Journal of Immunological Methods, 134 (1990) 51-59
51
Elsevier JIM 05731
Methods to quantitate human haptoglobin by complexation with hemoglobin S. C e r d a a n d S.K. O h Department of Microbiology, Boston University School of Medicine, Boston, MA 02118, U.S.A.
(Received 30 March 1990, revised received 3 July 1990, accepted 6 July 1990)
Native human haptoglobin isolated from normal human plasma by affinity chromatography on chicken hemoglobin -Sepharose was used as standard antigen. A direct sandwich ELISA for haptoglobin was developed, with human hemoglobin as a capturing agent. The peroxidase activity of the complex was measured as a means of detecting functional haptoglobin. The reactivities of monoclonal antibody vs. polyclonal antisera on the haptoglobin-hemoglobin complex were compared. Adopting a monoclonal antibody, clone 21.7, which is directed to the et chain of haptoglobin, a specific method to quantitate the native haptoglobin which can complex with hemoglobin has been developed. Key words: Haptoglobin; Hemoglobin; Affinity chromatography, Monoclonal antibody; Polyclonal antiserum; ELISA
Introduction Haptoglobin is a well defined plasma protein of a 2 acid glycoprotein (Smithies, 1959), which binds hemoglobin and forms a stoichiometric, practically irreversible non-covalent complex (Putnam, 1975; Hwang et al., 1979). H u m a n haptoglobin exhibits genetic polymorphism by the difference in
Correspondence to: S.K. Oh, Department of Microbiology S-413, Boston University School of Medicine, 80 E. Concord St., Boston, MA 02118, U.S.A. Abbreviations: Hpt, haptoglobin; Hb, hemoglobin; Ab, antibody; BSA, bovine serum albumin; PBS, phosphate-buffered saline; IgG,immunoglobulinG; G, goat; R, rabbit; M, mouse; ELISA, enzyme-linked immunosorbent assay; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; HRP, horseradish peroxidase; ABTS, 2,2'-azinobis(3-ethylbenzthiazoline sulfonic acid); PEG, polyethyleneglycol; DMEM, Dulbecco's modified Eagle's medium; HAT, hypoxanthine, aminopterine and tliymidine; HT, hypoxanthine and thymidine.
the types of light chains it contains (Smithies et al., 1956) and has three main genotypes of Hpl-1, Hp2-2, and Hp2-1. The genotype of H p l - 1 is a tetramer, composed of two a 1 type light chains (Kurosky et al., 1980) and two invariant heavy fl chains. Hp2-2 contains two a 2 type light chains and Hp2-1 has both a~ and a 2 type chains. In the genotypes of 2-1 and 2-2, a and fl subunits form unique patterns of polymers (Wejman et al., 1984), which have been linked together through disulfide bonds (Fuller et al., 1973). The a-fl dimer of hemoglobin binds irreversibly to the fl chain of haptoglobin with an equal binding affinity in all three genotypic forms (Valette et al., 1981). The formation of this complex is thought to prevent iron loss through urinary excretion (Allison, 1958). However, recent studies have shown that haptoglobin has i m m u n o m o d u l a t o r y properties, which may be more of a physiological importance than the conservation of hemoglobin (Oh et al., 1987).
0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
52 The aim of this study was to develop a rapid and quantitative method to detect native human haptoglobin from a mixture of both denatured and native forms of haptoglobin. The denatured haptoglobin refers to the haptoglobin which lost its functionality of binding hemoglobin due to the the disruption of its conformational structure by treatment with urea, or acid, or by multiple freezing and thawing. Quantitation of the complex by means of the putative peroxidase activity (Putnam, 1975) was found to be characteristic of only native h a p t o g l o b i n and not with the d e n a t u r e d haptoglobin. In this study, we also took advantage of the specificity of our monoclonal antibody, clone 21.7 which is directed to the a chain of human haptoglobin to develop a new method to quantitate haptoglobin by its complexation with hemoglobin. Quantitation of this complex using monoclonal antibody was compared with the conventional ELISA using polyclonal antisera.
Materials and methods
Isolation of human haptoglobin H u m a n haptoglobin was isolated according to the method of Rademacher and Steele (1987). Briefly described, human plasma was fractionated by affinity chromatography on chicken cyanomethemoglobin-Sepharose and eluted with 8 M urea, collecting the eluates in fractions to separate the active haptoglobin from its denatured form.
The urea-free, active haptoglobin fractions were then further purified by lectin affinity chromatography on Affi-Gel ConA to remove the non-glycoproteins, and the bound haptoglobin was eluted with 0.5 M glucose. Further purification was achieved by negative immunoadsorption chromatography on anti-chicken hemoglobin-bound protein A-Sepharose and the chicken hemoglobinhaptoglobin complex was removed. The material thus purified, was also characterized by SDSP A G E (Laemmli, 1970) for its molecular identification and, functional assay for hemoglobin binding property of haptoglobin was performed by ELISA (Table I).
Biochemicals and immunochemicals Purified human hemoglobin as well as the polyclonal antisera to human haptoglobin or hemoglobin were obtained from Sigma Chemical (St. Louis, MO). G o a t anti-rabbit I g G (H + L) conjugated with horseradish peroxidase was from BioRad Laboratories (Richmond, CA). 2,2'-Azinobis (3-ethylbenzthiazoline sulfonic acid) was also purchased from Sigma (A-1888) and was used as a chromogenic substrate for the peroxidase enzyme. Purified human cq acid glycoprotein (AGP) was a gift from John Walker from Natick A r m y Research and Development Center (Natick, MA). All other chemicals were reagent grade chemicals from Fisher Scientific Co. (Fair Lawn, N J).
Preparation of monoclonal antibody to haptoglobin Monoclonal antibodies were prepared by a cell fusion procedure as described by Gefter et al.
TABLE I SUMMARY OF ELISA METHODS USED ELISA method 1 2 3 4 5 6 7
Coating
Analyte
Hpt. Goat-anti-Hpt Goat-anti-Hpt Hb. Hpt. HPt. Hb.
21.7 Hpt. Hpt. Hpt. Hb Hb Hpt.
Detecting Ab Goat-antiM-HRP R-antiHpt-HRP 21.7 21.7 R-antiHb.
2nd Ab
Substrate
G-antiM-HRP G-antiM-HRP G-antiR-HRP -
ABTS ABTS ABTS ABTS ABTS ABTS ABTS
Key: Hpt, haptoglobin; Hb, hemoglobin; G, goat; M, mouse; R, rabbit; Ab, antibody; HRP, horseradish peroxidase; ABTS, 2,2'-azinobis(3-ethylbenzthiazoline sulfonic acid).
53 (1977). Briefly, ( B A L B / c x A / J ) F 1 mice were immunized with 50 /tg of suppressive E receptor (SER) haptoglobin, a polymeric variant of haptoglobin (Oh et al., 1989). Spleens were removed 3 days after the third immunization and 5 x 106/i111 cell suspensions depleted of red blood cells were mixed with 5 × 106/ml of S P 2 / 0 myeloma cells. Centrifugation and washing in serum-flee D M E M followed. Pellets were then resuspended in 30% (v/v) of polyethylene glycol (PEG) 1500 and cultured for 24 h in Dulbecco's modified Eagle's medium (DMEM) containing 20% fetal bovine serum (FBS) in petri dishes. Pelleted cells were resuspended in conditioned medium containing hypoxanthine, aminopterin and thymidine (HAT). Aliquots were cultured in 96 well flat bottom microtiter plates. 7 days later, they were fed with 0.1 ml of conditioned medium containing hypoxanthine and thymidine (HT). Clones were screened by ELISA on haptoglobin coated plates and the positive clones were subcloned by limiting dilution. Further characterization of the monoclonal antibodies was performed using SDS-PAGE analysis followed by Western blotting as described by Towbin et al. (1979).
General ELISA protocol 96 well polyvinyl assay plates (Costar, Cambridge, MA) were coated with 100 /tl of diluted capture antibody (goat anti-human haptoglobin) or hemoglobin at 100/~1 per well to saturate the binding capacity of the wells. Plates were incubated overnight at 37°C and were washed 3 x with phosphate buffered saline (PBS) containing 0.05% Tween 20 (Fisher, Fair Lawn, N J) followed by rinsing 3 x with PBS. Standard proteins were diluted to 1 /~g/ml with PBS and 100 /~1 were added to column 1 of the plate in quadruplicate. Serial dilution was carried out with PBS to generate concentrations ranging from 1000 n g / m l to 1 n g / m l . The last column served as blank. Plates were incubated at 4°C for 1 h, followed by washing and rinsing as above. The primary antibody specific to antigen was added next, i.e., monoclonal antibody, clone 21.7, or rabbit anti-human haptoglobin or rabbit antihuman hemoglobin at their optimum titers. Plates were incubated and washed similarly as above.
When assaying for haptoglobin function, addition of the substrate followed: 100 /xl/well of ABTS (at 0.04 g/l) in 12.5 ml of a 0.1 M sQdium phosphate + 1 mM citrate buffer (pH 4.0) and 1 /~1 of 30% hydrogen peroxide (Fisher, NJ). Peroxidase activity was quantitated by determining the OD of each well at 405 nm using a Titertek Multiskan Plus spectrophotometer (Flow Laboratories, McLean, VA). To assay haptoglobin or hemoglobin's binding affinities, 100 /~1 of the secondary antibody (goat anti-rabbit IgG (H + L) or goat anti-mouse IgG (H + L), both HRP-conjugated were added at appropriate titers, and incubation for 1 h at 4°C followed. Plates were then washed and rinsed as described above. The chromogenic substrate was then added and the plates were read at 405 nm.
SDS-PA GE S D S - P A G E gels were run on purified haptoglobin preparations under reducing conditions using the method of Laemmli (1970). Further characterization of the purified haptoglobin was performed by two dimensional gel electrophoresis as described by O'Farrell (1975). Characterization of antibody was performed by a Western blotting of the SDS-PAGE as described by Towbin et al. (1979).
Results
Determination of specificity of the monoclonal antibody to haptoglobin Normal human plasma haptoglobin was serially diluted and made to react with a polyclonal goat antiserum bound to the solid phase microtiter plate (Table I, method 3). Bound haptoglobin was then detected with the mouse monoclonal antibody, clone 21.7. This clone had previously been shown to react equally well with all three haptoglobin genotypes 1-1, 2-1, and 2-2 (Oh et al., 1989). As shown in the Western blot in Fig. 1, the monoclonal antibody, clone 21.7, specifically recognized the a subunits of the three human plasma haptoglobins separated by SDS-PAGE. The polymeric SER-haptoglobin did not show reactivity with the monoclonal antibody in this figure (lane a), due to the loss during reproduction of the gel.
54
Fig. 1. Western blot of monoclonal antibodies commonly reactive to SER and plasma haptoglobin analyzed on SDS-PAGE. Left and right panels show the Western blot of two monoclonal antibodies, clones CF10C3 and 21.7 directed to haptoglobins. Lanes b, c, d, represent Hpt types 2-2, 2-1 and 1-1. Lane a, represents purified SER. Numbers in the far right indicate the molecular weight standards expressed in kDa. Both monoclonals recognized the a subunits of the all three forms of human plasma haptoglobins.
However, our monoclonal has been shown to react with SER-haptoglobin (Oh et al., 1989).
Haptoglobin identity assays. The immunochemical identity of haptoglobin obtained b y affinity c h r o m a t o g r a p h y on chicken cyanomethemoglobin-Sepharose was confirmed by the conventional E L I S A using the commercial haptoglobin standard (Sigma Chemical) and the monoclonal antibody, clone 21.7 or rabbit-antih u m a n haptoglobin as detecting antibodies (Table I, methods 2 and 3). (results not shown). Further characterization of the purified haptoglobin was performed by two-dimensional gel electrophoresis (Fig. 2). The molecular weight standards shown on the left were loaded in a parallel gel run along
with the purified haptoglobin in the second dimension in S D S - P A G E gel. As shown in arrows on the right Hpt2-2 consisted of its fl and ot2 chains at 42 and 21 k D a respectively (the a chain exhibiting an anomalous electrophoretic mobility).
Development of an ELISA method to quantitate haptoglobin by its ability to complex with hemoglobin H a p t o g l o b i n has the capacity to bind b o t h the a and fl chains of hemoglobin through its/3 chain, giving a stoichiometric complex, which is virtually irreversible (Valette et al., 1981). We have develo p e d a new E L I S A m e t h o d to quantitate haptoglobin in serum samples which can bind and form stable complexes with hemoglobin. As de-
55
97,4 ~ 68 43 ¢--
25.7~
18.4 14.3
51
Elsevier JIM 05731
Methods to quantitate human haptoglobin by complexation with hemoglobin S. C e r d a a n d S.K. O h Department of Microbiology, Boston University School of Medicine, Boston, MA 02118, U.S.A.
(Received 30 March 1990, revised received 3 July 1990, accepted 6 July 1990)
Native human haptoglobin isolated from normal human plasma by affinity chromatography on chicken hemoglobin -Sepharose was used as standard antigen. A direct sandwich ELISA for haptoglobin was developed, with human hemoglobin as a capturing agent. The peroxidase activity of the complex was measured as a means of detecting functional haptoglobin. The reactivities of monoclonal antibody vs. polyclonal antisera on the haptoglobin-hemoglobin complex were compared. Adopting a monoclonal antibody, clone 21.7, which is directed to the et chain of haptoglobin, a specific method to quantitate the native haptoglobin which can complex with hemoglobin has been developed. Key words: Haptoglobin; Hemoglobin; Affinity chromatography, Monoclonal antibody; Polyclonal antiserum; ELISA
Introduction Haptoglobin is a well defined plasma protein of a 2 acid glycoprotein (Smithies, 1959), which binds hemoglobin and forms a stoichiometric, practically irreversible non-covalent complex (Putnam, 1975; Hwang et al., 1979). H u m a n haptoglobin exhibits genetic polymorphism by the difference in
Correspondence to: S.K. Oh, Department of Microbiology S-413, Boston University School of Medicine, 80 E. Concord St., Boston, MA 02118, U.S.A. Abbreviations: Hpt, haptoglobin; Hb, hemoglobin; Ab, antibody; BSA, bovine serum albumin; PBS, phosphate-buffered saline; IgG,immunoglobulinG; G, goat; R, rabbit; M, mouse; ELISA, enzyme-linked immunosorbent assay; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; HRP, horseradish peroxidase; ABTS, 2,2'-azinobis(3-ethylbenzthiazoline sulfonic acid); PEG, polyethyleneglycol; DMEM, Dulbecco's modified Eagle's medium; HAT, hypoxanthine, aminopterine and tliymidine; HT, hypoxanthine and thymidine.
the types of light chains it contains (Smithies et al., 1956) and has three main genotypes of Hpl-1, Hp2-2, and Hp2-1. The genotype of H p l - 1 is a tetramer, composed of two a 1 type light chains (Kurosky et al., 1980) and two invariant heavy fl chains. Hp2-2 contains two a 2 type light chains and Hp2-1 has both a~ and a 2 type chains. In the genotypes of 2-1 and 2-2, a and fl subunits form unique patterns of polymers (Wejman et al., 1984), which have been linked together through disulfide bonds (Fuller et al., 1973). The a-fl dimer of hemoglobin binds irreversibly to the fl chain of haptoglobin with an equal binding affinity in all three genotypic forms (Valette et al., 1981). The formation of this complex is thought to prevent iron loss through urinary excretion (Allison, 1958). However, recent studies have shown that haptoglobin has i m m u n o m o d u l a t o r y properties, which may be more of a physiological importance than the conservation of hemoglobin (Oh et al., 1987).
0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
52 The aim of this study was to develop a rapid and quantitative method to detect native human haptoglobin from a mixture of both denatured and native forms of haptoglobin. The denatured haptoglobin refers to the haptoglobin which lost its functionality of binding hemoglobin due to the the disruption of its conformational structure by treatment with urea, or acid, or by multiple freezing and thawing. Quantitation of the complex by means of the putative peroxidase activity (Putnam, 1975) was found to be characteristic of only native h a p t o g l o b i n and not with the d e n a t u r e d haptoglobin. In this study, we also took advantage of the specificity of our monoclonal antibody, clone 21.7 which is directed to the a chain of human haptoglobin to develop a new method to quantitate haptoglobin by its complexation with hemoglobin. Quantitation of this complex using monoclonal antibody was compared with the conventional ELISA using polyclonal antisera.
Materials and methods
Isolation of human haptoglobin H u m a n haptoglobin was isolated according to the method of Rademacher and Steele (1987). Briefly described, human plasma was fractionated by affinity chromatography on chicken cyanomethemoglobin-Sepharose and eluted with 8 M urea, collecting the eluates in fractions to separate the active haptoglobin from its denatured form.
The urea-free, active haptoglobin fractions were then further purified by lectin affinity chromatography on Affi-Gel ConA to remove the non-glycoproteins, and the bound haptoglobin was eluted with 0.5 M glucose. Further purification was achieved by negative immunoadsorption chromatography on anti-chicken hemoglobin-bound protein A-Sepharose and the chicken hemoglobinhaptoglobin complex was removed. The material thus purified, was also characterized by SDSP A G E (Laemmli, 1970) for its molecular identification and, functional assay for hemoglobin binding property of haptoglobin was performed by ELISA (Table I).
Biochemicals and immunochemicals Purified human hemoglobin as well as the polyclonal antisera to human haptoglobin or hemoglobin were obtained from Sigma Chemical (St. Louis, MO). G o a t anti-rabbit I g G (H + L) conjugated with horseradish peroxidase was from BioRad Laboratories (Richmond, CA). 2,2'-Azinobis (3-ethylbenzthiazoline sulfonic acid) was also purchased from Sigma (A-1888) and was used as a chromogenic substrate for the peroxidase enzyme. Purified human cq acid glycoprotein (AGP) was a gift from John Walker from Natick A r m y Research and Development Center (Natick, MA). All other chemicals were reagent grade chemicals from Fisher Scientific Co. (Fair Lawn, N J).
Preparation of monoclonal antibody to haptoglobin Monoclonal antibodies were prepared by a cell fusion procedure as described by Gefter et al.
TABLE I SUMMARY OF ELISA METHODS USED ELISA method 1 2 3 4 5 6 7
Coating
Analyte
Hpt. Goat-anti-Hpt Goat-anti-Hpt Hb. Hpt. HPt. Hb.
21.7 Hpt. Hpt. Hpt. Hb Hb Hpt.
Detecting Ab Goat-antiM-HRP R-antiHpt-HRP 21.7 21.7 R-antiHb.
2nd Ab
Substrate
G-antiM-HRP G-antiM-HRP G-antiR-HRP -
ABTS ABTS ABTS ABTS ABTS ABTS ABTS
Key: Hpt, haptoglobin; Hb, hemoglobin; G, goat; M, mouse; R, rabbit; Ab, antibody; HRP, horseradish peroxidase; ABTS, 2,2'-azinobis(3-ethylbenzthiazoline sulfonic acid).
53 (1977). Briefly, ( B A L B / c x A / J ) F 1 mice were immunized with 50 /tg of suppressive E receptor (SER) haptoglobin, a polymeric variant of haptoglobin (Oh et al., 1989). Spleens were removed 3 days after the third immunization and 5 x 106/i111 cell suspensions depleted of red blood cells were mixed with 5 × 106/ml of S P 2 / 0 myeloma cells. Centrifugation and washing in serum-flee D M E M followed. Pellets were then resuspended in 30% (v/v) of polyethylene glycol (PEG) 1500 and cultured for 24 h in Dulbecco's modified Eagle's medium (DMEM) containing 20% fetal bovine serum (FBS) in petri dishes. Pelleted cells were resuspended in conditioned medium containing hypoxanthine, aminopterin and thymidine (HAT). Aliquots were cultured in 96 well flat bottom microtiter plates. 7 days later, they were fed with 0.1 ml of conditioned medium containing hypoxanthine and thymidine (HT). Clones were screened by ELISA on haptoglobin coated plates and the positive clones were subcloned by limiting dilution. Further characterization of the monoclonal antibodies was performed using SDS-PAGE analysis followed by Western blotting as described by Towbin et al. (1979).
General ELISA protocol 96 well polyvinyl assay plates (Costar, Cambridge, MA) were coated with 100 /tl of diluted capture antibody (goat anti-human haptoglobin) or hemoglobin at 100/~1 per well to saturate the binding capacity of the wells. Plates were incubated overnight at 37°C and were washed 3 x with phosphate buffered saline (PBS) containing 0.05% Tween 20 (Fisher, Fair Lawn, N J) followed by rinsing 3 x with PBS. Standard proteins were diluted to 1 /~g/ml with PBS and 100 /~1 were added to column 1 of the plate in quadruplicate. Serial dilution was carried out with PBS to generate concentrations ranging from 1000 n g / m l to 1 n g / m l . The last column served as blank. Plates were incubated at 4°C for 1 h, followed by washing and rinsing as above. The primary antibody specific to antigen was added next, i.e., monoclonal antibody, clone 21.7, or rabbit anti-human haptoglobin or rabbit antihuman hemoglobin at their optimum titers. Plates were incubated and washed similarly as above.
When assaying for haptoglobin function, addition of the substrate followed: 100 /xl/well of ABTS (at 0.04 g/l) in 12.5 ml of a 0.1 M sQdium phosphate + 1 mM citrate buffer (pH 4.0) and 1 /~1 of 30% hydrogen peroxide (Fisher, NJ). Peroxidase activity was quantitated by determining the OD of each well at 405 nm using a Titertek Multiskan Plus spectrophotometer (Flow Laboratories, McLean, VA). To assay haptoglobin or hemoglobin's binding affinities, 100 /~1 of the secondary antibody (goat anti-rabbit IgG (H + L) or goat anti-mouse IgG (H + L), both HRP-conjugated were added at appropriate titers, and incubation for 1 h at 4°C followed. Plates were then washed and rinsed as described above. The chromogenic substrate was then added and the plates were read at 405 nm.
SDS-PA GE S D S - P A G E gels were run on purified haptoglobin preparations under reducing conditions using the method of Laemmli (1970). Further characterization of the purified haptoglobin was performed by two dimensional gel electrophoresis as described by O'Farrell (1975). Characterization of antibody was performed by a Western blotting of the SDS-PAGE as described by Towbin et al. (1979).
Results
Determination of specificity of the monoclonal antibody to haptoglobin Normal human plasma haptoglobin was serially diluted and made to react with a polyclonal goat antiserum bound to the solid phase microtiter plate (Table I, method 3). Bound haptoglobin was then detected with the mouse monoclonal antibody, clone 21.7. This clone had previously been shown to react equally well with all three haptoglobin genotypes 1-1, 2-1, and 2-2 (Oh et al., 1989). As shown in the Western blot in Fig. 1, the monoclonal antibody, clone 21.7, specifically recognized the a subunits of the three human plasma haptoglobins separated by SDS-PAGE. The polymeric SER-haptoglobin did not show reactivity with the monoclonal antibody in this figure (lane a), due to the loss during reproduction of the gel.
54
Fig. 1. Western blot of monoclonal antibodies commonly reactive to SER and plasma haptoglobin analyzed on SDS-PAGE. Left and right panels show the Western blot of two monoclonal antibodies, clones CF10C3 and 21.7 directed to haptoglobins. Lanes b, c, d, represent Hpt types 2-2, 2-1 and 1-1. Lane a, represents purified SER. Numbers in the far right indicate the molecular weight standards expressed in kDa. Both monoclonals recognized the a subunits of the all three forms of human plasma haptoglobins.
However, our monoclonal has been shown to react with SER-haptoglobin (Oh et al., 1989).
Haptoglobin identity assays. The immunochemical identity of haptoglobin obtained b y affinity c h r o m a t o g r a p h y on chicken cyanomethemoglobin-Sepharose was confirmed by the conventional E L I S A using the commercial haptoglobin standard (Sigma Chemical) and the monoclonal antibody, clone 21.7 or rabbit-antih u m a n haptoglobin as detecting antibodies (Table I, methods 2 and 3). (results not shown). Further characterization of the purified haptoglobin was performed by two-dimensional gel electrophoresis (Fig. 2). The molecular weight standards shown on the left were loaded in a parallel gel run along
with the purified haptoglobin in the second dimension in S D S - P A G E gel. As shown in arrows on the right Hpt2-2 consisted of its fl and ot2 chains at 42 and 21 k D a respectively (the a chain exhibiting an anomalous electrophoretic mobility).
Development of an ELISA method to quantitate haptoglobin by its ability to complex with hemoglobin H a p t o g l o b i n has the capacity to bind b o t h the a and fl chains of hemoglobin through its/3 chain, giving a stoichiometric complex, which is virtually irreversible (Valette et al., 1981). We have develo p e d a new E L I S A m e t h o d to quantitate haptoglobin in serum samples which can bind and form stable complexes with hemoglobin. As de-
55
97,4 ~ 68 43 ¢--
25.7~
18.4 14.3
