EP0140896A1 - Protection of antibody during chemical modification - Google Patents

Protection of antibody during chemical modification

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Publication number
EP0140896A1
EP0140896A1 EP19840900261 EP84900261A EP0140896A1 EP 0140896 A1 EP0140896 A1 EP 0140896A1 EP 19840900261 EP19840900261 EP 19840900261 EP 84900261 A EP84900261 A EP 84900261A EP 0140896 A1 EP0140896 A1 EP 0140896A1
Authority
EP
European Patent Office
Prior art keywords
antibody
organism
reacting
fragment
labelling
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP19840900261
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German (de)
French (fr)
Inventor
David H. Katz
Shung-Ho Chang
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QUIDEL
Original Assignee
QUIDEL
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Publication date
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Publication of EP0140896A1 publication Critical patent/EP0140896A1/en
Withdrawn legal-status Critical Current

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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.

Abstract

Réactifs améliorés et leurs procédés de préparation pour l'analyse de Streptococcus groupe A et d'autres organismes contenant des antigènes, où le fond important dérivant de la réaction croisée d'un groupe A anti-Streptococcique ou la réaction avec un Staphylococcus est éliminée, par modification chimique de l'anticorps. La réactivité de l'anticorps pour l'antigène est protégée pendant une telle modification. Les modifications comprennent le clivage de la partie Fc de l'anticorps et la liaison de l'anticorps ou d'un fragment de celui-ci sur un support à phase solide.Improved reagents and their methods of preparation for the analysis of group A Streptococcus and other antigen-containing organisms, where the important background derived from the cross-reaction of an anti-Streptococcal group A or the reaction with a Staphylococcus is eliminated, by chemical modification of the antibody. The reactivity of the antibody to the antigen is protected during such a modification. The modifications include cleavage of the Fc portion of the antibody and binding of the antibody or a fragment thereof to a solid phase support.

Description

PROTECTION OP ANTIBODY DURING CHEMICAL MODIFICATION
Background β-Hemolytic streptococci, Group A, are primarily responsible for streptococcal infections in man. It is, therefore, very desirable to be able to assay for
Streptococcus Group A reliably and with high sensitivity.
Notwithstanding the need for an assay which is definitive and highly sensitive, the prior art assays have exhibited a high background which reduces sensitivity and can lead to false positive readings.
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 and claim a method for overcoming this serious limitation on assays for Streptococcus Group A and other organisms in which the IgG anti-organism antibody includes a moiety which cross-reacts with the antibody.
Summary of the invention
An assay method specifically for Streptococcus Group A has been developed in which the background resulting from cross-reaction with the N-acetyl-D-glucosamine moiety on the Strep A antibody or reaction with
Staphylococcus A is reduced or eliminated. According a preferred embodiment of this invention, 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.
In a general sense, 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:
(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.
In a preferred form, 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 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.
In its preferred and exemplary, but non-limiting, form 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 test medium. In this exemplary useage, 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, and step (c) includes reacting F(ab')2 with N-acetyl-D-glucosamine followed by reacting the F(ab')2 complex with the labelling reagent.
Description of the Preferred Embodiment The following description will assure that all information necessary for those skilled in the art to carry the invention is given herein. This is the presently preferred embodiment, but no attempt is made here to circumscribe the invention, the reagents which may be used, the particular techniques which may be substituted and the variations which may be made without departing from the concept and the scope of the invention. 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). 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. 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')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.
In the preferred embodiment, labelled second antibody employs Anti-Strep A or the F(ab')2 fragment thereof labelled with the enzyme β-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 μl) containing equal number of antibody combining sites were incubated with IgG Anti-Strep A- α-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 β-galactosidase, o-nitrophenyl-galactopyranoside (ONPG), for 10 minutes. The β-galactosidase produces a yellow color when mixed with its substrate. The color formation may be visually detected or quantitatively determined spectro photometrically. Using 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. Thus, it has been shown that the present invention, using F(ab')2 Anti-Strep A, removes the self-binding of Anti-Strep A and provides a very much more sensitive and reliable ineLhod of assay for Strep A.
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% NaN3 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% NaN3 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% NaN3 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. The beads are then tested for their ability to bind 125I-strep Group A carbohydrate. Strep Group A carbohydrate (GAC) 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 125I-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.
The results were truly striking, quite surprising and unpredictable. The beads coupled in the presence of
0.1M N-acetyl-D-glucosoamine have approximately twice the binding capacity of beads prepared in the absence of N-acetyl-D-glucosamine.
In the above discussion, 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. Thus, 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.
The invention may be embodied in a kit 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. 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. For example, assuming β-galactosidase is used as the labelling indicator moiety, it is convenient to include a ONPG, a synthetic substrate for β-galactosidase, to bring about a color development which can be visually observed or measured quantitatively or semi-quantitatively using colorimetric or spectrophotmetric methods.
Discussion 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. β-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. For example, radioimmunoasεay (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. For example, 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. β-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.
Industrial Application 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.

Claims

WHAT IS CLAIMED IS:
1. In the assay of Streptococcus Group A which includes the steps of (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 Croup A but which will not cross-bind with the anti-Streptococcus A antibody itself 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 test medium, the step of: reacting 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.
2. In the assay of Streptococcus Group A which includes the steps of (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 the anti-Streptococcus A antibody itself 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 A; and (e) detecting thelabelling moiety on the reagent of step (c) as an indication of the presence of Streptococcus Group A in said test medium, the step of: 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 followed by removal of N-acetyl-D-glucosamine.
3. In the assay of Streptococcus Group A which includes the steps of (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 Croup A but which will not cross-bind with the Anti-Streptocpccus A antibody itself 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 test medium, the steps of (i) 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; (ii) 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; and (iii) reacting F(ab')2 with N-acetyl-D-glucosamine followed by reacting the F(ab')2-N-acetyl-D-glucosamine complex with the labelling reagent.
4. In the assay of 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 includes the steps of (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, the step of (i) 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.
5. In the assay of 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 which includes the steps (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, the step of (ii) 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.
6. In the assay of 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 which includes the steps of (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, the step of (iii) 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.
7. In the assay of an organism, using the IgG Anti-organism antibdoy, the Fc postion of which possesses a moiety to which the IgG Anti-organism antibody can self-bind which includes the steps of (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, the steps of (i) 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; (ii) 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 (iii) 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.
8. The method of protecting the chemical and immunological integrity of an antibody during chemical modification comprising the steps of: (a) reacting the antibody with the antigenic determinant of the complement antigen thereby bonding the antigenic determinant of the antibody to be protected; (b) chemically modifying the antibody with said antigen bound thereto; and (c) thereafter removing the antigen from the antigenic determinant of the protected antibody.
EP19840900261 1983-04-18 1983-12-14 Protection of antibody during chemical modification Withdrawn EP0140896A1 (en)

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US48573683A 1983-04-18 1983-04-18
US485736 1995-06-07

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US4794076A (en) * 1985-09-23 1988-12-27 Vxr, Inc. Simultaneous extraction of a ligand from a sample and capture by anti-ligands therefor in ligand/anti-ligand assays
DK289686A (en) * 1986-06-19 1987-12-20 Immuntech As PROCEDURE FOR PREPARING SURFACE BASED IMMUNAL ANALYSIS
US5447837A (en) * 1987-08-05 1995-09-05 Calypte, Inc. Multi-immunoassay diagnostic system for antigens or antibodies or both

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US3790447A (en) * 1972-07-05 1974-02-05 Abbott Lab Streptococci diagnostic method
US3896217A (en) * 1973-03-19 1975-07-22 Summa Corp Method and apparatus for radioimmunoassay with regeneration of immunoadsorbent
US4120945A (en) * 1976-07-06 1978-10-17 Becton, Dickinson & Company Substrate coated with receptor and labeled ligand for assays
GB2013688B (en) * 1978-01-26 1982-06-30 Technicon Instr Insolubilised proteins and immunoassays utilising them
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0258043A2 (en) * 1986-08-26 1988-03-02 Canon Kabushiki Kaisha Information record medium having a header having error correction code added for each block and a method for recording information on such a medium
EP0258043A3 (en) * 1986-08-26 1988-09-28 Canon Kabushiki Kaisha Information record medium having a header having error correction code added for each block and a method for recording information on such a medium

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