REMOVAL OF SELF-BINDING AND STAPH A CROSS-REACTIVITY OF ANTI-STREP ANTIBODY

Abstract: An improved diagnostic method and reagent for the assay of Streptococcus Group A, and other antigen containing organisms, in which the high background resulting from cross-reaction of Anti-Streptococcal Group A or the reaction with Staphylococcus is eliminated.

Description: REMOVAL OF SELF-BINDING AND STAPH A CROSS-REACTIVITY OF ANTI-STREP ANTIBODY Background of the Invention 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 a claim a method for overcoming this serious limitation on assays for Streptococcus Group A. 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- glucos- amine moiety on the Strep A antibody or reaction with Staphylococcus A is reduced or eliminated. According to a preferred embodiment of the invention, monoclonal Anti- Streptococcal Group A antibody is cleaved to remove the Fc portion which includes an N-acetyl-D-glucosamine moiety. An important feature of this invention is also the method of protecting the antibody during cleavage and during labelling to assure that the ability of the Anti- O PI Strep-A antibody to bind to Strep-A 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 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: (a) preparing F( b ' ) ? fragment of IgG Anti- organism antibody; (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; (c) labelling F(ab' )_ 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, the labelling moiety is an enzyme, and 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; 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. ■^J^E Q PI In its preferred and exemplary, but non-limiting, form the invention may be described in terms of an immunoassay for Streptococcus Group A, comprising the improvement set forth in the following steps: (a) preparing F( ab ' ) _ fragment of IgG Anti- Streptococcus Group A antibody, (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; (c) labelling F( ab' )_ 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 the exemplary embodiment, the labelling moiety is an enzyme, e.g. 6 -galactosidase. In a still more exemplary, but non-limiting sense, 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, and step (c) includes reacting F( ab ' )» with N-acetyl-D- glucosamine followed by reacting the F( ab ' ) -, 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 out 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') 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). 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 (BΞA), 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. In the preferred embodiment, labelled second antibody employs Anti-Strep A or the F(ab * ) 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 OR -viPI 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( a ' )~ or IgG coupled beads (50 μl) 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. 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 method of assay for Strep A. One of the features of this invention, which has independent significance in other applications, 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. The beads are then washed 6 times with PBS and incubated in IM 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 125 I-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 Biophysica 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.IM 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 r ) 2 antibody fragments, i.e., bead-F(ab') 2 -antigen-F(a ' )_ -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 * ) -Ag-Ab-label complex would be within the scope of the invention, as would the complex bead-Ab-Ag-F(a ' ) -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' ) , 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 B-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 ' ).,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. For example, 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. For example, 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 α very effective, reliable and highJy sensitive assay for Streptococcus Group A. The steps of stabilizing F( ab ' ) by reacting 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., β-galactosidase, provide an extremely efficient technique and high yield, with improved reliability and higher sensitivity when incorporated into 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. OMPI

Claims: WHAT IS CLAIMED IS: 1. In the assay of Streptococcus Group A, the improvement comprising the following 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 the Anti-Streptococcus A antibody, itself or Staphylococcus organism; (c) labelling F (ab ') 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 in indication of the presence of Streptococcus Group A in said test medium. 2. The Immunoassay of Claim 1 wherein the labelling moiety is an enzyme. 3. The immunoassay of Claim 1 wherein step (a) includes reacting 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. 4. The immunoassay of Claim 1 wherein step (c) includes reacting F( ab ' or IgG Anti-Streptococcus A antibody with N-acetyl-D-glucosam ne followed by reacting the resulting complex with the labelling reagent. 5. The immunoassay of Claim 1 wherein 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 substrate followed by removal of N-acetyl-D-glucosamine. 6. The immunoassay of Claim 1 wherein: step (a) includes reacting IgG Anti- Streptococcus Group A antibody with N-acetyl-D-glucosamine OMPI followed by a cleavage reaction to remove the N-acetyl-D- glucosamine containing moiety of said antibody; step (b) includes reacting F(ab' )_ 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; and step (c) includes reacting F(ab' L with N- acetyl-D-glucosamine followed by reacting the F(ab' )_ -N- acetyl-D-glucosamine complex with the labelling reagent. 7. The immunoassay of Claim 6 wherein the labelling moiety is B-galactosidase. 8. 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 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. 9. The immunoassay of Claim 8 wherein the labelling moiety is an enzyme. 10. The immunoassay of Claim 8 wherein: step (a) includes reacting IgG Anti-organism antibody with the antigenic determinant of the antigen for said antibody followed by α 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. O PI

Inventors: CHANG SHUNG-HO, KATZ DAVID H, CHANG, SHUNG-HO, KATZ, DAVID, H.
Applicants: QUIDEL
Application year: 1983
Main classification: G01N33
IPC codes: G01N33