Reaction Of Complete Identity Research Articles

Implementation of immunohistochemical methods in diagnostics of bovine sarcocystosis

Bovine sarcocystosis was examined in three age categories of cattle using direct and immunohistochemical methods. The direct methods of compression, trypsin digestion and a histological method were applied, and the applied immunohistochemical methods included peroxidase antiperoxidase (PAP), the highly sensitive streptavidin biotin immunoperoxidase complex technique (LSAB+) and indirect immunofluorescence (IF). Primary rabbit antiserum obtained for the zoites Sarcocystis cruzi syn. S. bovicanis was used for all immunochemical and immunohistochemical examinations. Cross reactivity was determined using primary rabbit antiserum to antigens of S. ovifelis zoites. Both primary antiserums were produced in the laboratory, as no commercial preparations were available on the market. The evaluation of the sensitivity and efficacy of the immunohistochemical methods was performed on the grounds of comparisons with the direct methods: compression, trypsin digestion and a histological method. The applied immunohistochemical analyses are very specific in proving sarcocystosis in cattle, especially the LSAB+ technique, and, according to their sensitivity in detecting this infection, they are similar to the method of trypsin digestion. The obtained rabbit antiserum against antigens of S. bovicanis zoites yielded good results in the diagnosis of the homologous specie. Proven cross reactivity of the homologous and heterologous antiserums and antigens, as an immunochemical reaction of complete identity in one part to the antigen structure, was established in primary rabbit antiserum to S. ovifelis zoites, which indicates that heterologous antiserums can also be used in immunohistochemical reactions in the diagnostics of bovine sarcocystosis. .

Monoclonal rat antibodies to rat carcinoembryonic antigen.

An IgM-class rat monoclonal antibody, designated 5B1, was produced against rat carcinoembryonic antigen. This antibody, which can be considered an autoantibody, formed a single precipitation line in a gel diffusion test with an extract of the CEA-positive rat colonic adenocarcinoma, RCA-1. This line gave a reaction of complete identity with a line formed by a rabbit antiserum specific for rat CEA. 5B1 also bound to extracts of the RCA-1 tumor in EIA, but did not bind at all to extracts of the CEA-negative rat colonic adenocarcinoma, RCA-2, nor to extracts of normal rat tissues including liver, kidney and intestine. Inhibition tests showed that the activity of the monoclonal antibody in EIA was abolished by extracts of RCA-1 tumor, but was hardly affected by extracts of the other tissues.

Electrophoretic migration of Paracoccidioides brasiliensis specific antigenic fraction.

Using the technique of immunoelectroosmophoresis-immunodiffusion (IEOP-ID), two antigenic fractions of Paracoccidioides brasiliensis one of them species-specific, produced precipitin bands in both the cathodic and anodic zones. Reactions of complete identity among the bands formed in these zones were demonstrated by a modification of the technique, employing additional wells around the central antigen well. Such bands would correspond to a simple diffusion of the corresponding antigenic fractions rather than to active electrophoretic migration.

Physicochemical and immunochemical properties of γ1 heavy chain disease protein baz

The physicochemical and antigenic properties of a γ1 heavy chain disease protein (γl HCD BAZ) are described. Protein BAZ is positive for the Glm(1) determinant and gives a reaction of identity with Fc fragment, whether derived from Cohn Fraction II or from an IgG1 myeloma protein. It also gives a reaction of complete identity with the γHCD proteins CRA and ZUC. The mol. wt of the native protein was determined from (1) sedimentation-diffusion, (2) sedimentation-viscosity, and (3) sedimentation-equilibrium measurements to be approx 60,000 daltons. The mol. wt of the unreduced protein in 6 M guanidine hydrochloride was found to be 30,000 daltons, which was identical to that of the reduced-alkylated form in 6 M guanidine. These results established that γ1 HCD BAZ is a noncovalently linked dimer and suggested the possibility of a missing hinge region.

Half-Life of Platelet Factor 4 (PF-4) in Plasma and Platelets from Macaca Mulatta

Human platelet factor 4 (PF-4) showed a reaction of complete identity with PF-4 from Macaca mulatta when tested against rabbit anti-human-PF-4. Such immunoglobulin was used for quantitative precipitation of in vivo labelled PF-4 in monkey serum. The results suggest that the active protein had an intra-platelet half-life of about 21 hours. In vitro 125I-labelled human PF-4 was injected intravenously into two monkeys and isolated by immuno-precipitation from platelet-poor plasma and from platelets disrupted after gel-filtration. Plasma PF-4 was found to have a half-life of 7 to 11 hours. Some of the labelled PF-4 was associated with platelets and this fraction had a rapid initial disappearance rate and a subsequent half-life close to that of plasma PF-4. The results are compatible with the hypothesis that granular PF-4 belongs to a separate compartment, whereas membrane-bound PF-4 and plasma PF-4 may interchange.

Large scale separation of protease inhibitors from malignant human breast tissue.

α1-Antitrypsin was isolated as an electrophoretically homogeneous protein in preparative quantities from malignant human breast tissue by a two-step procedure of affinity chromatography on Sepharose-chymotrypsin, followed by polyacrylamide gel electrophoresis. Additionally, α2-macroglobulin and α1-antichymotrypsin were bound to, and subsequently eluted from the column. Antithrombin III and α1-acid glycoprotein, which were also components of the tissue extracts, were not bound, and appeared in the breakthrough peak. α1-Antitrypsin appeared to be partially modified immunologically by Sepharose-chymotryppin chromatography. However, α2-macroglobulin remained immunologically unaltered. Upon subsequent polyacrylamide gel electrophoresis, the α1-antitrypsin regained its capacity to give an immunological reaction of complete identity. Inhibitory activity toward proteases is also observed in filtrates of 1000 molecular weight cut-off membrane filters. Endogenous caseinolytic activity was observed in several eluate fractions. However, only N-benzoyl-L-tyrosine-ethyl ester, of the model compounds tested, was cleaved. The esterase activity is attributed to a breast tissue component since the column was free from dissociable chymotrypsin. Affinity chromatography on Sepharose-chymotrypsin represents a means of rapidly separating 37.5% of the total antichymotryptic activity from breast tissue extracts in high purity, in a single step.

Studies of mycobacterial antigens, with special reference to Mycobacterium leprae.

Eight individual antigens were detected in soluble antigen preparations from Mycobacterium leprae bacilli by using pools of serum samples from lepromatous leprosy patients as antibody reagents in crossed immunoelectrophoresis. Two of these antigens were analyzed further. Antgent no. 1 gave an elution pattern on Sephadex G-200 corresponding to a molecular weight of 285,000. This antigen was also present in three slow-growing and eight fast-growing mycobacterial species. There was a reaction of complete identity in immunological tests using lepromatous serum pools as well as with rabbit antisera raised against M. leprae and M. smegmatis. Antigen no. 21 of M. leprae showed antigenic heterogeneity when compared with other species. Three types of antigenic determinants were detected; one, called 21A, was shared by all mycobacteria, another, called 21B, was limited to antigen no. 21 of M. leprae; a third, called 21C, was present in all mycobacteria except the leprosy bacillus. This submolecular heterogeneity may indicate a separate taxonomic position of M. leprae among the mycobacteria.

Production of antiserum to human properdin and demonstration of antigenic differences between the native and activated protein.

Antisera were raised in rabbits to human properdin in the precursor form (P) and in the activated state (P). On Ouchterlony analysis using the anti-P, reactions of complete identity were obtained between P, P, and properdin in twofold concentrated serum (NHS). However, when anti-P was used, a reaction of identity was obtained only between P and NHS, and a reaction of partial identity was formed between P and P and between properdin in NHS and P, suggestive of the fact that certain antigenic determinants in P may be lacking in P. The results indicate that activation of precursor properdin may involve proteolytic cleavage and/or conformational alterations of the molecule.

A fetuin-like antigen from human nephroblastoma.

An antigen was detected in pooled human nephroblastomas using antiserum prepared in rabbits against an ethylemediaminetetra acetic acid (EDTA) extract of the tumors. This antigen was not found in normal human plasma or kidney extracts, and was not related to the ABO or Forssman blood groups. The antigen was detected in extracts of cultured nephroblastoma cells, but was not present in extracts of normal human fetal kidney cell cultures. The antigen is believed to be present at the cell surface, as cell viability was not significantly lowered during the extraction procedure. A reaction of complete identity was demonstrated by Ouchterlony double diffusion experiments with this antigen and purified bovine fetuin. The antigen was not found in extracts of human fetal spleen, thymus or kidney, nor in human fetal serum. Furthermore, the antigen does not possess determinants in common with the human alpha-fetoprotein of hepatomas, nor was it detected in human renal clear cell carcinoma. Initial characterization of the antigen showed it to be nondialysable, not sedimentable at 100,000 times g for 2 h, stable to repeated freeze-thawing and to incubation at 56 degrees C for 1 h, and water soluble over a wide pH range. The antigen was susceptible to digestion with pronase and trypsin and possibly hyaluronidase, but not to ribonuclease or neuraminidase. The protein portion is therefore of major importance to the structural integrity of this antigen. The relationship between this antigen and other abnormal materials reported previously in nephroblastoma patients is being studied.

Immunologic relationship of purified human skin collagenase to other human and animal collagenases

Antibodies prepared to a number of animal and human collagenases have been used to investigate the relationships between these enzymes. The human skin collagenase used as an immunogen was one of two chromatographically separable peaks of collagenolytic activity obtained from culture media of normal human skin. On the basis of their electrophoretic mobility on polyacrylamide gel, they have been designated as fast-moving or slow-moving human skin collagenase. The fast moving enzyme was further purified and used to produce an anti-human skin collagenase antibody. Anti-fast-moving human skin collagenase antibody gives a reaction of complete identity on immunodiffusion with slow-moving skin collagenase as well as with preparations of human gingival and rheumatoid synovial collagenases. In addition, anti-fast-moving human skin collagenase antibody produces complete inhibition of these other human collagenases, suggesting that these enzymes are closely related proteins. Antibodies to tadpole and rat uterine collagenases are immunologically not identical when compared with one another or to the human skin enzyme. Crustacean hepatopancreas and bacterial collagenases also fail to react with anti-fast-moving human skin collagenase antibody, further indicating species specificity among collagenases.

Reactions of Newcastle Disease Virus with Infectious Mononucleosis Sera in Agar Gel

Summary A gel diffusion test with infectious mononucleosis (IM) sera and Newcastle disease virus (NDV) is described. IM serum diffusing against concentrated virus preparation was found to form a distinct reaction line. Lines formed by various IM sera merged in reactions of complete identity. Positive results were obtained with the VIC strain of NDV but not with the B1 strain of NDV or with other myxoviruses. Of 65 IM sera, 43 were positive in gel diffusion tests but only 2 of 125 non-IM sera gave positive results in this test. IM serum diffusing simultaneously against NDV and bovine erythrocyte stromata added to opposite wells formed reaction lines which crossed, pointing to complete nonidentity of the antigens and antibodies participating in the test with NDV and in the heterophile test. The immunizing stimuli which are possibly responsible for the formation of heterophile antibodies and antibodies combining with NDV are discussed. The data suggest that the gel diffusion test would be of value for diagnostic purposes.