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Heat shock protein detection by flow cytometry
Abstracts
12 5
C-7.2 #237
B-7.3 #238
M O N O C L O N A L A N T I B O D I E S FOR EPITOPE TYPING OF HLA-DP. WH Marshall, S Drover, D Codner, J Ga~berg, D Copp, HW Liu and HB Younghusband, Immunology Laboratory, Faculty of Medicine, Memorial University of Newfoundland, St. John's, Newfoundland, AlE 3V6. We have made a series of mouse monoclonal antibodies for typing polymorphisms of HLA-DP moleoules. Using either L cell transfectants, or B cell lines, or purified DP molecules as immunogens we have produced monoclonal antibodies that are specific for DP molecules. Some of these antibodies are monomorphic for DP or sometimes for DP and DR; others recognize polymorphic variation on the DP molecules. Epitopes have been located (i) at the two ends of the alpha helix formed by the beta chain, (ii) on the alpha chain (not yet localized), (iii) in more complex patterns involving combinations of alpha helix and the floor of the groove (presumably with overlying peptide). Some epitopes are more immunogenic for mouse B cells than others; thus we have duplicates or up to five or six for some reg%ons including antibodies with overlapping but sllghtly variable epitopes. Surprisingly, there have been no antibodies to recognize specifically the PLT-defined specificities DPwl through 6. None of these antibodies is allele-specific and in this they resemble oligonucleotide probes in their reaction patterns, which tend to cover several alleles. The antibodies can be used for epitope typing and these can usually be haplotyped when families are atudiad.
HEAT SHOCK PROTEIN DIETECTION BY FLOW CYTOMETRY. BF" D u f f y , D Osborne, I Karl and R Hotchkiss HI_A Laboratory, Barnes Hospital and Departments of Medicine and Anesthesiology, Washington University School of Medicine, St. Louis, MO Heat shock proteins (HSP) are a family of proteins produced in response to stress, i . e . , heat, cytokines and heavy metals. A flow cytometry assay for detection of HSP 72 in peripheral blood leukocytes (PBL) obtained f r o m healthy and septic patients i s presented and confirmed by Western Blot analysis. HSP 72 was induced in v i t r o by heating PBL suspended in IOX FBS in a 42°C Nater bath for 30 min followed by various recovery periods at 37°C. PBL were also incubated at 3 7 t during the entire induction and recovery periods. Cells were fixed in paraformaldehyde and permeabilized in n-octyl BD-glucopyranoside. Anti-HSP 72 monoclonal antibody or IgG control was added and l a b e l l i n g occurred with g o a t anti-mouse Ig FITC. The 42~ heated lymphocytes showed peak expression (40X-75X) a f t e r 4-8 hr recovery while expression in the 37~ samples remained in a narrow range (lO~-aSX) (n=b). Repeat flow cytometry and Western Blot Analysis confirmed the highest expression of HSP ?a in 4E~C induced samples a f t e r 16 hr recovery period (n=3). Cells from three septic patients were tested in p a r a l l e l with c e l l s from healthy donors for HSP 72 by flow cytometry and Western Blot. The septic patients showed increased expression (a5X-B5X) r e l a t i v e to the control c e l l s (1~-28X). Western Blot also sho~=d a higher HSP 72 concentration in patient than in the control c e l l s . These studies demonstrate heat shock protein induction by heat and sepsis can be detected by flow cytometry and Western Blot analysis.
12 5
C-7.2 #237
B-7.3 #238
M O N O C L O N A L A N T I B O D I E S FOR EPITOPE TYPING OF HLA-DP. WH Marshall, S Drover, D Codner, J Ga~berg, D Copp, HW Liu and HB Younghusband, Immunology Laboratory, Faculty of Medicine, Memorial University of Newfoundland, St. John's, Newfoundland, AlE 3V6. We have made a series of mouse monoclonal antibodies for typing polymorphisms of HLA-DP moleoules. Using either L cell transfectants, or B cell lines, or purified DP molecules as immunogens we have produced monoclonal antibodies that are specific for DP molecules. Some of these antibodies are monomorphic for DP or sometimes for DP and DR; others recognize polymorphic variation on the DP molecules. Epitopes have been located (i) at the two ends of the alpha helix formed by the beta chain, (ii) on the alpha chain (not yet localized), (iii) in more complex patterns involving combinations of alpha helix and the floor of the groove (presumably with overlying peptide). Some epitopes are more immunogenic for mouse B cells than others; thus we have duplicates or up to five or six for some reg%ons including antibodies with overlapping but sllghtly variable epitopes. Surprisingly, there have been no antibodies to recognize specifically the PLT-defined specificities DPwl through 6. None of these antibodies is allele-specific and in this they resemble oligonucleotide probes in their reaction patterns, which tend to cover several alleles. The antibodies can be used for epitope typing and these can usually be haplotyped when families are atudiad.
HEAT SHOCK PROTEIN DIETECTION BY FLOW CYTOMETRY. BF" D u f f y , D Osborne, I Karl and R Hotchkiss HI_A Laboratory, Barnes Hospital and Departments of Medicine and Anesthesiology, Washington University School of Medicine, St. Louis, MO Heat shock proteins (HSP) are a family of proteins produced in response to stress, i . e . , heat, cytokines and heavy metals. A flow cytometry assay for detection of HSP 72 in peripheral blood leukocytes (PBL) obtained f r o m healthy and septic patients i s presented and confirmed by Western Blot analysis. HSP 72 was induced in v i t r o by heating PBL suspended in IOX FBS in a 42°C Nater bath for 30 min followed by various recovery periods at 37°C. PBL were also incubated at 3 7 t during the entire induction and recovery periods. Cells were fixed in paraformaldehyde and permeabilized in n-octyl BD-glucopyranoside. Anti-HSP 72 monoclonal antibody or IgG control was added and l a b e l l i n g occurred with g o a t anti-mouse Ig FITC. The 42~ heated lymphocytes showed peak expression (40X-75X) a f t e r 4-8 hr recovery while expression in the 37~ samples remained in a narrow range (lO~-aSX) (n=b). Repeat flow cytometry and Western Blot Analysis confirmed the highest expression of HSP ?a in 4E~C induced samples a f t e r 16 hr recovery period (n=3). Cells from three septic patients were tested in p a r a l l e l with c e l l s from healthy donors for HSP 72 by flow cytometry and Western Blot. The septic patients showed increased expression (a5X-B5X) r e l a t i v e to the control c e l l s (1~-28X). Western Blot also sho~=d a higher HSP 72 concentration in patient than in the control c e l l s . These studies demonstrate heat shock protein induction by heat and sepsis can be detected by flow cytometry and Western Blot analysis.