Isolation and characterization of a human liver and kidney-specific protein: the hepato-renal (H-R) antigen.

This paper reports the isolation and characterization of a soluble antigen shared by the liver and kidney of human and some other animal species. Homogenates of human liver in saline were centrifugated at 27,000 g and the supernatants were fractionated by preparative polyacrylamide gel electrophoresis. The gels were divided in sections and each was injected into rabbits; after absorption with polymerized normal human serum, the antiserum obtained by injecting one of the sections reacted only with saline extracts of human liver and kidney when tested against a variety of human tissue extracts. The absorbed antiserum, polymerized and insolubilized with glutaraldehyde, was used to purify the antigen by affinity chromatography. The purified antigen proved to be a glycoprotein containing 19 percent carbohydrate, had a molecular weight of 5.8-6.0 x 10(4) Daltons and a pI of 7.2-7.4. The antigen, relatively thermostable, was precipitated by 35-55 percent ammonium sulphate; its antigenic activity was not affected by extraction with 0.6 N perchloric acid or by incubation with ribonuclease, deoxyribonuclease or neuraminidase but was destroyed by incubation with ttypsin or chymotrypsin. Immunoperoxidase studies showed that the antigen appeared concentrated in the neclei of liver and kidney glomerular epithelial and tubular epithelial cells in humans and rats. The antigen could not be detected in human hepatomas or hypernephromas or in the rat Morris hepatoma 5123.

Isolation and characterization of a human pancreas-specific protein.

Homogenates of human pancreas in saline were centrifuged at 27 000 X g and the supernates were fractionated by preparative polyacrylamide gel electrophoresis. The gels were divided into sections and each section was injected into rabbits; after absorption with polymerized serum from apparently normal humans, the antiserum obtained by injecting one of the sections was tested against a variety of human tissue extracts but reacted only with saline extracts of human pancreas. The absorbed antiserum, polymerized and made insoluble with glutaraldehyde, was used to purify a pancreas-specific antigen by immunoaffinity batch technique. The purified antigen proved to be a protein with some carbohydrate content (180 mg/g by weight) and a molecular mass of about 2.25 X 10(5) daltons. The antigen is relatively thermostable, and precipitates in the range of 245.64-340.2 g/L saturated ammonium sulfate; its antigenic activity is not affected by incubation with ribonuclease or deoxyribonuclease, but is destroyed by incubation with trypsin or neuraminidase and by extraction with perchloric acid. Immunofluorescence studies show that the antigen is diffusely present in the cytoplasm of pancreatic acinar cells.

Radioimmunoassay for detection of latent chronic alcoholic pancreatitis, an unrecognized clinical syndrome.

We developed a radioimmunoassay procedure in which we used an antibody monospecific for pancreatic acinar antigen, to sensitively and specifically test for the presence of subclinical (latent) alcoholic pancreatitis. The antigen was isolated, purified, and partially characterized. Results of testing appropriate populations of patients support the idea that chronic alcoholism is associated with chronic, subclinical damage to the pancreas and concomitant continuous release of a pancreatic acinar protein into the peripheral circulation, where it can be detected and quantitated. About 40% of the samples from chronic alcoholics (whether during a bout of acute alcoholism or during abstinence) demonstrated the circulating antigen, strongly suggesting that those alcoholics who will develop symptomatic, chronic alcoholic pancreatitis can be readily detected during the non-symptomatic stage.

Isolation and characterization of canine venereal tumor-associated inhibitory and blocking factors.

Spontaneous regression of the canine venereal tumor is associated with the production of a serum factor which inhibits in vitro tumor colony-forming units in agar. Logarithmic or persistent tumor growth, on the other hand, is characterized by a serum factor which protects cells against in vitro inhibition (blocking factor). These factors have been characterized by immunochemical methods. Whole regressor and blocking sera were fractionated by Sephadex G-200 filtration and immunoabsorption with rabbit antiserum specific for canine immunoglobulin G2a. Fractions were characterized by immunoelectrophoresis, radial immunodiffusion, and disc gel electrophoresis. In vitro inhibitory and blocking activity of the whole serum was accounted for by the purified immunoglobulin G2a. Blocking activity was also found in protein eluted from logarithmically growing tumors. Preparative polyacrylamide electrophoresis revealed five major fractions with blocking activity only in the immunoglobulin G fraction. Tumor eluates and immunoglobulin G isolated from serially removed tumors demonstrated with the clinical course of the tumor. Using ultrafiltration and sodium dodecyl sulfate electrophoresis of tumor-associated immunoglobulin G at low pH, it was not possible to identify an antigen complexed to the blocking antibody.

Cerebrospinal fluid IgG, IgA, IgM, IgD, and IgE levels in central nervous system disorders.

Using a radioimmunoassay, we evaluated the cerebrospinal fluid content of IgG, IgA, IgM, IgD, and IgE in multiple sclerosis, viral meningitis, and tumors. Abnormal immunoglobulin levels occurred in all conditions, reaching 13 times the norm for IgM in multiple sclerosis and 5 times the norm in aseptic meningitis. Striking increases also occurred with IgA, IgM, and IgE in benign and malignant (both primary and metastatic) tumors of the CNS. Furthermore, the immunoglobulin-protein ratio was calculated. In aseptic meningitis and multiple sclerosis, the ratio was significant as compared to the norms.

Hematological response of rabbits to chronic, repetitive, severe bleedings for the production of antisera.

A study was undertaken to determine whether large amounts of blood (antiserum) could be rapidly removed from rabbits over a two-month period using a simple suction technique. It was found that half a blood volume could be readily removed each week (over 900 ml/rabbit/54 days) without a single fatality. The rabbits efficiently replaced the essential components of their blood during the experimental period as shown by a study of their blood parameters. They moderately overcompensated in replacing some of their blood components after a 6-week rest period. The study demonstrates that rabbits are satisfactory (in lieu of larger animals) for supplying relatively large amounts of antisera.

Insoluble human plasma protein immunoadsorbents. Large-scale production and storage.

Methods for producing and preserving large volumes of insoluble immunoadsorbents (for removing unwated antibodies to serum proteins) from surplus blood bank plasma by glutaraldehyde were evaluated by quantitative and qualitative means using radioactive 125I and immunoelectrophoresis, respectively. Some of the factors affecting the desired physical characteristics and antibody-absorbing properties of the imjunoadsorbent studied were: plasma acidification, varying concentrations of glutaraldehyde, addition of small amounts of formalin, storage under varying conditions of temperature, and exposure to preservatives in the wet and lyophilized state for periods up to 2.5 years. The best preservation of antibody-adsorbing properties (under storage conditions) was obtained in the washed state at 4 degrees C, but good preservation was also obtained at room temperature in the presence 10% formalin and in the unwashed state at room temperature in the presence of unreacted glutaraldehyde. Lyophilization destroyed about 70% of an adsorbent's activity.

Radioimmunoassays for Ig classes G, A, M, D, and E in spinal fluids: normal values of different age groups.

Radioimmunoassay procedures of sufficient sensitivity (IgG, 0.5 mug per 100 mul; IgA, 25.0 ng. per 100 mul; IgM, 10.0 ng. per 100 mul; IgD, 0.5 U. per 100 mul; and IgE, 1.0 U. per 100 mul) were developed detect and quantitate all 5 immunoglobulin classes in the cerebrospinal fluid on small aliquots (1 ml.) of unconcentrated cerebrospinal fluid. All 5 immunoglobulin classes were routinely detected in normal individuals for the first time, the levels varying with different age groups for IgG and A but not for the remaining immunoglobulin classes. Race and sex had no effect. Standardization of techniques and establishment of normal values for different age groups sets the stage for determination of immunoglobulin changes related to central nervous system disease.

Characterization of monoclonal gammopathies, a new approach.

A new approach to the characterization (typing) of L- and H-chains of monoclonal immunoglobulins and Bence-Jones protein utilizing elution of a monoclonal immunoglobulin in conjunction with solid-support electrophoresis and the reverse Mancini procedure of radial immunodiffusion on Mylar-backed cellulose acetate membranes is described. A number of benefits are derived, particularly the use of only a fraction (1/200) of the antisera used with agar immunoelectrophoresis. The importance of "standardizing" commercial antisera (and a method for carrying this out) with the techniques described is emphasized.

Sequential blood samples from the tail vein of rats and mice obtained with modified Liebig condenser jackets and vacuum.

A simple method for obtaining multiple, sizeable blood samples rapidly and sequentially from mice and rats without the use of anesthesia utilizing an adapted Liebig condenser jacket connected to a vacuum line is described. The method permits the removal and insertion of sample tubes during the procedure so that anticoagulated and clotted specimens can be obtained during a single bleeding.