Human platelet-derived growth factor: radioimmunoassay and discovery of a specific plasma-binding protein.

The platelet-derived growth factor (PDGF) is the principal mitogen in serum for cultured cells of mesenchymal origin. PDGF also is a potent chemotactic protein for inflammatory cells and for cells required for wound repair. Because activity levels of PDGF in biological fluids are difficult to measure, we attempted to develop a radioimmunoassay for PDGF. Rabbits were immunized with purified PDGF; the antiserum obtained was monospecific for PDGF in immunodiffusion analysis against concentrated platelet lysates, serum, and plasma. A radioimmunoassay for PDGF was developed with a sensitivity of congruent to 0.2 ng/ml. Levels of PDGF in plasma/serum were measured and compared with PDGF levels determined by a receptor-competition assay and by a standard biological assay measuring incorporation of [3H]thymidine into 3T3 cells. Radioimmunoassay showed apparent PDGF levels of 50 ng/ml in human plasma and 103 ng/ml in serum. The 50 ng/ml PDGF in plasma was unexpected because the plasma samples contained little or no platelet release products as determined by very low levels of platelet factor 4. We therefore sought an immunologically reactive PDGF molecule in human plasma. No immunologically reactive protein was detected by immunodiffusion analysis or when plasma was treated with an immunoaffinity gel. Subsequently, a 125I-PDGF-binding protein was identified; the 125I-PDGF-plasma-binding protein complex was not reactive with anti-PDGF immunoglobulin. Correction for 125I-PDGF bound by the plasma-binding protein established serum levels of PDGF of congruent to 50 ng/ml; congruent to 50 ng/ml PDGF was found in serum by radioreceptor-competition assays and by mitogenic assays as well. The plasma-binding protein may serve to clear PDGF released in the circulation, thereby limiting PDGF activity to its local interactions at the site of blood-vessel injury.

Immunochemical characterization of monoamine oxidase from human liver, placenta, platelets and brain cortex

1. Antiserum raised to purified human liver monoamine oxidase was used to characterize the monoamine oxidase from human liver, brain cortex, placenta and platelets. 2. Antibodies to monoamine oxidase were purified by adsorption with a mitochondrial preparation. 3. Monoamine oxidase was present in liver particle-free supernatant as measured by enzyme activity and immunodiffusion. 4. Multiple precipitin lines were obtained on immunodiffusion analysis against the purified liver enzyme. It is proposed that this is due to either aggregation or to differential lipid binding. 5. The results suggest that the functionally different enzymes found in liver, brain cortex, platelets and placenta are immunochemically related and may be identical.

Conformational Alterations of α2-Antiplasmin Induced By Complex Formation With Plasmin

The conformational and structural changes induced in the α2-antiplasmin (AP) molecule by complex formation with plasmin have been analyzed utilizing quantitative radioimmuno-chemical analysis. Complexes prepared in plasmin excess (PAP-P) and therefore subjected to limited proteolysis and complexes prepared in AP excess (PAP-A) have been compared with free AP. With AP antiserum. PAP-A, PAP-P and AP yielded reactions of complete identity by immunodiffusion analysis. In radioimmunoassay, however, these were clearly distinguished, and four distinct sets of antigenic determinants were delineated. Set I determinants were expressed equivalently by PAP-P, PAP-A and AP and were, therefore, not altered by complex formation. This set was recognized by 90% of the antibodies, and the determinants Were all included within a large fragment of Mr 60,000 derived from the NH2-terminal region of AP. The other three sets of determinants were modulated by complex formation. Set II was expressed by PAP-A and AP but not by PAP-P, and these were sensitive to proteolysis by plasmin. Set III determinants were expressed only by AP and were localized to a peptide of Mr 8,000 derived from the COOH-terminal region of AP. Set IV determinants were also present only on AP but were not present in the peptide and required an intact reactive site in AP for expression. Thus, there is evidence for multiple conformational modulations in AP induced by complex formation, and these modulations can be pinpointed to specific loci within the AP molecule.

Conformational Alterations of α2-Antiplasmin Induced by Complex Formation with Plasmin

The conformational and structural changes induced in the α2-antiplasmin (AP) molecule by complex formation with plasmin have been analyzed utilizing quantitative radioimmuno-chemical analyses. Complexes prepared in plasmin excess -(PAP-P) and therefore subjected to limited proteolysis and complexes prepared in AP excess (PAP-A) have been compared with free AP. With AP antiserum, PAP-A, PAP-P and AP yielded reactions of complete identity by immunodiffusion analysis. In radioimmunoassay, however, these were clearly distinguished, and four distinct sets of antigenic determinants were delineated. Set I determinants were expressed equivalently by PAP-P, PAP-A and AP and were, therefore, not altered by complex formation. This set was recognized by 90% of the antibodies, and the determinants were all included within a large fragment of Mr 60,000 derived from the NH2-terminal region of AP. The other three sets of determinants were modulated by complex formation. Set II was expressed by PAP-A and AP but not by PAP-P, and these were sensitive to proteolysis by plasmin. Set III determinants were expressed only by AP and were localized to a peptide of Mr 8,000 derived from the COOH-terminal region of AP. Set IV determinants were also present only on AP but were not present in the peptide and required an intact reactive site in AP for expression. Thus, there is evidence for multiple conformational modulations in AP induced by complex formation, and these modulations can be pinpointed to specific loci within the AP molecule.

Comparative virulence and immunodiffusion analysis of Petriellidium boydii (Shear) Malloch strains isolated from feedlot manure and a human mycetoma

The virulence to mice of a Petriellidium boydii strain isolated from feedlot manure was compared with that of a strain from a human mycetoma. Washed spores suspended in 5% hog gastric mucin were injected intraperitoneally into non-inbred female albino Swiss mice. In 21 days, the mycetoma strain killed 95% of the test animals whereas injection of eight times as many feedlot strain spores resulted in only 28% mortality. A progressive, exclusively sinistral (left-sided) torticollis was the most obvious symptom of experimental petriellidiosis. Mycelia bearing annellospores were observed in PAS-stained sections of most internal organs, including the brains, of the test animals. Immunodiffusion analyses were conducted on soluble antigen preparations with concentrated antisera produced in rabbits using live spore suspensions. The two strains-contained common antigens but the proportions of two were significantly different.

Immunochemical studies on burro antibodies to Histoplasma capsulatum

Burro antiserum to Histoplasma capsulatum was studied from the viewpoint of precipitating antibody distribution among euglobulins, pseudoglobulins, and immunoglobulin classes. By immunodiffusion analysis it was determined that throughout immunization most of the antibody was euglobular, but there was an increase in pseudoglobular antibody as immunization progressed. By immunoelectrophoretic and immunodiffusion analyses of specifically purified antibody, and antibody non-specifically fractionated by column chromatography and ultracentrifugation, it was established that there was no demonstrable IgM antibody, that most of the antibody appeared to be IgG, and that a γ1 immunoglobulin, probably IgA or T-globulin, may have been responsible for some of the activity. No major changes in the distribution of antibody among immunoglobulin classes seemed to occur during immunization.