Immunoaffinity Procedure Research Articles

Immunoaffinity purification of creatine kinase-MB from human, dog, and rabbit heart with use of a monoclonal antibody specific for CK-MB.

We describe a simple, rapid immunoaffinity procedure for purifying the MB isoenzyme of creatine kinase. Immunoaffinity gel is prepared by linking a monoclonal antibody ("Conan-MB"), specific for this isoenzyme, to Sepharose 4B. Heart tissue is homogenized and fractionated with 40-70% saturated ammonium sulfate before it is applied to the immunoaffinity gel. CK-MB activity, retained on the gel, is then eluted with a high-pH diethylamine buffer (0.1 mol/L, pH 10.5). The purified CK-MB isoenzyme is stabilized by collection directly into tubes containing glycerol (to prevent dissociation of the enzyme subunits) and pH-neutralizing buffer. This procedure compares favorably in yield, specific activity, and technical ease with a multi-column purification method previously used in our laboratory. We have used the immunoaffinity procedure to purify to homogeneity CK-MB from human, dog, and rabbit heart, with yields of 50.0%, 53.1%, and 49.3% and specific activities of 540, 477, and 377 kU/g, respectively. The preparations are pure as judged by protein staining with silver nitrate after electrophoresis on sodium dodecyl sulfate/polyacrylamide gel.

Expression of a synthetic bovine rhodopsin gene in monkey kidney cells.

We report here the high-level expression of a synthetic gene for bovine rhodopsin in transfected monkey kidney COS-1 cells. Rhodopsin is produced in these cells to a level of 0.3% of the cell protein, and it binds exogenously added 11-cis-retinal to generate the characteristic rhodopsin absorption spectrum. We describe a one-step immunoaffinity procedure for purification of the rhodopsin essentially to homogeneity. The COS-1 cell rhodopsin activates the GTPase activity of bovine transducin in a light-dependent manner with the same specific activity as that of purified bovine rhodopsin. Electron microscopy of immunogold-stained cells indicates that rhodopsin is located in the plasma membrane of the transfected cells and is oriented with the amino terminus on the extracellular side of the membrane. This orientation is analogous to that of rhodopsin in the disk membranes of photoreceptor cells in the bovine retina.

Monoclonal antibodies to hydroxyindole O-methyltransferase from bovine pineal gland.

Hybridomas that produce monoclonal antibodies to bovine hydroxyindole O-methyltransferase have been established by immunizing a mouse with hydroxyindole O-methyltransferase partially purified from bovine pineal glands followed by fusion of the spleen cells with myeloma NS-1 cells. Twenty-three clones have been isolated by screening the medium by two different methods. These clones produced various antibodies with different binding properties. Most of the antibodies belonged to IgG1 subtype, while 7 clones produced IgG2 alpha or IgG2 beta antibodies. Three antibodies inhibited enzymatic activity, whereas others did not. Antibodies of 7 clones recognized enzyme protein denatured with sodium dodecyl sulfate, while other antibodies reacted only with native enzyme protein. Thus a variety of monoclonal antibodies will offer us a good tool for immunohistochemical localization of the enzyme, immunoaffinity purification of hydroxyindole O-methyltransferase, and isolation of the cDNA clones encoding it. We also report here on the immunohistochemical demonstration of hydroxyindole O-methyltransferase in bovine pineal gland and a new immunoaffinity procedure to purify the enzyme to homogeneity by use of monoclonal antibodies.

Monoclonal antibodies to hydroxyindole O-methyltransferase from bovine pineal gland

Hybridomas that produce monoclonal antibodies to bovine hydroxyindole O-methyltransferase have been established by immunizing a mouse with hydroxyindole O-methyltransferase partially purified from bovine pineal glands followed by fusion of the spleen cells with myeloma NS-1 cells. Twenty-three clones have been isolated by screening the medium by two different methods. These clones produced various antibodies with different binding properties. Most of the antibodies belonged to IgG1 subtype, while 7 clones produced IgG2α or IgG2β antibodies. Three antibodies inhibited enzymatic activity, whereas others did not. Antibodies of 7 clones recognized enzyme protein denatured with sodium dodecyl sulfate, while other antibodies reacted only with native enzyme protein. Thus a variety of monoclonal antibodies will offer us a good tool for immunohistochemical localization of the enzyme, immunoaffinity purification of hydroxyindole O-methyltransferase, and isolation of the cDNA clones encoding it. We also report here on the immunohistochemical demonstration of hydroxyindole O-methyltransferase in bovine pineal gland and a new immunoaffinity procedure to purify the enzyme to homogeneity by use of monoclonal antibodies.

Characterization of human thyroid peroxidase purified by monoclonal antibody-assisted chromatography.

A murine monoclonal antibody (mAb) to human thyroid peroxidase (TPO) was produced and used for purification of the enzyme. This report describes studies done to characterize the mAb and the purified human TPO. The mAb bound to human TPO at a molar ratio of 1:2 or 1:1 and to human thyroid microsomes with a dissociation constant of 1.2 nM. The mAb had no effect on TPO activity to catalyze guaiacol oxidation. As immunoglobulin G, the mAb consisted of a light chain (kappa) of 26 kilodaltons (kDa) and a heavy chain (gamma 1) of 53 kDa; its pI value was 5.9. Using an mAb immunoaffinity column, 85% of TPO activity was recovered from an ammonium sulfate precipitate of a human thyroid microsomal preparation solubilized with deoxycholate. The purified TPO had a specific activity of 164 guaiacol U/mg protein and an A413 nm/A280 nm ratio of 0.26. The enzyme showed bands only at 107 and 100 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified TPO was very stable at alkaline pH, and its ability to catalyze iodide oxidation and tyrosine iodination was increased to the same extent as its ability to catalyze guaiacol oxidation. For comparison with a conventional method, the immunoaffinity-purified TPO was trypsinized and rechromatographed on the immunoaffinity column. The trypsinized enzyme had a specific activity of 336 U/mg and an A413 nm/A280nm ratio of 0.65. The absorption spectrum of the enzyme suggested that the prosthetic group of human TPO is protoheme IX. These results indicate the value of the immunoaffinity procedure for human TPO purification. The major advantages of this purification procedure are not only high yield and speed, but also recovery of intact enzyme.

A procedure for the rapid purification in high yield of human glucocerebrosidase using immunoaffinity chromatography with monoclonal antibodies

A novel chromatographic immunoaffinity procedure is described for the purification of Form I glucocerebrosidase (see J. M. F. G. Aerts, W. E. Donker-Koopman, M. K. Van der Vliet, L. M. V. Jonsson, E. I. Ginns, G. J. Murray, J. A. Barranger, J. M. Tager, and A. W. Schram, 1985, Eur. J. Biochem. 150, 565–574) from extracts of human tissues. The affinity support consists of two monoclonal anti-(glucocerebrosidase) antibodies immobilized by covalent coupling to CNBr-activated Sepharose 4B. After adsorption of the enzyme from a crude detergent extract, the column is washed successively with 30% ethylene glycol in citrate buffer (pH 6). 1% Triton X-100 in citrate phosphate buffer (pH 5.2), and 50% ethylene glycol in citrate buffer. The enzyme is eluted with 90% ethylene glycol in citrate buffer. After dilution to 30% ethylene glycol, the immunoaffinity purification is repeated. The procedure can be completed within less than 18 h. The final preparations have a high specific activity (50 U/mg protein ( n = 4) for the placental enzyme) and contain no detectable impurities after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The yield is high (81 ± 8% for the placental enzyme). The immunoaffinity column has a high capacity, can be regenerated easily, and can be utilized repeatedly without loss of activity.

Immunoreactive corticotropin-releasing factor in human plasma.

Plasma immunoreactive corticotropin-releasing factor (I-CRF) levels were determined by using a human CRF radioimmunoassay and an immunoaffinity procedure. The basal plasma I-CRF level in normal subjects was 6 +/- 0.5 pg/ml (mean +/- SD). We found that most plasma I-CRF levels were affected by stress, negative feedback, and circadian rhythm. Basal I-CRF levels were high in patients with Addison's disease, Nelson's syndrome, hypopituitarism stemming from pituitary macroadenoma, and CRF- and adrenocorticotropic hormone-producing tumors. A very low, but significant, amount of I-CRF was detected (1-3 pg/ml) in patients with Cushing's syndrome, in corticosteroid-treated patients, and in a patient with hypothalamic hypopituitarism. These results suggest that a major component of plasma I-CRF is of hypothalamic origin, however, other extrahypothalamic tissues cannot be ruled out as a minor source of plasma I-CRF.

Affinity purification of β-endorphin-like material from NG108CC15 hybrid cells by means of the monoclonal β-endorphin antibody 3-E7

Neuroblastoma × glioma hybrid cells (NG108CC15) were examined for the presence of β-endorphin-like material. In order to differentiate this β-endorphin-like material from crude cell extract, a procedure for immunoaffinity chromatography was developed. The monoclonal antibody 3-E7 employed possesses the unique property of recognizing the N-terminal sequence of virtually all endogenous opioid peptides, but not their precursors. By means of this immunoaffinity procedure about 90% of exogenous β-endorphin was recovered from 10 ml phosphate buffered saline samples. Affinity chromatography served as first-step purification of crude NG108CC15 cell extract for the separation and concentration of β-endorphin-like material. The eluate of the immunoaffinity gel was subjected either to Sephadex gel filtration or to high pressure liquid chromatography. Under either condition, immunoreactive β-endorphin which eluted with synthetic β-endorphin was detected. The concentration in six different batches varied from 4 to 17 fmol/10 8 cells. This would be 10–200-fold lower than that observed for the enkephalins or dynorphin A/α-neo-endorphin. It is concluded that the utilization of the monoclonal antibody 3-E7 for a first-step purification of cell extracts was an essential pre-requisite for the separation of β-endorphin-like material from the hybrid cells. The presence of enkephalin-like material, of dynorphin A/α-neo-endorphin-like material and of β-endorphin immunoreactive material suggests that NG108CC15 cells are able to generate opioid peptides related to the precursors pre-proenkephalin A, pre-proenkephalin B and pro-opiomelanocortin.

DNA binding activity of polyoma virus large tumor antigen.

Polyoma virus large tumor antigen from productively infected mouse cells has been purified to greater than 50% homogeneity by a simple immunoaffinity procedure using monoclonal antibodies. A radioimmunoreaction was devised for assaying purity. The purified large tumor antigen retained its antigenicity and its ability to bind DNA specifically. The regions on the polyoma virus genome recognized by the protein were characterized. Three binding regions were localized within the portion of the genome between the viral origin of DNA replication and the protein coding sequence, overlapping the early promoter and the sites of initiation of mRNAs that specify the viral tumor antigens. The binding regions each contain direct repeats of the pentanucleotide sequence G-R-G-G-C.