Effect of chemical modification on the cryoprecipitation of monoclonal human cryoglobulin M

The effect of the chemical modification of lysine, histidine, arginine, tyrosine, tryptophan residues and carboxylic groups on the cryoproperties of monoclonal human cryoglobulin M has been studied. The modification of 35-40 lysine residues and that of 42-45 arginine residues in the molecule of cryo-IgM has been shown to result in practically complete inhibition of the cryoprecipitation. The same effect is observed on the modification of 60 histidine residues per molecule and on modification of 50 or 51 carboxylic groups. At the same time the modification of practically all the reagent-exposed tryptophan (10 residues per molecule) and tyrosine residues (55 residues per molecule) does not lead to any noticeable decrease in the cryoprecipitation. The conformations of the modified and native proteins are identical according to the circular dichroism data.     

Spectroscopic and kinetic analysis of a monoclonal IgG cryoglobulin. Effect of mild reduction on cryoprecipitation.

The precipitation of a monoclonal IgG2 crystalline cryoglobulin (WEB) is shown to be highly dependent on temperature and concentration. Below a critical concentration of 0.6 mg/mL there is no cryoprecipitation. The kinetics of the aggregation exhibits a concentration-dependent lag time. This evidence suggests that a nucleation event is important in the precipitation. Circular dichroism (CD) was used to investigate the conformational properties of the protein. At a low concentration (0.12 or 0.15 mg/mL), no detectable spectral changes in the far- and near-UV range were noted between 40 and 3 degrees C. However, at higher concentrations (1.21 mg/mL), a small and rapid CD change was observed in the 250-280-nm region at 3 degrees C. This indicates an intermolecular interaction that precedes the precipitation. Cryoprecipitation of WEB was also shown to be dependent on maintenance of intact interchain disulfide bonds. Only one or two interchain disulfides need be cleaved to abolish cryocrystallization and to significantly diminish the CD change at 3 degrees C. The evidence is consistent with the formation of an initial intermediate that involves interactions near the disulfide bonds in the hinge region of the cryoimmunoglobulin. In this model, cleavage of these disulfides prevents this interaction and abolishes cryoprecipitation.     

Immunochemical characterization of human plasma fibronectin.

Human plasma fibronectin has been purified by a non-denaturing affinity chromatography procedure [Vuento & Vaheri, (1979) Biochem.J. 183, 331--337], and antisera have been raised by immunizing rabbits with the native protein. The antisera reacted strongly with native fibronectin, but only weakly with reduced and alkylated fibronectin or with heat-denaturated fibronectin. Denaturation also affected the haemagglutinating and gelatin-binding activities of fibronectin and increased its susceptibility to proteolytic degradation. The antisera reacted with fragments of fibronectin obtained by proteolysis with plasmin. Large fragments (mol.wt. 180000--200000), lacking the region harbouring the interchain disulphide bridges but containing the sites responsible for gelatin-binding and haemagglutinating activity, showed as intense a reaction with the antisera as intact fibronectin. Smaller peptides showed a weaker reaction. All fragments tested showed sensitivity to denaturation in their reaction with the antisera. The results were interpreted as showing that: (1) native fibronectin has an ordered conformation that is easily perturbed by denaturation; (2) most of the antigenic determinants of the protein are dependent on conformation; (3) the region of the fibronectin molecule containing the interchain disulphide bridges has only few antigenic determinants; and (4) covalent interaction of the two subunits does not contribute to the antigenic structure recognized by rabbit antisera. The observed correlation between the antigenic activity and a structural and functional intactness of fibronectin suggests that the antibodies to native fibronectin could be used as a conformational probe in studies on this protein.     

Studies on Mv red cells. II. Immunochemical investigations

The properties of the Mv antigen, a low incidence receptor of the MNSs blood group system, were investigated by serological tests with protease treated red cells and inhibition assays with glycoproteins or peptides from normal and Mv erythrocytes. Our data demonstrate that the Mv receptor represents an allelomorphic form of the 'N' antigen on the Ss sialoglycoprotein, rather than variant of the M receptor on the MN sialoglycoprotein. Anti-Mv plus -N (serum Arm.) reacts with the N, 'N' and Mv antigens, whereas anti-Mv (serum Arch.) is specifically directed against the latter receptor.     

Conformational stability of a human cryoglobulin

Studies on a single component human cryoimmunoglobulin (cryo-IgG) (gamma 1 : lambda, Gm 4) were undertaken to gain a better understanding of the conformational stability of macromolecular interfaces essential for self-association of cryo-IgG leading to the formation of visible gel mass. Changes in the gross and localized conformation of cryo-IgG and a monoclonal IgG (gamma 1 : lambda, Gm 4) isolated from a myeloma patient (Hy) (Hy IgG) (gamma 1 : lambda, Gm 4) in alkaline media were determined by analytical ultracentrifugation, fluorescence characteristics, tyrosine ionization and H+ titration. Ultracentrifugal studies revealed that major transition in gross conformation took place at pH 11.4 for cryo-IgG and pH 11.7 for Hy IgG, whereby the number of charges and tyrosine residues exposed to aqueous environment was 110 and 26 for cryo-IgG, and 111 and 48 for Hy IgG, respectively. Beyond this transition pH fragmentation of both the proteins occurred and cryo-IgG lost its capacity for gel formation. Self-association of cryo-IgG was observed upto pH 11.4 in decreasing order with increase in denaturation pH. Cryo-IgG renatured from exposure to higher alkaline pH upto pH 11.4, showed the capability for forming gel, in spite of the irreversible local conformational changes as established by direct and reverse fluorimetric titration and tyrosine ionization studies. Cryo-IgG could be maintained in the optically clear sol phase at pH 10.5, at which pH 12 out of 62 tyrosine residues became exposed to aqueous media. There are distinct differences in the accessibility of tyrosine residues of cryo-IgG and Hy IgG as reflected in their tyrosine ionization profiles.     

Immunochemical characterization of human lung epoxide hydrolases

Immunochemical techniques were used to investigate the biochemical properties of human lung epoxide hydrolases. Two epoxide hydrolases with different immunoreactive properties were identified. These two epoxide hydrolases were found in both cytosolic and microsomal cell fractions. Immunotitration of enzyme activity showed that enzymes that catalyze the hydration of benzo(a)pyrene 4,5-oxide react with antiserum to rat microsomal epoxide hydrolase; those that hydrate trans-stilbene oxide do not. Immunotitration and Western blot experiments showed that microsomal and cytosolic benzo(a)pyrene 4,5-oxide hydrolases have significant structural homology. Immunohistochemical staining of human lung benzo(a)pyrene 4,5-oxide hydrolase showed that the enzyme is localized primarily in the bronchial epithelium. No cell type-specific localization was observed. An enzyme-linked immunosorbent assay was developed which allows direct quantitation of benzo(a)pyrene 4,5-oxide hydrolase protein. Levels of enzyme protein detected by this assay correlated well with enzyme levels determined by substrate conversion assays.     

Antigenic determinants on human choriogonadotropin alpha-subunit. II. Immunochemical mapping by a monoclonal antipeptide antibody

In order to study antigenic site(s) present in the carboxyl-terminal part of the alpha-subunit of human choriogonadotropin (hCG-alpha), we attempted to produce site-specific antibodies directed against a 34-residue synthetic peptide analogous to region 59-92 of hCG-alpha. From a fusion experiment performed with a mouse injected with hCG-alpha-(59-92)-peptide conjugated to tetanus toxoid as immunogen, we selected a monoclonal antipeptide antibody (designated FA36) which has high binding activity for 125I-hCG-alpha but not for 125I-hCG in a radioimmunoassay. This antibody is of the IgG1 subclass and displays an affinity constant for 125I-hCG-alpha of 3.1 x 10(8) M-1. Hapten inhibition experiments performed by either radioimmunoassay or enzyme-linked immunosorbent assay with synthetic peptides spanning different portions of the region (59-92) demonstrated that the binding site of FA36 resides on (minimally) the six COOH-terminal amino acids of hCG-alpha, namely Cys-Tyr-Tyr-His-Lys-Ser, and that FA36 binds preferentially to peptides containing a carboxyl group on the COOH-terminal residue. Monoclonal immunoradiometric assays were established to determine the location of antigenic regions recognized by FA36, by antibody AHT20 (which binds only to hCG-alpha), and by antibody HT13 (which binds to both hCG and hCG-alpha). FA36 has the capacity to bind to hCG-alpha bound to either AHT20 or HT13, demonstrating that both AHT20 and HT13 antibodies are directed against antigenic regions distinct from the epitope of FA36. Monoclonal immunoradiometric assays were also carried out to study the binding of FA36 to hCG, the ovine and equine lutropin alpha-subunit, or hCG-alpha minus the 5 COOH-terminal residues (hCG-alpha core). Whereas significant binding of 125I-FA36 was observed with the ovine lutropin alpha-subunit, no binding was found with the equine lutropin alpha-subunit. As expected, FA36 did not bind to hCG-alpha core. Binding was also not detected with hCG, confirming that FA36 is specific for free hCG-alpha and that the COOH-terminal part of hCG-alpha is either weakly or (more likely) not at all accessible in the alpha/beta-dimer for antibody binding. Finally, immunoblots performed on hCG-alpha-(59-62)-peptide and various denatured alpha-subunits indicated that, with the exception of the equine lutropin alpha-subunit, FA36 detected various denatured alpha-subunits and particularly the alpha-subunit of carp gonadotropin-thyrotropin. This latter observation suggests a high degree of homology between the COOH-terminal regions of the alpha-subunits of fish gonadotropin and analogous mammalian hormones.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunochemical characterization of a 150 000 dalton human fibroblast surface glycoprotein

The characterization of a human fibroblast surface glycoprotein, visualized by crossed immunoelectrophoresis using rabbit antibodies against whole fibroblasts, is described. The antigen is synthesized by fibroblasts in culture and was localized both intracellularly and at the cell surface. It was highly antigenic and was detected only in human cells of mesenchymal origin.The glycoprotein occurred in two different forms with α₂ and β electrophoretic mobility. The slow migrating amphiphilic β form was localized at the cell surface and showed a single protein band with an apparent molecular weight of 150 000 in SDS-polyacrylamide gel electrophoresis. By external papain treatment of intact viable cells, a water-soluble molecule was released with a reduced molecular weight (140 000) and an increased electrophoretic mobility as compared to the native membrane component. This hydrophilic form was also present intracellularly in fibroblasts not treated with exogeneous proteases. The observation that the detergent-solubilized β form was irreversibly converted to a more anodic form by incubation of whole cell extract at acidic pH, suggested that the intracellular protein represented a lysosomal degradation product of native internalized fibroblast surface glycoprotein.     

Immunochemical characterization of aldo-keto reductases from human tissues

Aldose reductase, aldehyde reductase and carbonyl reductase constitute a family of monomeric NADPH-dependent oxidoreductases with similar physical and chemical properties. Characterization of the enzymes from human tissues by immunotitration and an enzyme immunoassay indicated that, despite their apparent likeness, the three reductases do not cross-react immunochemically.     

Immunochemical and functional characterization of a polyclonal antibody to human sperm antigen

A polyclonal antibody was raised against a 16 kDa human sperm protein identified by a monoclonal antibody to human sperm. The antibody showed significant reactivity with mouse spermatozoa as seen by ELISA. Immunohistochemical analysis showed that the antibody reacted with antigens from mouse testis, prostate as well as seminal vesicle. In both mouse and human testis the antibody localized antigens in round as well as elongated spermatids and mature spermatozoa. By SDS-PAGE and Western blot analysis the antibody reacted with a 16 kDa protein in the testis and seminal vesicle, whereas in the prostate it identified two proteins, one at 20 kDa and another at 25 kDa. Immunofluorescent localization by the antibody showed reactivity with acrosomal and/equatorial and midpiece region of human spermatozoa. The antibody showed extensive agglutination both in mouse and human spermatozoa. The results indicate that the antigen may be a conserved antigen. Cross reactivity of the antibody with mouse spermatozoa enabled us to carry out antifertility trials. Passive immunization of female mice with this antibody caused 67% reduction in fertility. It is likely that the antifertility effect could be partly due to agglutinating nature of the antibody which may have caused inhibition of all processes that depend on forward motility such as cervical mucus penetration and possibly preventing sperm egg interaction. Such well characterized and functionally relevant antibodies will enable to identify sperm antigens relevant for fertility. Identification of such antigens may also help in diagnosis of immuno infertility.     

Immunochemical characterization of a haemagglutinating antigen of Arcobacter spp.

Abstract The Arcobacter haemagglutinin has been identified by Western immunoblot to be an immunogenic protein of about 20 kDa. The haemagglutinating activity is sensitive to proteolytic enzyme digestion and heat treatment of 80 °C and above. The Arcobacter haemagglutinin is possibly a lectin-like molecule binding to erythrocytes via a glycan receptor containing d-galactose as part of its structure.     

Immunochemical characterization of human red cell acid phosphatase isozymes

An immunological study was performed on human red cell acid phosphatase (ACP1) isozymes encoded by different alleles, each of which is expressed as an electrophoretically fast (f) isozyme and a slow (s) isozyme. These isozymes reacted as two immunochemically different groups. Allele-specific reactions were not detected between either the f isozymes or the s isozymes. Quantitation of ACP1 isozymes in red cells by crossed immunoelectrophoresis revealed a phenotype-dependent variation in the concentration of isozyme protein. A simple gene dosage effect was indicated and the ordering of the ACP1 alleles (ACP1*A < ACP1*B < ACP1*C < ACP1*E) was identical to that found for enzyme activity levels. Also, an allele effect on the proportion between s and f isozymes (s/f) was observed; the ordering here was ACP1* B < ACP1*A < ACP1*, which is the same as that reported for the susceptibility to modulation with purines. These variations in isozyme protein levels appear to account for the phenotypic differences in the intensity of the isozyme bands, when activity-stained after electrophoresis, and in the red cell enzyme activity levels. Investigation of two carriers of a Null allele showed no evidence of an aberrant protein product, and half-normal concentrations of enzyme protein were observed in the red cells of these individuals.