Enzyme-linked Immunosorbent Assay Activity Research Articles

Production and Characterization of Monoclonal Antibodies That React to the Nucleocapsid Protein of Avian Metapneumovirus Subtype C

Monoclonal antibodies (MAbs) were prepared against avian metapneumovirus (aMPV) subtype C (aMPV/ Minnesota/turkey/1a/97). Six MAbs were selected based on enzyme-linked immunosorbent assay activities and characterized by isotyping, neutralization test, western blot analysis, and immunohistochemistry assay. The results showed that three MAbs (3E, 9D, and 12C) belonged to the IgG1 subclass, whereas the other three (5D, 8E, and 16E) were of the IgG2a subclass. None of the six MAbs neutralized aMPV infectivity at a detectable level, but all reacted with both denatured and nondenatured forms of the nucleocapsid (N) protein of aMPV, suggesting that these MAbs may recognize structurally independent epitopes of the N protein. These MAbs provide new tools and methods for investigating aMPV infection and pathogenesis, as well as diagnosis of aMPV disease.

Generation and characterization of putative monoclonal and polyclonal antibodies against tartrate-resistant acid phosphatase.

The generation of monoclonal antibodies (MAbs) specific for tartrate-resistant acid phosphatase (TRAP) may aid development of a serological immunoassay for this marker of bone resorption. The lack of MAbs to TRAP largely reflects the difficulty in obtaining sufficient antigen for in vivo immunization strategies. We have circumvented this problem by using in vitro immunization, requiring a small amount of TRAP isolated from osteoclastoma tumor. Two MAbs designated 312D and 310A were generated that exhibited weak anti-TRAP activity in enzyme-linked immunosorbent assay and immunocytochemistry. A polyclonal antibody to TRAP (Ab8023) was also raised in rabbit, using synthetic peptide. Ab8023 and MAbs 310A and 312D exhibited no activity against TRAP in dot-blotting experiments. Further characterization in enzyme-liked immunosorbent assay showed that Ab8023 was remarkably specific for TRAP whereas MAb 312D cross-reacted with another metalloenzyme, human prostatic acid phosphatase.

Use of chelating agents as terminators of alkaline phosphatase activity in Enzyme-Linked Immunosorbent Assay (ELISA) tests

For a long time chelating agents have been known to be inhibitors of alkaline phosphatase activity. However, the use of chelating agents in stopping alkaline phosphatase activity in ELISA reactions is a novel application with several advantages over conventionally used acids or bases.

Enterobacterial common antigen: isolation from Shigella sonnei, purification and immunochemical characterization.

In the studies presented the effective procedure of isolation and purification of enterobacterial common antigen from Shigella sonnei has been elaborated. The method is based on sonification of bacterial suspension in the presence of lysozyme and EDTA and subsequent extraction of the pellet with boiling water. The crude extract of common antigen was purified by fractionation with ethanol and chromatography on silica gel and Sephadex LH-20. The comparison of several extraction procedures of enterobacterial common antigen from Shigella sonnei proved that the method described above is most effective. The purified enterobacterial common antigen preparation obtained preserved full biological activity: antigenicity (precipitation and activity in enzyme-linked immunosorbent assay), immunogenicity in rabbits, ability to coat erythrocytes (passive hemagglutination) and inhibitory activity in passive hemagglutination. The pure enterobacterial common antigen was identified to 90% as a polymer of N-acetyl-D-mannosaminuronic acid and N-acetyl-D-glucosamine (2:1, molar ratio), O-acetylated and containing 3.2% fatty acids (C16:0 and C18:1, not oleic). It contains 5.3% nitrogen, less than 4% protein, less than 0.5% phosphorus and less than 1.6% neutral sugar; glycerol and RNA were not found in the preparation.