Immunoadsorbent Chromatography Research Articles

Purification of C1 inhibitor A new approach for the isolation of this biologically important plasma protease inhibitor

C1 inhibitor (C1-INH) acts to inhibit active enzymes of both the classical complement and Hageman factor-dependent pathways. Previously reported C1-INH purification procedures were multistep and most have been associated with significant loss in specific functional activity. We have developed a simple chromatographic procedure which yields a pure C1-INH protein from normal human plasma with a specific activity equal to or greater than the starting sample. Briefly, protease inhibitor-treated, pooled human citrated plasma was fractionated with polyethylene glycol (PEG 4000); the supernatant fraction that remained soluble at 16% was obtained. The inhibitor was precipitated with 45% PEG. The resulting precipitate was solubilized and chromatographed on DEAE Sephacel using a linear salt gradient. The eluted fractions containing the C1-INH and other contaminants were pooled and dialyzed against the starting buffer of the next chromatographic step. A unique separation procedure using zinc ion chelate-coupled agarose was employed as the second chromatographic step. The eluted C1-INH, after zinc ion chromatography, displayed a significant enhancement in purity and maintained a specific functional activity twice that of plasma. The final procedure utilized immunoadsorption chromatography using an anti-contaminant column. Under reducing conditions on sodium dodecyl sulfate polyacrylamide gel electrophoresis, the purified C1-INH migrated as a single band with an apparent molecular weight of 90 000–105 000, but under non-reducing conditions, a doublet with apparent molecular weigths of 94 000–100 000 and 85 000–93 000 was seen. C1-INH antigenic concentrations were measured and shown to be correlated in serum, citrate plasma, and EDTA plasma from 16 normal subjects.

Subunit interface of triosephosphate isomerase: site-directed mutagenesis and characterization of the altered enzyme.

We have replaced asparagine residues at the subunit interface of yeast triosephosphate isomerase (TIM) using site-directed mutagenesis in order to elucidate the effects of substitutions on the catalytic activity and conformational stability of the enzyme. The mutant proteins were expressed in a strain of Escherichia coli lacking the bacterial isomerase and purified by ion-exchange and immunoadsorption chromatography. Single replacements of Asn-78 by either Thr or Ile residues had little effect on the enzyme's catalytic efficiency, while the single replacement Asn-78----Asp-78 and the double replacement Asn-14/Asn-78----Thr-14/Ile-78 appreciably lowered kcat for the substrate D-glyceraldehyde 3-phosphate. The isoelectric point of the mutant Asn-78----Asp-78 was equivalent to that of wild-type yeast TIM that had undergone a single, heat-induced deamidation, and this mutant enzyme was less resistant than wild-type TIM to denaturation and inactivation caused by elevated temperature, denaturants, tetrabutylammonium bromide, alkaline pH, and proteases.

Immunoreactive anionic trypsin and anionic elastase in human milk.

Immunoreactive anionic trypsin and anionic elastase have been demonstrated in human milk, by immunodiffusion and immunoelectrophoresis. Cationic trypsin and cationic elastase could not be detected by these methods. The anionic trypsin is probably present in complex with IgA. No benzoyl-DL-arginine-p-nitroanilide (BAPNA) splitting activity was found in human skim milk. Anionic trypsin was isolated by immunoadsorption chromatography. The purified enzyme had a BAPNA-splitting activity.

Similarity among Organ Region Protein Antigens of Pea

Summary Immunodiffusion, two-dimensional crossed rocket immunoelectrophoresis and immunoadsorbent column chromatography were used in an attempt to isolate an organ region-specific antibiody against organ region total protein extracts of pea. Although organ region-specific differences could by detected by immunodiffusion, it was not found possible to isolate an organ region-specific antibody.

Protein X, a proteolysis product of human pancreatic juice. Immunological relationship with trypsinogen 1.

Protein X (PX) previously isolated from human pancreatic juice is an inactive protein of 14 kDa which has been shown to be a degradation product liberated by proteolysis of 19 kDa precursors. Polyclonal antibodies against P19 and PX were prepared in rabbits by injection of the two proteins purified by SDS polyacrylamide gel electrophoresis. These antibodies reacted with a form of trypsin 1 (DFP-trypsin 1) which was shown to be partly proteolysed. Immunological studies were performed with pancreatic juice proteins and partially purified trypsinogen 1 using antibodies directed against PX, P19 and trypsin 1. The results of immunoprecipitation and immunoadsorbent chromatography show that these different antisera recognized a protein of 25 kDa. Immunoblotting has permitted to characterize this protein as a trypsinogen 1-like molecule which would be a form of inert protein generated by uncontrolled trypsinogen activation.

A general method for the isolation of haptoglobin 1-1, 2-1, and 2-2 from human plasma

A general method for the isolation of haptoglobin 1-1, 2-1, and 2-2 from human plasma is described. Plasma is fractionated by affinity chromatography on chicken hemoglobin-Sepharose using the full capacity of the column; then after washing the column thoroughly, haptoglobin is eluted with 8 m urea and the eluate is collected in fractions to separate active and denatured haptoglobin. The urea-free, active fractions of haptoglobin are fractionated by affinity chromatography on Affi-Gel Con A to remove nonglycoproteins, principally apolipoprotein A-I, and the haptoglobin is eluted with 0.5 m glucose. Then the haptoglobin-containing fractions are fractionated by negative immunoadsorption chromatography on anti-chicken hemoglobin-protein A-Sepharose to remove chicken hemoglobin-human haptoglobin complexes. Haptoglobin prepared by this three-step procedure is biologically active and nearly homogeneous. The recovery is approximately 70%, irrespective of phenotype. The procedure can be completed in 3 days. A partial purification of apolipoprotein A-I is obtained simultaneously by this method.

Subunit composition of plasma von Willebrand factor in patients with the myeloproliferative syndrome

In order to evaluate the role of proteolysis in acquired von Willebrand's disease (vWD) associated with the myeloproliferative syndrome, we have determined the relative quantity of von Willebrand factor (vWF) fragments as compared with the intact 225 kDa subunit in four patients. The plasma vWF of each individual lacked large multimers; each had a prolonged bleeding time; and both platelet and leukocyte counts were elevated. Plasma was obtained from blood drawn into 1 mmol/L leupeptin, 6 mmol/L N-ethylmaleimide, and 5 mmol/L EDTA to prevent in vitro proteolysis. vWF was isolated from plasma by immunoadsorbent chromatography, reduced, subjected to SDS-5% polyacrylamide gel electrophoresis, and immunoblotted with a mixture of 55 anti-vWF monoclonal antibodies. In three patients with essential thrombocytosis (ET) the 176 and 140 kDa fragments were increased in proportion to the intact 225 kDa subunit indicating increased proteolysis. Treatment of one ET patient with CCNU (Lomustine) decreased the platelet count and, to a lesser extent, the white blood cell count. This was associated with a correction of the bleeding time, a partial correction of the multimeric abnormality, and a lessening of vWF cleavage. In a patient with polycythemia rubra vera (PRV) the proportion of the 176 kDa fragment was increased to the upper limit of normal but there was no definite evidence of increased proteolysis. These studies provide evidence that proteolysis plays a role in the acquired von Willebrand's disease associated with the myeloproliferative syndrome. However, other mechanisms must also be considered.

Subunit Composition of Plasma von Willebrand Factor in Patients With the Myeloproliferative Syndrome

In order to evaluate the role of proteolysis in acquired von Willebrand's disease (vWD) associated with the myeloproliferative syndrome, we have determined the relative quantity of von Willebrand factor (vWF) fragments as compared with the intact 225 kDa subunit in four patients. The plasma vWF of each individual lacked large multimers; each had a prolonged bleeding time; and both platelet and leukocyte counts were elevated. Plasma was obtained from blood drawn into 1 mmol/L leupeptin, 6 mmol/L N-ethylmaleimide, and 5 mmol/L EDTA to prevent in vitro proteolysis. vWF was isolated from plasma by immunoadsorbent chromatography, reduced, subjected to SDS-5% polyacrylamide gel electrophoresis, and immunoblotted with a mixture of 55 anti-vWF monoclonal antibodies. In three patients with essential thrombocytosis (ET) the 176 and 140 kDa fragments were increased in proportion to the intact 225 kDa subunit indicating increased proteolysis. Treatment of one ET patient with CCNU (Lomustine) decreased the platelet count and, to a lesser extent, the white blood cell count. This was associated with a correction of the bleeding time, a partial correction of the multimeric abnormality, and a lessening of vWF cleavage. In a patient with polycythemia rubra vera (PRV) the proportion of the 176 kDa fragment was increased to the upper limit of normal but there was no definite evidence of increased proteolysis. These studies provide evidence that proteolysis plays a role in the acquired von Willebrand's disease associated with the myeloproliferative syndrome. However, other mechanisms must also be considered.

Association of the Y hapten with glycoproteins, glycolipids and carcinoembryonic antigen in colorectal carcinoma

The expression of the Y hapten (Fucα1→2Galβ1→4[Fucα1→3]GlcNAc −) defined by the monoclonal antibody C14/1/46/10 (abbreviated to C14), is frequently elevated in colo-rectal tumours in comparison to adjacent, apparently normal tissues [2,3]. This determinant was shown to be expressed upon glycoproteins of approximately 200 kDa, which were isolated by immunoadsorbent chromatography from detergent-solubilized subcellular membranes and cytosol from colo-rectal carcinoma. In addition, the Y hapten was expressed upon purified carcinoembryonic antigen (CEA, 180 kDa). However, monoclonal antibodies specifically reactive with CEA or cross-reactive with CEA and NCA-1 (normal cross-reacting antigen) failed to react with the C14 defined antigens of 200 kDa. Further diversity in the expression of molecules bearing the Y hapten was established following its identification in the glycolipid fraction of colo-rectal carcinoma.

Epitope analysis of monoclonal antibody NCRC-11 defined antigen isolated from human ovarian and breast carcinomas

NCRC-11 is an IgM monoclonal antibody which defines an antigen found in most epithelial malignancies. The antigen has previously been shown to be a high mol. wt. glycoprotein (greater than 400,000) and in this study, antigen preparations were isolated by immunoadsorbent chromatography from ovarian mucinous and ovarian serous cyst adenocarcinoma and from breast carcinoma. Other monoclonal antibodies, against products in normal human milk, and antibodies of the Ca series (Bramwell et al., 1985) reacted with all three antigen preparations. Tests involving epitope mapping were performed to probe the relationships of the various epitopes to that defined by the NCRC-11 antibody, and, of note, the three antigen preparations from different tumour sources were remarkably similar with respect to their relative levels of epitope expression and to their topographical distribution of epitopes. The major differences in epitope expression could be attributed to the degree of sialylation in the three antigens. The antigens from ovarian tumours expressed I(Ma) blood group determinants (defined by the antibody LICR-LON-M18) which were partially masked by sialic acid. With NCRC-11 defined antigen from breast carcinoma, this determinant was totally masked by sialic acid although neuraminidase treatment clearly exposed epitopes reactive with M18 antibodies.

The enzyme-linked immunosorbent assay for detecting antibodies against Dirofilaria immitis in dogs

Antisera prepared in mice by injection of antigens from Dirofilaria immitis, Toxocara, canis, Dipylidium canium and Fasciola hepatica and sera from Dirofilaria-infected and non-infected dogs were tested at different dilutions using an enzyme-linked immunosorbent assay (ELISA). For this ELISA, adult D. immitis antigen was fractionated by gel filtration methods and then absorbed with immunoadsorbent-fixed IgG fractions from mouse sera immunized with various parasites. The results indicated satisfactory discrimination between antisera to D. immitis and those to other parasites. A significant ELISA O.D. value was considered to be ⩾ 0.30 with a 1 : 250 dilution of the dog sera. However, the lack of significant differences between the O.D. value of microfilaraemic and amicrofilaraemic infections was observed. These facts suggest that the use of immunoadsorbent chromatography for canine dirofilariasis is especially useful for purifying antigens and eliminating cross-reactions against other parasitic infections, when immunological methods are used for serodiagnosis.

Rat hepatic bile acid sulfotransferase: identification of the catalytic polypeptide and evidence for polymeric forms in female rats.

A monoclonal antibody, PK1B, directed against rat liver bile acid sulfotransferase was used for the purification and characterization of the enzyme. Incubation of rat liver supernatant with the antibody followed by immunoprecipitation with Staphylococcus aureus cells demonstrated that PK1B reacted with 90% of the enzymatic activity present in the liver supernatant from female rats and 40 to 50% of the activity in male liver preparations. Immunoadsorption chromatography with PK1B bound to Sepharose isolated active enzyme which was purified greater than 75-fold. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of this preparation in the presence of 2-mercaptoethanol demonstrated three polypeptides: Mr 29,500; 32,500, and 34,000. Western blot analysis indicated that PK1B recognized an epitope which was found only on the Mr 29,500 polypeptide. Two-dimensional gel electrophoresis associated the enzymatic activity with this Mr 29,500 band. High-pressure liquid chromatographic analysis of immunopurified enzyme defined three distinct, enzymatically active protein populations: I (Mr 400,000 to 170,000); II (Mr 130,000), and III (Mr 43,000). An Mr 29,500 polypeptide was the sole constituent of Peaks I and III and a major constituent of Peak II. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and absence of 2-mercaptoethanol indicated that in Peak II, catalytically active Mr 29,500 protein is associated with the other two polypeptides by disulfide bonds. In contrast, Peak I consists of a polymer of Mr 29,500 polypeptide which is independent of disulfide interaction.

Monoclonal antibodies to bovine UDP-galactosyltransferase. Characterization, cross-reactivity, and utilization as structural probes.

A series of mouse monoclonal antibodies has been developed against a soluble form of bovine UDP-galactose:N-acetylglucosamine galactosyltransferase purified to apparent chemical homogeneity by a combination of affinity and immunoadsorption chromatography. The purified enzyme consists of two molecular mass variants of 42 and 48 kDa. Individual monoclonal antibodies were selected for by their ability to recognize immobilized affinity-purified galactosyltransferase and were not reactive against bovine alpha-lactalbumin and bovine immunoglobulins. Based on competitive binding assays and Western blot analysis with either galactosyltransferase or lactose synthetase (covalently cross-linked alpha-lactalbumin galactosyltransferase), these monoclonal antibodies can be subdivided into four groups. Group A (3 clones) recognize an epitope at or near the alpha-lactalbumin binding site. In addition, this group is cross-reactive with soluble galactosyltransferase from human milk and pleural effusion. Group B (6 clones) and D (1 clone) appear to recognize two different epitopes on the 6-kDa fragment which is released when the 48-kDa galactosyltransferase polypeptide is converted to the 42-kDa form, apparently by proteolysis. Groups A and C (1 clone) recognize epitopes found on both the 48- and 42-kDa polypeptide. Interestingly, immunofluorescence studies indicate that only two monoclonal antibody groups (C and D) are able to decorate membrane-bound galactosyltransferase (Golgi-associated) in formalin-fixed, methanol-, or detergent-permeabilized cells. Thus, these groups of monoclonal antibodies appear to identify four separate structural/functional domains on soluble galactosyltransferase, two of which are not readily accessible for binding in situ.

Characterization and fibrin-binding properties of different molecular forms of pro-urokinase from a monkey kidney cell culture

Culture fluid of a monkey kidney cell culture was harvested every two days, for a two week period, in order to obtain urokinase in the zymogen form. Pro-urokinase was isolated by immunoadsorption chromatography and gel filtered on Sephadex G-150, which resulted in three peaks with pro-urokinase activity. SDS-polyacrylamide gel electrophoresis showed that the first peak contained 55 kd pro-urokinase, aggregated with high molecular weight contaminants, whereas the second and third peaks consisted of almost pure 55 kd and 30 kd pro-urokinase, respectively. The latter form represented a relatively unknown and inactive precursor of low molecular weight urokinase, which was, like 55 kd pro-urokinase, activatable with plasmin. In comparison with tissue-type plasminogen activator, 55 kd and 30 kd pro-urokinase only bound weakly to purified fibrin clots and fibrin-sepharose columns. The extent of binding of the two pro-urokinases and their plasmin-activated forms to fibrin-sepharose decreased in the following order: 55 kd pro-urokinase 30 kd pro-urokinase 55 kd urokinase 30 kd urokinase. These results indicate that the two precursors exhibited stronger binding to fibrin-sepharose than the corresponding active enzymes, and the two 55 kd forms exhibited stronger binding than the corresponding 30 kd forms. This indicates the importance of both the zymogen nature and an intact NH2-terminal part of the molecules for binding to fibrin.

Collagenase synthesis in clonal rabbit odontoblast-like cells (RP cells)

Post-confluent cultures of cloned rabbit odontoblast-like cells (RP: R abbit P ulp cells) produced latent collagenase, which was isolated from serum-containing culture media by heparin-Sepharose affinity chromatography. RP collagenase was purified 4,420-fold from serum-containing medium to a specific activity of 1,313 units/mg with a yield of 14% by heparin-Sepharose affinity chromatography, carboxymethyl-cellulose ion-exchange chromatography, and by immunoadsorption chromatography. The RP cell culture appears to be a suitable model to use for studying collagenase synthesis in mineralized tissue-forming cells and for elucidating the mechanism of collagen degradation in pulp tissue and dentin matrix.

Multiple epitopes on a human breast‐carcinoma‐associated antigen

The monoclonal antibody (MAb) NCRC-11 identifies an epitope expressed variably in human breast cancer. The degree of expression of this epitope in primary operable tumours is closely related to the subsequent clinical course of the disease (Ellis et al., 1985). The target antigen for NCRC-11 was isolated from subcellular membranes of breast carcinomas and purified by immunoadsorbent chromatography. NCRC-11 epitopes were expressed upon a large glycoprotein of more than 400 kd. This material was susceptible to degradation by pronase and papain and contained N-acetylglucosamine, as indicated by its binding to wheat-germ agglutinin. The NCRC-11-defined antigen expressed epitopes for the anti-human milk-fat globule membrane antibodies HMFG-1 and HMFG-2, and other antibodies against epithelial membrane antigens (EMA, LICR-LON-M8). The reactivity of these antibodies with tumour membranes was also similar, but not identical, to that of the NCRC-11 antibody. In competitive binding-inhibition assays, these antibodies partially inhibited the binding of 125I-NCRC-11 antibody to antigen, suggesting that the epitopes involved are topographically closely associated. Sandwich immunoassays demonstrated that NCRC-11 epitopes are likely to represent repeated structures of the NCRC-11 antigen. The findings presented are interpreted as indicating that the NCRC-11 antigen expresses a variety of epitopes which are associated with normal differentiation and malignant change.

Bacteriophage T7 DNA polymerase: cloning and high-level expression.

Phage T7 DNA polymerase consists of a 1:1 complex of the viral T7 gene 5 protein and the host cell thioredoxin. A 3.25-kilobase T7 DNA fragment containing the complete coding sequence of gene 5, and the nearby genes 4.7 and 5.3, was cloned in the BamHI site of the plasmid pBR322. Transformation of the thioredoxin-negative (trxA-) Escherichia coli strain BH215 with the recombinant plasmid pRS101 resulted in large overproduction of gene 5 protein corresponding to a level about 60-fold higher than in T7-infected cells. Transcription of gene 5 probably originates from a previously unknown E. coli RNA polymerase promoter located immediately upstream of the structural gene. Contrary to expectation, pRS101 could be maintained also in E. coli trxA+ cells despite the in vivo formation of active T7 DNA polymerase. However, the expression of gene 5 was lower by a factor of 5-10 than in trxA- cells. Since the plasmid copy number in the two strains was the same, a gene dosage effect can be excluded. The observed difference suggests an autoregulatory interaction of T7 DNA polymerase holoenzyme on the expression of T7 gene 5. The trxA- strain BH215/pRS101 is an excellent source of gene 5 protein and T7 DNA polymerase. After in vitro reconstitution of holoenzyme by addition of excess thioredoxin, highly active T7 DNA polymerase was purified to homogeneity by a simple antithioredoxin immunoadsorbent chromatography technique.

Affinity Chromatography for Purification of Antibodies to the Serotype 15 and 16 Proteins of Meningococci

Immunoadsorbent chromatography is described as a method to purify from rabbit antisera antibodies specific for protein serotype antigens of meningococci, exemplified by antibodies to the serotype 15 and P1.16 proteins, respectively.

Purification of recA-based fusion proteins by immunoadsorbent chromatography. Characterization of a major antigenic determinant of Escherichia coli recA protein.

Monoclonal antibodies to Escherichia coli recA protein were prepared, characterized, and used as affinity reagents for the purification of recA and recA:somatostatin fusion proteins. The monoclonal antibodies recognize an antigenic determinant or determinants located between amino acids 260 and 330 of recA. Addition of a fragment of the recA gene coding for these amino acids to an unrelated gene (beta-galactosidase) allowed the resulting beta-galactosidase fusion protein to be recognized by the recA monoclonal antibodies.

Antigenic and immunogenic characteristics of bovine herpesvirus type-1 glycoproteins GVP [formula omitted] and GVP 6/11a/16, purified by immunoadsorbent chromatography

Glycoproteins GVP 3/9 and GVP 6/11a/16, two of the major glycosylated proteins specified by bovine herpesvirus type-1 (BHV-1), were purified on immunoadsorbents consisting of the appropriate monoclonal antibodies linked to Affigel-10. Each glycoprotein, whether purified from virus-infected cells or from virus, retained antigenic activity and induced high titers of monospecific antibodies in rabbits. These antibodies could neutralize virus and mediate complement-dependent lysis of virus-infected cells. Denatured glycoproteins GVP 3, GVP 6, GVP 11a, and GVP 16, which were purified by immunoadsorbent chromatography, followed by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, also retained antigenicity and immunogenicity, though to a lesser extent than the native glycoproteins. Antibodies induced by GVP 9, GVP 6, and GVP 11a could also neutralize and mediate immune lysis. Even though GVP 16 induced high levels of antibody, these antibodies could not neutralize virus or participate in antibody and complement-mediated cytolysis. These results may suggest that the orientation of GVP 6/11a/16 in the membrane is such that GVP 11a is better exposed on the virion envelope and the cell surface than GVP 16. Cross-reactivity between monospecific antibodies against GVP 3 and GVP 9, as well as GVP 6, GVP 11a, and GVP 16 supported the previously proposed hypothesis that GVP 3 (180K) is a dimer of GVP 9 (91K) and that GVP 6 exists in two forms: one being a 130K polypeptide and the other composed of GVP 11a (74K) and GVP 16 (55K) linked by disulfide bonds. These data suggest that, thus far, either GVP 6/11a/16 or GVP 3 9 may be a potential candidate for a subunit vaccine against BHV-1 infection.