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Preparation of monoclonal antibodies to discriminate T-cell defined epitopes of HLA-DR4
Abstracts
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THE i CREG COMPRISES MULTIPLE SETS OF PUBLIC EPITOPES. DR Lee, KM Amszynski, TD Lee, RA Bray, JE Bray, and GE Rodey, Dept Pathology and Winship Cancer Ctr, Emory University, and The American Red Cross, Bethesda, Md. Recurring serological crossreactivity patterns are used to define major crossreactive groups or CREGs. The i CREG (AI, 36, 3, 9, i0, II, 28, 29, 30, 31, 32, and 33) is among the most complex. We studied 9 broadly reactive i CREG alloantisera, using extensive differential adsorptions of sera, analysis of eluates, and microadsorption of eluates with cells bearing each of the defined specificities. The purpose of the study was to determine how many discrete, operationally monospecific antibodies could be identified within the alloantisera. Adsorptions were performed using well-characterized B cell lines. Acid eluates were obtained from the adsorbing cells for reanalysis and further microadsorptions. Testing of antisera and eluates was performed by two-color fluorescence, using AHG-CDC. 21 separate alloantibodies, found in the 9 sera, defined 7 distinct public epitope patterns shared by: I) AI,36,3,9,10,II,28,29,30,31,32,33 (the full creg); 2) I0,ii; 3) AI,36,10,II; 4) AI,36,3,11; 5) AI,36,9,11; 6) AI,36,10,II,29; 7) A28,10. A9 crossreactivity occurs primarily with AI,3,11, but not AI0. A28 crossreactivity is primarily with AI0 members. Other known CREG patterns include 30,31; and AI0 members with A32 and 33. Thus, there are at least 9 discrete public epitopes within the i CREG, not including the "broad" specificities that include splits (e.g., 9, i0 ,28). Many, but not all of these putative public epitopes correlate with known linear amino acid sequences of the alpha i and 2 heavy chain domains. We conclude that the serological complexity of the i CREG is due the existence of multiple public epitopes that are differentially shared by among members of the i CREG.
PREPARATION OF MONOCLONALANTIBODIES TO DISCRIMINATE T-CELL DEFINED EPITOPES OF HLA-DR4. S. Drover, D. Codner, J. Gamberg, R. Karr, R. Sekaly and W.H. Marshall, Faculty of Medicine, Memorial University of Newfoundland, St. John's, Newfoundland, Canada. The HLA-DR4 specificity has been s p l i t by T-cell cloning and DNA typing into several different subtypes, which vary by only one to three amino acid residues. Several groups have shown that susceptibility to Rheumatoid A r t h r i t i s is associated with two of the subtypes (Dw4 and Dwl4) and with a different serotype, DRI, which has sequence homology to Dw14 in the third hypervariable region (HVR). The purpose of this work was to produce monoclonal antibodies (moab) to differentiate serologically the various subtypes of DR4 and to look for conformationally-equivalent epitopes shared by DR4 and non-DR4 molecules. C3H mice were immunized with DR4-expressing transfectants and subsequent specificity analysis of the resulting moabs was done on class ll-expressing transfectants and homozygous B-cell lines. The panel of anti-DR4 antibodies characterized using the transfectants as target cells include the following s p e c i f i c i t i e s : (I) w4 + w14; (2) w4 + w14 + w13; (3) w4 + wI4 + w13 + w]5; (4) w4 + w14 • w13 + w10; (5) w4 + w14 + w13 + w15 + wIO. However the patterns for the f i r s t three moabs were longer when the target cells were B-cell lines. In addition to the overlapping DR4 subtype s p e c i f i c i t i e s some moabs recognize epitopes on DR molecules such as DRI and DR14 (subtype w16) which have sequence homology in the third HVR with one or more of the DR4 subtypes. They also react with some DR2 molecules which do not have equivalent sequence homology. This is similar to the phenomena observed using T-cell clones in which DR4 restricted clones crossreact with DR molecules that in most cases, but not always, have shared sequences in the third HVR.
#I 1.1
#2 1.3
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THE i CREG COMPRISES MULTIPLE SETS OF PUBLIC EPITOPES. DR Lee, KM Amszynski, TD Lee, RA Bray, JE Bray, and GE Rodey, Dept Pathology and Winship Cancer Ctr, Emory University, and The American Red Cross, Bethesda, Md. Recurring serological crossreactivity patterns are used to define major crossreactive groups or CREGs. The i CREG (AI, 36, 3, 9, i0, II, 28, 29, 30, 31, 32, and 33) is among the most complex. We studied 9 broadly reactive i CREG alloantisera, using extensive differential adsorptions of sera, analysis of eluates, and microadsorption of eluates with cells bearing each of the defined specificities. The purpose of the study was to determine how many discrete, operationally monospecific antibodies could be identified within the alloantisera. Adsorptions were performed using well-characterized B cell lines. Acid eluates were obtained from the adsorbing cells for reanalysis and further microadsorptions. Testing of antisera and eluates was performed by two-color fluorescence, using AHG-CDC. 21 separate alloantibodies, found in the 9 sera, defined 7 distinct public epitope patterns shared by: I) AI,36,3,9,10,II,28,29,30,31,32,33 (the full creg); 2) I0,ii; 3) AI,36,10,II; 4) AI,36,3,11; 5) AI,36,9,11; 6) AI,36,10,II,29; 7) A28,10. A9 crossreactivity occurs primarily with AI,3,11, but not AI0. A28 crossreactivity is primarily with AI0 members. Other known CREG patterns include 30,31; and AI0 members with A32 and 33. Thus, there are at least 9 discrete public epitopes within the i CREG, not including the "broad" specificities that include splits (e.g., 9, i0 ,28). Many, but not all of these putative public epitopes correlate with known linear amino acid sequences of the alpha i and 2 heavy chain domains. We conclude that the serological complexity of the i CREG is due the existence of multiple public epitopes that are differentially shared by among members of the i CREG.
PREPARATION OF MONOCLONALANTIBODIES TO DISCRIMINATE T-CELL DEFINED EPITOPES OF HLA-DR4. S. Drover, D. Codner, J. Gamberg, R. Karr, R. Sekaly and W.H. Marshall, Faculty of Medicine, Memorial University of Newfoundland, St. John's, Newfoundland, Canada. The HLA-DR4 specificity has been s p l i t by T-cell cloning and DNA typing into several different subtypes, which vary by only one to three amino acid residues. Several groups have shown that susceptibility to Rheumatoid A r t h r i t i s is associated with two of the subtypes (Dw4 and Dwl4) and with a different serotype, DRI, which has sequence homology to Dw14 in the third hypervariable region (HVR). The purpose of this work was to produce monoclonal antibodies (moab) to differentiate serologically the various subtypes of DR4 and to look for conformationally-equivalent epitopes shared by DR4 and non-DR4 molecules. C3H mice were immunized with DR4-expressing transfectants and subsequent specificity analysis of the resulting moabs was done on class ll-expressing transfectants and homozygous B-cell lines. The panel of anti-DR4 antibodies characterized using the transfectants as target cells include the following s p e c i f i c i t i e s : (I) w4 + w14; (2) w4 + w14 + w13; (3) w4 + wI4 + w13 + w]5; (4) w4 + w14 • w13 + w10; (5) w4 + w14 + w13 + w15 + wIO. However the patterns for the f i r s t three moabs were longer when the target cells were B-cell lines. In addition to the overlapping DR4 subtype s p e c i f i c i t i e s some moabs recognize epitopes on DR molecules such as DRI and DR14 (subtype w16) which have sequence homology in the third HVR with one or more of the DR4 subtypes. They also react with some DR2 molecules which do not have equivalent sequence homology. This is similar to the phenomena observed using T-cell clones in which DR4 restricted clones crossreact with DR molecules that in most cases, but not always, have shared sequences in the third HVR.