Monoclonal antipeptide antibodies: affinity and kinetic rate constants measured for the peptide and the cognate protein using a biosensor technology.

The interaction of antipeptide antibodies with the corresponding peptide and the cognate protein has been compared using a novel biosensor technology (BIAcore, Pharmacia). The peptide corresponds to residues 110-135 of the coat protein of tobacco mosaic virus, known to encompass an alpha-helical region reactive with antiprotein antibodies. A panel of 33 monoclonal antibodies raised against the peptide was studied and the epitope recognized by these antibodies was determined by the pepscan method. Further discrimination between the antibodies was performed by measurements of association and dissociation kinetic constants. Several antibodies showed an heterogeneous binding profile when reacting with the 25 residue long peptide but not with a shorter 10 residue peptide suggesting that they recognized different conformational states in the longer peptide. Equilibrium affinity constants were calculated for five of the antibodies and were found to be 10-50 times higher for the peptide than for the protein, the difference being caused mainly by a lower association rate constant.

Differentiation of peanut clump virus serotypes by monoclonal antibodies.

A panel of monoclonal antibodies (mAb) produced against peanut clump virus (PCV) was used to characterize five serotypes of the virus. Four different formats of enzyme-linked immunosorbent assays (ELISA) were compared to establish the most suitable one for diagnosis of infected plants and for serotype differentiation. Since most mAb retained their activity when used for coating microtitre plates, a dual mAb-type assay was found to be most suitable. The same mAb could be used in ELISA as coating and as biotinylated antibody. Because of the ability of mAb to recognize subtle conformational alterations in the viral antigen, it is important to carefully select the ELISA format used for comparing different viral isolates.

Specificity of antibodies raised against triacetylated histone H4.

Ten monoclonal antibodies (mAbs) were obtained by immunizing animals with triacetylated histone H4 from cuttle-fish. The fine specificity of these antibodies was studied using various populations of acetylated H4, (H3H4)2 tetramers and histone octamers as well as with acetylated and nonacetylated peptides of H4. None of these mAbs were found to recognize triacetylated H4. Only five of them bound to diacetylated, monoacetylated and nonacetylated H4. One antibody was specific for H4 associated in the form of histone octamers and did not bind to any nonacetylated or acetylated form of H4 monomers. Eight of the antibodies were specific for residues situated in the region 9-23 of H4. None of the mAbs was completely specific for acetylated forms of H4. In contrast, antisera raised in rabbits against triacetylated H4 reacted strongly with tri and diacetylated H4, weakly with monoacetylated H4 and barely or not at all with nonacetylated H4.

Temporal arteritis in polymyalgia rheumatica: immune complex deposits and the role of the leukocyte elastase in the pathogenesis.

Immunohistochemical studies were performed on the temporal artery of 34 patients with clinically established polymyalgia rheumatica (PR) or temporal arteritis, 6 patients with vasculitis, and 25 patients with various diseases. The combined immunofluorescence and peroxidase-anti-Peroxidase Methode zeigte Immunoglobulin- und C3-Ablagerunin histologically affected and to some degree also in unaffected arteries of patients with PR and in all patients with temporal arteritis. The deposits were found both inter- and intracellularly, and contained IgA and to a lesser extend IgG, IgM, and C3. Linear deposits of leukocyte elastase were found along the fragmented internal lamina, and decaying polymorphonuclear (PMN) leukocytes surrounded by elastase-containing inclusions were found in the neighborhood of zones rich in elastic material. These findings suggest that immune complex deposition is a prominent feature of temporal arteritis and that the PMN elastase is probably involved in the destruction of elastic fibers. The combined immunohistochemical investigation appears to increase the diagnostic value of temporal artery biopsy.

Degradation in vivo of articular cartilage in rheumatoid arthritis by leucocyte elastase from polymorphonuclear leucocytes.

Using a specific substrate, no leucocyte elastase activity could be detected in 55 synovial fluids, including 29 from patients with rheumatoid arthritis (RA). However, a high percentage of samples contained phagocytic inclusions of elastase, alpha 1-proteinase inhibitor (alpha 1-PI) and alpha 2-macroglobulin (alpha 2-MG) in both the polymorphonuclear (PMN) and mononuclear phagocytes. Immunofluorescence and indirect peroxidase-antiperoxidase staining of articular cartilage (ACA) from 52% of 21 patients with RA and one with juvenile RA (JRA) showed presence of elastase in the superficial layer of microscopically intact but proteoglycan depleted pannus-free ACA. In histologically altered pannus-free RA-ACA superficial elastase deposits were found in 24% of the cases. Adjacent ACA sections contained IgG, C3, alpha 1-PI and rarely alpha 2-MG. RA-ACA below or surrounded by pannus showed close contact with intact and decaying PMN in 62% and 48% of the cases, respectively. ACA specimens from patients with degenerative disease and systemic lupus were negative. These findings strongly suggest that PMN leucocyte elastase is operative in the degradation of RA-ACA and JRA-ACA, and that this activity is largely dependent upon the presence of entrapped immune complexes in such cartilage.

[Chronic polyarthritis: role of polymorphonuclear leukocytes in the destruction of pannus-free articular cartilage]. / Die chronische Polyarthritis: Rolle der polymorph-kernigen Leukozyten bei der Zerstoerung von Pannus-freiem Gelenkknorpel.

The synovial fluid (SF) of RA patients contains large amounts of PMN which are well equipped with neutral enzymes to degrade articular cartilage: elastase and cathepsin G, which both destroy proteoglycans and native collagen, as well as 2 types of collagenoases. Indirect evidence suggests that PMN might be important in the destruction of RA articular cartilage. In 19 SF of RA patients no free elastase or collagenase was found. Using immune histochemical methods, we observed that PMN and macrophages of SF contain both elastase and alpha 1-anti-trypsin and alpha 2-macroglobulin. Peripheral PMN - but not monocytes - contain elastase, however both types of cells lack alpha 1-antitrypsin and alpha 2-macroglobulin. Elastase is demonstratable in the superficial layer of pannus free RA articular cartilage. These findings suggest that neutral proteinases from PMN in RA SF are generally neutralized by physiologic inhibitors and removed by phagocytes. The enzyme-inhibitor interaction might be bypassed during "frustrated phagocytosis" so that enzymes like PMN elastase can damage RA articular cartilage.