PROTECTION OF ANTIBODY DURING CHEMICAL MODIFICATION

Abstract: Improved reagents and methods of preparing the same 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, by chemically modifying the antibody wherein the reactivity of the antibody for the antigen is protected during such modification. Modifications include cleaving the Fc portion of the antibody and binding 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-glucoεamine, 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 i munoglobulins. 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 GroupA and other organisms-in which the IgG anti-organism antibody includes a moiety which cross-reacts with th-e antibody. Summary j__f £]____ 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 inven ion 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 self- bind; the improvement comprising" the following steps: (a) preparing F(ab * >2 fragment of IgG Anti-organism antibody; (b) binding (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 P(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 P(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 P(ab')2 with N-acetyl-D- glucosamine followed by reacting the F(ab')2 complex with the labelling reagent. Description nL 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 whichmay 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(db') 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/ l is dialyzed against 0.05M NaOAc, 0.07M NaCl, pH 4.8. Pepsin is added to give a 3 wt/wt percent concentra ion 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-glucosaπune - Sep arose 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 with bovine 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 Ffab' antibody and 0.1% glutaraldehyde at 4°C overnight. Thereafter, the beads are again washed 5 times with PBS and incubated with 1M ethanola ine, 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 a-galactosidase, although the label per se is not critical and the method is fully suitable for radioim unoassay 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 E ab' °" IgG coupled beads (50 μl) containing equal number of antibody combining sites were incubated with IgG Anti-Strep A- -galactosidase conjugate 5 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 o-galactosidase, o-ni rophenyl-galactopyranoside (ONPG), for 1U minutes. The a-galactosidase produces a yellow color when mixed with its substrate. The color formation 0 may be visually detected or quan itatively determined spectro photometrically. Using a Beckman spectrophotomete , 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 5 coupled to them. Thus, it has been shown that the present invention, using F(ub')., 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. 0 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 25 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% a 3 at room temperature for 2 hours, the beads are washed 4 times with PBS and then mixed with PBS 30. containing 2% glutaraldeyhde and 0.02% aN3 for 30 hours at room temperature. The beads are then washed 6 times with PBS andmixed 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 35 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. O PI The beads are then tested for their ability to bind 125ι-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 ££ Biophysics Acta 2:177, 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. The results were truly striking, quite surprising and unpredictable. The beads coupled in the presence of 0.1M N-acetyl-D-glucoβoamine 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 1 )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 complexwould 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 e--galactosidase, to bring about a color development which can be visually observed or measured quantitatively or semi-quantitatively using colorimetric or εpectrophotmetric 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. ø-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, radioi munoasεay (RIA) procedures may be used but present a number of disadvantages, not the least of which is the necessity for handling radioactive reagents andwastes. 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- O PI 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. ø-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 5 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 Λ antibody itself or Staphylococcus 10 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 15 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 20 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 25 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 30 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 35. 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 OMH- lϋ 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-Streptoc ccu≤ 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 reac ng the F( b' ) 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 An i-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 An i-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 _ i l antibody followed by coupling the resulting F(ab' ) 2 -an igen 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' -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. ■" _ .v.?.

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