Protein A-Sepharose Column Research Articles

Monoclonal antibodies to rabbit sperm autoantigens

Rabbits were immunized with homologous spermatozoa to investigate their autoimmunogenic nature. Major sperm autoantigens that elicit antisperm antibodies were analyzed molecularly by the sodium dodecylsulfate (SDS) gel/protein blot radioimmunobinding method. IgG fractions of the autoimmune sera were purified by a protein A-Sepharose column, immobilized on Sepharose as affinity ligands, and utilized to purify major sperm autoantigens from rabbit testes. The autoimmunogenicity of the purified autoantigens was verified by reimmunizations in rabbits. BALB/c mice were immunized with the affinity-purified autoantigens to raise monoclonal antibodies by modified hybridoma techniques. Following fusions and clonings, we have established more than 100 hybrid cell lines that were shown to secrete antibodies to purified autoantigens and to rabbit sperm. A variety of techniques was employed to characterize these monoclonal antibodies. By the SDS gel/protein blot radioimmunobinding method, some were found to react with unique proteins of rabbit spermatozoa. By indirect immunofluorescent assay, about one third were shown to bind different cytologic regions of spermatozoa from rabbit, man, and/or mouse. Six were found to inhibit rabbit sperm binding to rat ova in vitro. In addition, agglutinating and immobilizing activities of these antibodies on live sperm were observed.

Use of protein A to remove immunoglobulins from serum in hybridoma culture media

The levels of protein A-reactive immunoglobulin (PA-Ig) in foetal bovine serum were measured in commercial batches. For tissue culture media incorporating 10% foetal bovine serum, the levels of bovine PA-Ig were of a similar order to those of mouse monoclonal antibodies produced by hybridomas grown in such media. The equilibrium constants were calculated for the binding to protein A-Sepharose of a number of mouse monoclonal antibodies, and of PA-Ig in foetal bovine serum and normal mouse serum. The average affinity of the mouse PA-Ig was 10 times higher than that of the bovine PA-Ig, suggesting that the two could be separated by affinity chromatography on protein A-Sepharose. The mouse monoclonal antibodies, however, displayed a range of affinity 1.5–100 times that of the bovine PA-Ig, indicating that such separation could not be generally applied. The optimal technique involved removing PA-Ig from bovine serum before its inclusion in the culture medium and then purifying the monoclonal antibody on a second protein A-Sepharose column.

Homocytotropic and heterocytotropic activity of mouse IgG1 antibodies.

Mouse anti-ovalbumin antibodies were isolated by affinity chromatography and fractionated in a column of protein A-Sepharose. Three peaks were obtained: peak I that did not bind to protein A, peak II eluted with 0.5 M NaSCN and peak III eluted with 2.0 M NaSCN. Peak I and peak II contained IgG1 but no detectable IgG2 or IgG3, whereas peak II contained IgG2 and IgG3 but no detectable IgG1. Peak I, but not peak II, showed heat-resistant passive cutaneous anaphylactic (PCA) activity in rats, which was absorbed by anti-IgG1 serum but not by anti-IgE. Both peak I and peak II showed heat-resistant PCA activity in mice, that was absorbed by anti-IgG1. Peak III showed PCA activity in guinea pigs but not in mice or rats. These findings suggest that mouse IgG1 can be divided into 2 populations that differ in physicochemical and biological properties.

Use of a monoclonal antibody to purify the tetrodotoxin binding component from the electroplax of Electrophorus electricus.

The tetrodotoxin binding component of the voltage-sensitive sodium channel from Electrophorus electricus electroplax was purified by using a monoclonal antibody. An impure preparation of tetrodotoxin binding component was mixed with the pure monoclonal antibody, and the immune complex so formed was isolated by affinity chromatography on a protein A-Sepharose column. Excess antibody was removed by ion-exchange chromatography. The purified material has a specific activity of over 1,800 pmol of [3H]tetrodotoxin bound per mg of protein. By assuming that the immune complex has a stoichiometry of 1:1, this specific activity then represents an actual specific activity of 3,000 pmol of [3H]tetrodotoxin bound per mg of eel electroplax protein, or 75% of the theoretical specific activity expected for a pure toxin binding component of Mr 250,000. The peptide composition of the purified material was simple with the predominant species present being of Mr approximately equal to 250,000. Minor components were also present with Mrs of approximately equal to 95,000, approximately equal to 44,000, and approximately equal to 23,000.

Purification of low-abundance messenger RNAs from rat liver by polysome immunoadsorption.

We have purified three low-abundance hepatic mRNAs to near homogeneity by polysome immunoadsorption. The mRNAs coding for the precursor of ornithine transcarbamoylase [carbamoylphosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3], the precursor of the beta-subunit of propionyl-CoA carboxylase [propionyl-CoA:carbon dioxide ligase (ADP-forming), EC 6.4.1.3], and cystathionine beta-synthase [L-serine hydro-lyase (adding homocysteine), EC 4.2.1.22], representing approximately 0.20, 0.02, and 0.015% of total hepatic mRNA, respectively, were purified 450- to 6,300-fold. We used the following steps: interaction of rat liver polysomes with an IgG fraction of monospecific antisera raised against each polypeptide; immobilization of polysome-antibody complexes on a protein A-Sepharose column; removal of the bulk of polysomes by extensive washing; dissociation of ribosomal subunits and elution of specific mRNA with EDTA; and isolation of the eluted mRNA by chromatography on an oligo(dT)-cellulose column. It seems likely that this procedure will permit isolation of other low-abundance mRNAs and subsequent cloning of their respective cDNAs.

Separation of guinea pig IgG subclasses by affinity chromatography on protein A-Sepharose

Guinea pig serum was chromatographed on a column of protein A-Sepharose equilibrated in citrate-phosphate buffer at pH 7.3. The bulk of serum proteins eluted in the starting buffer. Two peaks of protein A-bound serum proteins were eluted by a decreasing pH gradient, a smaller peak centered at pH 4.7 and a larger peak centered at pH 4.3. IgG contained in the peak eluted at pH 4.7 had fast γ immunoelectrophoretic mobility and IgG in the peak eluted at pH 4.3 had slow γ mobility. Antiserum to guinea pig IgG, when absorbed with the pH 4.7 peak, reacted only with the pH 4.3 peak. Antiserum to guinea pig IgG 1, when absorbed with the pH 4.3 peak, reacted only with the pH 4.7 peak. The IgG in the pH 4.7 peak had the immunoelectrophoretic and antigenic characteristics of IgG 1 and the IgG in the pH 4.3 peak had the characteristics of IgG 2. The two subclasses were efficiently separated by pH-dependent affinity chromatography on protein A-Sepharose. The IgG 1 in the pH 4.7 peak was contaminated with 10.8% IgG 2, and the IgG 2 in the pH 4.3 peak was contaminated with 2.7% IgG 1.

Passive allograft enhancement by subclasses of polyclonal antibodies directed toward restricted regions of the major histocompatibility complex.

Two parameters of enhancing major histocompatibility complex (MHC) antibodies, previously separately studied, namely, Ig class and antigen specificity, have been treated simultaneously. In the experimental model used, Sa 1 tumor cells, indigenous of A/J (H-2a) were grafted on CBA (H-2k) or C57BL/Ks (H-2d) mice. Immune sera specific for the H-2 K/D- or H-2 I coded antigens of the A/J haplotype (anti-Kk, or IAk, IBk, IJk, IEk, or ICd, Sd, Gd, or Dd) and their immunoglobulin fractions (separated on protein A-Sepharose columns) were injected either i.v. or locally as mixture with the challenging Sa 1 cells. Within the limits of the studied system, the following results were obtained: (1) Sa 1 cells do possess Iak antigens at their surface detected by C-dependent cytotoxicity; no ICd, Sd, or Gd products were detected. (2) The bulk of enhancing activity is concentrated in IgG1 anti-K/D antibodies (anti-Dd when Sa 1 was grafted on CBA mice and anti-Kk, on C57BL/Ks). (3) Anti-Iak antibodies have some activity on Sa 1 cells grafted on C57BL/Ks mice. This activity is significant for IgG1 anti-Iak and suggestive for IgG2 of the same specificity. (4) No enhancing activity was detected in the other antibodies: IgG2 anti-Dd, IgG2 anti-Kk, IgG1, or IgG2 anti-ICd, Sd, Gd as well as in fractions containing IgM and IgA antibodies directed against any studied portion of the MHC products. This results are discussed in terms of the mechanisms involved in enhancement.

Chromatographic fractionation of rhesus monkey ( Macaca mulatta) IgG subclasses using DEAE cellulose and protein A-Sepharose

Rhesus monkey serum was applied to a protein A-Sepharose column at pH 7.3 IgG was not detectable in the unbound effluent. Bound protein was eluted with a pH gradient of citrate-phosphate buffer. Two major elution peaks reproducibly resulted at pH 4.85–4.9 and pH 4.35–4.43, respectively, with only slight variability among individuals. Since the immunoelectrophoretic mobility of IgG in the peak at pH 4.85–4.9 was slower than that of the peak at pH 4.35–4.43, serum was first fractionated by ion exchange chromatography into 2 fractions: the first did not bind to DEAE cellulose in 0.01 M phosphate buffer pH 7.8, the second did bind and was eluted by an NaCl gradient. Each DEAE fraction was then further fractionated on protein A-Sepharose. IgG in the first DEAE fraction bound to protein A at pH 7.3 and eluted in a single peak at pH 4.72–5.0. IgG in the second DEAE fraction also bound to protein A, but eluted in 2 peaks at pH 4.65–5.1, and 4.25–4.6, respectively. All 3 IgG fractions eluted in the same position from an A5m column, had immunodiffusion reactions of identity with anti-human γ chain and were composed of similar heavy and light chains judged by SDS polyacrylamide gel electrophoresis. However, IgG eluting from protein A at the low pH (4.25–4.6) had a reaction of partial identity with other IgG fractions when tested by immunodiffusion against rabbit anti-rhesus γ chain, suggestive of an antigenically different subclass. Analysis of rabbit antisera prepared against the 3 IgG fractions confirmed the occurrence of at least 3 antigenically distinct rhesus monkey IgG subclasses. These 3 subclasses have been provisionally designated IgG I, IgG II and IgG III.

Detection of monoclonal antibody to high-mobility-group protein 17 from chick oviduct.

Total chromosomal HMG (high-mobility-group) proteins have been isolated from oestrogen-stimulated chick oviduct. The antibodies against these proteins were induced in mice and subsequently their spleen cells were fused with myeloma cells to form hybridomas. A highly purified HMG protein, 17, was used to select for the hybridomas that produce antibody against HMG protein 17. The hybridomas were cultured and injected into mice to produce ascites. The antibody against HMG protein 17 in the IgG (immunoglobulin G) fraction of the ascites fluid was obtained by Protein A-Sepharose column chromatography. We have devised a solid-phase radioimmunoassay and enzyme-linked serological assay for the detection and characterization of this antibody directed against HMG protein 17. This anti-(HMG protein 17) IgG interacted only with HMG protein 17, but not with other chromosomal proteins, e.g. histone H1, "95K protein' (a chick oviduct-specific chromosomal protein) and HMG proteins 1, 2 and 14. The monospecific nature of this anti-(HMG protein 17) IgG fraction is confirmed.

CDNA clones for the heavy chain of HLA-DR antigens obtained after immunopurification of polysomes by monoclonal antibody.

A monoclonal antibody (HC 2.1) directed against the separated heavy chain of HLA-DR has been prepared. By binding HC 2.1 to polysomes from human B lymphoblastoid cells followed by the use of a protein A-Sepharose column as an immunoadsorbent, we have purified the mRNA coding for the HLA-DR heavy chain nearly to homogeneity. The immunopurified mRNA has been used to prepare labeled cDNA with which to probe cDNA libraries. Double-stranded cDNA was also made from the immunopurified mRNA and cloned directly into pBR322. Two clones, one from each of the above procedures, positively selected DR heavy chain message as assayed by cell-free translation and immunoprecipitation. One clone, pDRH-2 [500 base pairs plus 75 base pairs of poly(A)] contains the entire 3' untranslated region as well as coding information for the carboxy-terminal hydrophilic intracellular domain and part of the hydrophobic transmembrane region. Results of carboxypeptidase digestion of the heavy chains from detergent-solubilized (p34) and papain-treated (p33) HLA-DR antigen were consistent with the predicted protein sequence. Specific immunopurification of polysomes by defined monoclonal antibodies followed by direct cloning of cDNA to the highly purified mRNA is a powerful method for obtaining identified cDNA clones.

The use of a protein a-sepharose column in the detection of platelet-associated IgG

The use of a protein a-sepharose column in the detection of platelet-associated IgG

An immunochemical method for mRNA purification. Application to messenger RNA encoding trypanosome variable surface antigen.

A simplified procedure for isolation of specific mRNA using Staphylococcus aureus protein A immunoadsorbent chromatography has been developed. This procedure has been applied to the mRNA encoding Trypanosoma brucei variable surface antigen. Trypanosome polyribosomes were reacted with antibodies isolated from an anti-variable antigen-specific serum by protein A-Sepharose column chromatography. Antibody-bound variable antigen-synthesizing polyribosomes were then separated from unbound polyribosomes also by protein A-Sepharose column chromatography. This simple protocol gave a greater than 50% yield of variable antigen-specific mRNA which appeared to be very highly purified as determined by translation in an mRNA-dependent reticulocyte lysate assay. The mRNAs encoding three different T. brucei surface antigens have been purified. The procedure described here should be useful in purification of other mRNAs.

Skin testing in farmers' lung disease

Intradermal skin tests with a culture filtrate antigen of Micropolyspora faeni grown on a synthetic medium were performed on patients with farmers' lung disease (FLD) and well farmers with and without antibodies to a panel of FLD antigens. Seventy-five percent of the FLD patients, 79% of the well farmers with M. faeni antibody, and 5% of well farmers without M. faeni antibody had a 2+ or greater intradermal immediate skin-test reaction. Prausnitz-Küstner (P-K) reactions were positive using serum of M. faeni immediate skin test-positive FLD patients. IgG-rich fractions from a staphylococcal protein A-Sepharose column of such serum contained the sensitizing factor whereas IgG-depleted fractions did not. M. faeni—specific IgE could not be detected in serum by a polystyrene radioimmunoassay. Positive late-onset (6-hr) skin tests occurred only in FLD patients and farmers with precipitating antibody. Biopsy specimens of the 6-hr reactions revealed a generalized dermal and perivascular polymorphonuclear infiltrate with deposits of immunoglobulin and complement about blood vessels. The skin-sensitizing factor noted in FLD patients and well farmers with antibody is not disease specific. This factor appears to be associated with the IgG-rich fraction of serum, and its role in the pathogenesis of FLD is unclear.

The interactions of porcine and ovine, serum and colostral immunoglobulins with staphylococcal Protein A.

The purpose of these studies was to determine the proportion of each immunoglobulin class/subclass in blood and colostrum of the pig and sheep, which would bind to staphylococcal Protein A. The concentrations of porcine IgG, IgM, and IgA were determined for serum and colostral whey from five sows. Similar measurements were made on two fractions produce by elution of the sample through a Protein A-Sepharose column: fraction 1, immunoglobulins which did not bind to Protein A, and fraction 2, immunoglobulins which bound to Protein A. The concentrations of ovine IgG1, IgG2, IgM, and IgA were measured for serum and colostral whey from six ewes, and again similar measurements were made after elution of each ovine sample through Protein A-Sepharose. All classes/subclasses of porcine and ovine serum and colostral immunoglobulins bound to Protein A to some extent. More than 90% of IgG from both porcine colostral whey and serum bound to Protein A. Ovine IgG1 from most ewes possessed a low affinity for Protein A whereas ovine IgG2 generally possessed a high affinity; 100% of the IgG2 in ovine colostral whey samples bound to Protein A. There was remarkable variation between individuals in the binding capacity of porcine IgM and each of the ovine immunoglobulins. For the ovine samples, in particular there were distinct differences between Protein A binding capacity of serum and colostral immunoglobulins of the same class/subclass.

Studies of the IgM and IgA contamination obtained by eluting IgG from protein A-sepharose column with pH steps

The IgG fraction obtained from pooled human plasma by eluting a protein A column with a buffer at pH 2.5 was contaminated with approximately 30% of the IgM originally present in the sample. Both the IgM and the IgA contamination can be reduced and the IgG recovery maintained at 90% of the bound IgG when elution of the column is performed at pH 4.0.

PH gradient elution of human IgG1, IgG2 and IgG4 from protein A-Sepharose

Pooled human serum having a normal IgG subclass content was chromatographed on a column of protein A-Sepharose. The immunoglobulins that bound to the column at pH 7.0 were eluted with a pH gradient generated by 3 equal volumes of citrate/phosphate buffer at pH 5.0, 4.5 and 2.2. The elution pattern consisted of two major overlapping peaks centered 0.4 pH units apart in the pH gradient; on average the first peak centered at pH 4.7, and the second centered at pH 4.3. Upon second passage of each component, single major peaks centered at the appropriate pH were seen. The subclass distributions of the re-chromatographed peaks were as follows: the high pH-eluting IgG contained <1% IgG1, 95% IgG2 and 5% IgG4; the low pH-eluting IgG contained 90% IgG1, 6% IgG2 and 5% IgG4. IgG3 does not bind to protein A and was thus absent from the pH grdient fractions. Chromatography on protein A-Sepharose provides a means for separating normal human IgG1 from IgG2 and may therefore prove useful as an additional tool for studying the relative biological role of these IgG subclasses.

Simple purification of goat IgG1 and IgG2 subclasses by chromatography on protein A-sepharose at various pH

Two ml of goat serum are applied on a 2 × 20 cm column of protein A-Sepharose at pH 9.1. Elution at the same pH yields two protein peaks, the second being highly purified IgG1. A minor Ig fraction, eluted at pH 6.5, is discarded. Pure IgG2 is then eluted at pH 5.9. Yields of both subclasses average 70 %.

Interactions between mouse immunoglobulins and staphylococcal protein A.

When mouse serum or ascites is applied on protein A-Sepharose columns and washed with enough phosphate-buffered saline, a second protein peak is often eluted with the same buffer after the first major peak of unbound proteins. This second peak is almost pure Ig G1. More IgG1 plus IgG2a, IgG2b and IgG3 are thereafter eluted with acid saline. 90% of the IgG1 whichhad been eluted with neutral buffer could be re-eluted at the same retarded position with the same buffer. When a gradient from 0 to 3 M sodium thiocyanate was started after the major peak of unbound proteins, all IgG1 was eluted before IgG2 and IgG3. These results suggest that IgG1 has a much lower affinity for protein A than IgG2 or IgG3 and that normal mouse serum IgG1 can be purified by such a simple procedure.

ACTH-like activity in immune complexes of patients with oat-cell carcinoma of the lung

Immune complexes could be isolated from sera of 7 patients with oat-cell carcinoma of the lung, but not from 5 normal controls, using zonal ultracentrifugation. After ultracentrifugation, fractions containing macromolecular IgG were absorbed on a protein A-sepharose column and the immune complexes were eluted and dissociated by glycin-HCl buffer at pH 3.5. The eluates were tested for the presence of tumour-associated proteins as carcinoembryonic antigen (CEA), non-specific crossreacting antigen (NCA), alpha2 pregnancy associated antigen (alpha2PAG) and isoferritin. Whereas none of these tumour-associated antigens could be demonstrated, an ACTH-like activity was detected in the immune-complex fractions of 4 patients with oat-cell carcinoma, by radioimmuno- and bioassay. Polyacrylamide electrophoresis of an immune-complex fraction from a patient with Cushing syndrome showed ACTH-like activities, with mol. wt of 110,000, 75,000, 30,000 and less than 20,000 (all glycoproteins) indicating the presence of different subfractions of big ACTH.

Staphylococcal protein A-sepharose columns and the characterization of measles virus-specific polypeptides in persistently infected cells

Viral polypeptides in extracts prepared from [ 35S]methionine-labeled human epithelial carcinoma cells persistently infected with measles virus were reacted with virus-specific antisera. Microcolumns of staphylococcal protein A linked to Sepharose were used to isolate antigen-antibody complexes that contained few contaminating host polypeptides. Measles virus polypeptides in these complexes could be identified readily on sodium dodecyl sulfate-polyacrylamide gels even when antiserum with low reactivity toward the viral antigens was used.