2 mg/ml in PBS , for 2 hours at room temperature.
• BSA body serum albumin
• 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.
• the beads are incubated in PBS solution containing 1 mg/ml anti-Strep A F(ab') 2 antibody and 0.1% glutaraldehyde at 4°C overnight. Thereafter, the beads are again washed 5 times with PBS and incubated with 1M ethanolamine, pH 8.0 for 2 hours at 4°C. Finally, the beads are then washed 5 times with PBS.
• labelled second antibody employs Anti-Strep A or the F(ab') 2 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. In one particular determination a thirteen-fold decrease in background due to self-binding of Anti-Strep A was accomplished. This dramatic new and surprising result makes the assay for Strep A several times more sensitive and reduces the chance for a false positive significantly.
• Equal volumes of F(ab') 2 or IgG coupled beads (50 ⁇ l) containing equal number of antibody combining sites were incubated with IgG Anti-Strep A- ⁇ -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 ⁇ -galactosidase, o-nitrophenyl-galactopyranoside (ONPG), for 10 minutes.
• the ⁇ -galactosidase produces a yellow color when mixed with its substrate.
• the color formation may be visually detected or quantitatively determined spectro photometrically.
• a Beckman spectrophotometer at a wavelength of 420 nm, the beads which have IgG Anti-Strep A coupled to them were found to produce approximately 13 times the absorbance than the beads which have F(ab') 2 Anti-Strep A coupled to them.
• This invention which has independent significance in other applications, is exemplified in the preferred non- limiting embodiment as preparing F(ab') 2 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 3 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 3 for 30 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% NaN 3 with 0.1M N- acetyl-D-glucosamine for 24 hours at 4°C.. The beads are then washed 6 times with PBS and incubated in 1M ethanolamine, pH 8, for 5 hours at 4° C., and, finally, washed 5 times in PBS.
• Strep Group A carbohydrate is prepared according to the method described by Salton, M.R.J. and Home, R.W. Biochim et Biophysics Acta 7:177, 1951. Different volumes of 1:1 suspension of the beads in borate buffer saline (BBS) , pH 8.2, containing 5% newborn calf serum are reacted with a constant amount of 125 I-GAC for 30 minutes at room temperature. The beads are washed 4 times with PBS-0.2 NP-40 and counted in a gamma counter.
• BBS borate buffer saline
• 0.1M N-acetyl-D-glucosoamine have approximately twice the binding capacity of beads prepared in the absence of N-acetyl-D-glucosamine.
• the preferred embodiment of the invention has been described as including, in the reacted form in which the antigen is bound by an antibody or antibody fragment to a solid support material, e.g. the surface of a bead, and a labelled antibody or antibody fragment is bound to the antigen, two F(ab') 2 antibody fragments, i.e. bead-F(ab') 2 -antigen-F(ab') 2 -label. It should be clearly recognized that the label could also be attached to a complete antibody, since it would not cross- react with F(ab') 2 .
• a bead-F(ab') 2 -Ag-Ab-label complex would be within the scope of the invention, as would the complex bead-Ab-Ag-F(ab') 2 -label, although the latter would be of questionable value as compared with the preferred embodiment.
• the kit below is described in terms of the preferred embodiment.
• 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') 2 .
• Most conveniently the kit would also contain a developer.
• ⁇ -galactosidase is used as the labelling indicator moiety
• the principle of the invention involves the combined steps of providing F(ab') 2 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.
• ⁇ -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.
• radioimmunoas ⁇ ay (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 immunoglobulin.
• the sandwich enzyme immunoassay is a well-established technique, and the present invention permits the use of this general assay technique in a very effective, reliable and highly sensitive assay for Streptococcus Group A.
• the steps of stabilizing F(ab') 2 by reacting the F(ab') 2 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 in to the overall method of this invention.
• 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
  • 1983-12-14 EP EP19840900261 patent/EP0140896A1/de not_active Withdrawn
  • 1983-12-14 WO PCT/US1983/001953 patent/WO1984004170A1/en not_active Application Discontinuation

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