Subject(s)
Polymerase Chain Reaction/veterinary , Toxoplasma/genetics , Toxoplasma/isolation & purification , Toxoplasmosis, Animal/diagnosis , Animals , Base Sequence , Birds/parasitology , DNA Primers/genetics , DNA Probes/genetics , DNA, Protozoan/genetics , Evaluation Studies as Topic , Mammals/parasitology , Molecular Sequence Data , Polymerase Chain Reaction/methods , Polymerase Chain Reaction/statistics & numerical data , Reptiles/parasitology , Sensitivity and Specificity , Toxoplasmosis, Animal/parasitologyABSTRACT
CD63 is a 53-Kd lysosomal membrane glycoprotein that has been identified as a platelet activation molecule. The current study presents evidence that CD63 and Pltgp40, a platelet membrane glycoprotein identified in this laboratory, are the same molecule and that CD63/Pltgp40 is identical to the well-characterized, stage-specific melanoma-associated antigen ME491. Identity of CD63 and Pltgp40 was demonstrated by immunoprecipitation and sequential immunodepletion studies, which showed that the anti-Pltgp40 monoclonal antibody (MoAb) H5C6 and an anti-CD63 MoAb CLB/Gran 12 recognized the same 40- to 55-Kd platelet glycoprotein. In addition, the anti-CD63 MoAb specifically recognized immunoaffinity-purified Pltgp40. Amino acid sequences obtained from NH2-terminal and tryptic fragment peptides of Pltgp40 were used to generate complementary DNA (cDNA) probes using the polymerase chain reaction (PCR) technique. A 386-bp cDNA probe partially encoding CD63/Pltgp40 and a full length cDNA probe were 100% identical to the corresponding sequence of ME491. Antibodies H5C6 and CLB/Gran12 immunoprecipitated a 30- to 60-Kd heterodisperse glycoprotein from G361 melanoma cells, as had previously been reported for antibodies recognizing ME491. These data, taken together with the extensive homology recently reported between ME491, the Schistosoma mansoni membrane antigen SM23, CD37, the tumor-associated antigen CO-029, and the target of an antiproliferative antibody-1, suggest that CD63 is a member of a new family of related molecules.
Subject(s)
Antigens, CD/analysis , Antigens, Neoplasm/analysis , Platelet Activation , Platelet Membrane Glycoproteins/analysis , Amino Acid Sequence , Antibodies, Monoclonal , Antigens, CD/genetics , Antigens, Neoplasm/genetics , Cell Line , Humans , Melanoma , Molecular Sequence Data , Molecular Weight , Platelet Membrane Glycoproteins/genetics , Polymerase Chain Reaction , Sequence Homology, Nucleic Acid , Tetraspanin 30ABSTRACT
Three leukocyte adhesion receptors have been described which mediate intercellular binding of leukocytes: LFA-1 (CD11a/CD18), Mac-1 (CD11b/CD18), and p150/95 (CD11c/CD18). We have previously reported the production of several monoclonal antibodies against the common subunit of these receptors (CD18). We have describe the production of monoclonal anti-idiotype antibodies against one of the anti-CD18 antibodies (H52) which has been shown to inhibit potently the function of leukocyte adhesion receptors. Three IgG1 and two IgM anti-idiotype antibodies were derived which recognized private idiotopes on the H52 molecule. Two of these antibodies blocked the binding of H52 to purified LFA-1 and to cell surface expressed antigen. One of the antibodies (AIM.6) was shown to be an internal image-type (Ab2 beta) antibody based on inhibition of its binding to H52 by purified LFA-1 and by its ability to induce Ab3 which recognize LFA-1 when used as immunogen. The AIM.6 Ab2 beta antibody was tested for recognition of leukocyte adhesion ligands in LFA-1-mediated leukocyte adhesion and activation assays. The AIM.6 antibody did not block intercellular adhesion of leukocytes or mitogen stimulation of T cells, functions which were completely inhibited by low concns of H52. AIM.6 Ab2 beta antibody bound to H52 very well at 0 degrees C but bound very poorly or not at all at 37 degrees C. Binding studies on a panel of anti-CD18 monoclonal antibodies showed that the idiotope defined by AIM.6 was unique to H52 and an antibody recognizing the same epitope on CD18 (H5B9). This result showed that inhibitory anti-CD18 monoclonal antibodies utilize at least two distinct paratopes in binding to CD18. The above results are in contrast to those obtained in other systems in which Ab2 beta antibodies against receptor-specific Ab1 antibodies recognize receptor ligands and are discussed in the context of ligand recognition by leukocyte adhesion receptors.
