Biochemical and functional characterization of a new murine monoclonal antibody against human platelet glycoprotein IIIa.

A new murine monoclonal antibody, MDP-1, specific for human platelet glycoprotein IIIa has been produced and characterized. Following SDS-polyacrylamide gel electrophoresis, MDP-1 reacted with a 94kDa protein immobilized on a nitrocellulose membrane. Upon reduction, MDP-1 no longer bound to the 94kDa protein indicating an epitope requiring at least one disulfide bond.

Separate clones in concomitant multiple myeloma and a second B-cell neoplasm demonstrated by molecular and immunophenotypic analysis.

The occurrence of multiple myeloma (MM) and a second B-cell neoplasm in the same patient is a rare event. We present 2 such patients, and provide evidence to support the presence of separate clones in these coexisting neoplasms. In the first case, MM became evident 14 months after the diagnosis of chronic lymphocytic leukemia (CLL).

A flow cytometric method to detect anti-pyruvate dehydrogenase antibody in primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is an autoimmune disease characterized by the presence of anti-mitochondrial antibodies specifically directed against the M2 group of mitochondrial antigens. Recently, the E-1, the E-2, and protein X components of pyruvate dehydrogenase enzyme complex have been identified as the major antigens within the M2 group of autoantigens. An immunoassay using pyruvate dehydrogenase enzyme complex as a specific antigen for the diagnosis of PBC was developed.

Quantification of effector/target conjugation involving natural killer (NK) or lymphokine activated killer (LAK) cells by two-color flow cytometry.

Precise estimates of the frequency of NK- and LAK-target conjugates were obtained by two-color flow cytometry using hydroethidine and calcein as intracellular labels for target cells and effector cells, respectively. These two dyes can easily be used with a standard single-laser flow cytometer with excellent signal separation and dye retention. Hydroethidine labeling did not alter target susceptibility, and calcein labeling did not significantly alter NK function.

Monoclonal antibody to human cartilage cells and its reactivities to chondrocytic tumors.

A murine monoclonal antibody (E10) was made against cultured cartilage cells. The E10 antibody binding is localized to the surface of cultured cartilage cells in suspension and is present in the cytoplasm in paraffin embedded sections. There is no reactivity with cartilage matrix, or with the matrix of cartilaginous tumors.

Characterization of effector-target conjugates for cloned human natural killer and human lymphokine activated killer cells by flow cytometry.

The present studies demonstrate that the intracellular fluorochromes calcein and hydroethidine can be used for quantification of effector-target conjugates involving cloned human natural killer (NK) or interleukin-2 (IL-2) activated human lymphokine activated killer (LAK) cells by dual color flow cytometry without potential artifacts that might result from extensive modification of effector and/or target cell membranes. Cloned NK cells and LAK cells form conjugates with cultured cell lines regardless of susceptibility to lysis. The strength of the interactions in these conjugates was investigated using a variable speed vortexer.

Cytoplasmic androgen binding protein of rat liver: molecular characterization after photoaffinity labeling and functional correlation with the age-dependent synthesis of alpha 2u-globulin.

The liver of the mature male rat contains a moderate affinity (Kd = 10(-8)M), low-capacity, cytoplasmic androgen binding protein (CAB) whose appearance during puberty and disappearance during senescence correlate with the androgen-dependent synthesis of alpha 2u-globulin. Molecular properties of CAB were examined by photoaffinity labeling with tritiated methyltrienolone (R-1881), a synthetic androgen, and by its localization within the hepatocytes which are competent to produce alpha 2u-globulin. Photoaffinity labeling of the liver cytosol derived from postpubertal male rats, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, showed a predominant androgen binding band corresponding to Mr 31,000.

Polymerization of intact beta 2-microglobulin in tissue causes amyloidosis in patients on chronic hemodialysis.

Systemic amyloidosis with a predilection for bone and synovium may complicate the course of patients on long-term hemodialysis. This form of amyloidosis can be typed as distinct from other amyloid diseases by using small tissue samples obtained by bone biopsy and at postmortem. Immunoblot analysis of two-dimensional gels of partially solubilized amyloid fibrils established that tissue deposits are composed of monomers, dimers, and higher polymers of beta 2-microglobulin (beta 2m) and that amyloid P component was also present.

Normal and abnormal aspects of proteinuria. Part I: Mechanisms, characteristics and analyses of urinary protein. Part II: Clinical considerations.

Part I highlights the mechanisms of glomerular filtration and tubular reabsorption of plasma proteins, selected characteristics of urinary proteins based upon electrophoretic properties and recent advances in clinical laboratory analysis of proteinuria. Both structural characteristic of the glomerular capillary wall and molecular properties of plasma proteins are important determinants of glomerular filtration. Proteins filtered by the glomerulus subsequently appear in urine only after escaping the efficient mechanisms of tubular reabsorption.

A collaborative study of a preparation of normal human serum for use as a reference in the assay of beta 2 microglobulin.

A biological standard for beta 2 microglobulin (beta 2m) determination has been prepared from pooled human serum by sampling and freeze-drying. A preliminary study of parallelism between the dose-response curves of the preparation and pure beta 2m or biological fluids was made with four different methods of assay and gave satisfactory results. In a collaborative study in five laboratories in five countries using a common method of assay, evidence was obtained that the preparation coded 80-12-3200 was suitable to serve as a standard for the assay of beta 2 microglobulin.

Monoclonal antibodies to alpha 2u-globulin and immunocytofluorometric analysis of alpha 2u-globulin-synthesizing hepatocytes during androgenic induction and aging.

Stable hybridomas generated by fusion of spleen cells from hyperimmunized mice and mouse myeloma cells were cloned to prepare monoclonal antibodies to alpha 2u-globulin, an androgen-dependent urinary protein of hepatic origin. One of these monoclonal antibodies was used as a probe for immunocytofluorometric analysis of alpha 2u-globulin producing hepatocytes during androgenic induction and aging through fluorescence-activated cell sorting (FACS). FACS patterns of hepatocytes from mature male rats that produce high levels of alpha 2u-globulin showed tow distinct peaks, arbitrarily designated as peak I (weakly fluorescent) and peak II (brightly fluorescent).

Post-gamma globulin. II. Radioimmunoassay determination of levels of post-gamma globulin and beta 2-microglobulin.

Post-gamma globulin previously isolated and partially sequenced in this laboratory was used for production of polyclonal and monoclonal (hybridoma) antibodies. A radioimmunoassay method was developed for quantitation of post-gamma globulin with either antibody. The titration curves obtained were treated statistically and found practically indistinguishable.

Turkey beta 2-microglobulin--II. Physiological parameters investigated by radioimmunoassay.

A competitive binding radioimmunoassay (RIA) was developed utilizing 125I-labeled turkey beta 2-microglobulin (beta 2m), rabbit anti-turkey beta 2m serum and polyethylene glycol for separation of bound and free antigen. The antibody-antigen reaction was demonstrated to be monospecific by immunofixation electrophoresis. Characteristic parameters of the RIA were: nonspecific binding, 5.

Turkey beta 2-microglobulin--I. Isolation, properties and amino acid analysis.

Turkey beta 2-microglobulin (beta 2m) was purified from pooled serum by successive steps of ultrafiltration, gel filtration chromatography, lectin affinity chromatography, anion exchange chromatography, isoelectric focusing and a second step of gel filtration. Identification of turkey beta 2m was based upon NH2-terminal primary structure analysis. The NH2-terminal primary structure of turkey beta 2m is: NH2-Lys-Ile-Glu-Val-Tyr-Ile-Lys-.

Post-gamma globulin : tissue distribution and physicochemical characteristics of dog post-gamma globulin.

An animal model (dog) was established to investigate physicochemical and biological parameters of a basic low molecular mass protein---post-gamma globulin (p-gamma). In this study the distribution of this 11 000 daltons (11 kdaltons) protein was determined in extracts of the number of tissues. Extracts of parotid gland, brain and kidney contained high amounts of "free" p-gamma globulin (11 kdaltons).

Association of human and bovine beta 2-microglobulins with detergent solubilized HLA-A,B antigens.

The association of human and bovine beta 2-microglobulins with detergent solubilized HLA-A,B antigens was analyzed by a direct binding assay using radiolabeled beta 2-microglobulin and an immunoadsorbent containing a monoclonal antibody to the HLA-A,B heavy chains. Binding of beta 2-microglobulin to HLA-A,B heavy chains could be saturated with respect to the amount of membrane glucoprotein in the system and reached steady state after 6 h at 37 degrees C. Inhibition of [125I]beta 2-microglobulin binding to HLA-A,B heavy chains by beta 2-microglobulin purified from human urine or bovine colostrum resulted in identical inhibition curves and apparent dissociation constants of 1 X 10(-8) M.

Statistical analysis of beta 2-microglobulin levels in sera of lung and GI tract cancer patients.

Two radioimmunoassay methods were used to determine levels of beta 2-microglobulin in sera of lung and GI tract tumor patients (benign and malignant), as well as of normal individuals. Two screening sera panels were provided by the National Cancer Institute for this purpose. Statistical analysis of the results (performed by an independent institution) of the two panels provided discrepant results.

beta 2-Microglobulin: methods and clinical applications.

beta 2-Microglobulin is a low molecular weight protein that is found in most biological fluids. It was originally isolated from urine of cadmium-poisoned patients. Its amino acid sequence was established and shown to be structurally related to immunoglobulin constant domains.

Suppression of lymphocyte proliferation by a nonspecific factor produced by the Burkitt lymphoma-derived lymphoblast cell line.

The DAUDI lymphoblast cell line derived from a patient with Burkitt lymphoma was obtained from two different sources. One of these (DAUDI-I) produced a factor that inhibited lymphocyte proliferation in both human and mouse regardless of the stimulator, i.e.