Detection of antibody responses and delayed dermal hypersensitivity with Blastomyces dermatitidis yeast and mycelial lysate antigens

Yeast cell lysate and mycelial lysate antigens prepared from one strain (T-58) of Blastomyces dermatitidis were evaluated with respect to the detection of antibodies and delayed dermal hypersensitivity. Comparable ELISA sensitivity values were evidenced with the two antigens when assayed against serum specimens from dogs with blastomycosis, sera from non-infected dogs residing in endemic and nonendemic areas for blastomycosis and sera from rabbits that were hyperimmunized with B. dermatitidis antigens. Specificity determinations with anti -Histoplasma capsulatum rabbit sera indicated that both reagents exhibited only minimal cross-reactivity; the mycelial antigen was slightly more specific than the yeast phase reagent. Similar sensitivity and specificity results were experienced when the two antigens were used to detect delayed dermal hypersensitivity in guinea pigs previously sensitized with B. dermatitidis or H. capsulatum.     

Mouse monoclonal antibodies to swine blood group antigens

Eight mouse hybridomas with haemagglutination capacity to swine blood group antigens were obtained, three of them producing antibodies capable of being used as blood group reagents. Two detected the Ba factor and another the Fa factor. The others gave non-specific and weak reactions or cross-reaction with antigens present in more than one system. We conclude that mouse monoclonal antibodies are also suitable for use in swine as a complement of polyclonal reagents.     

Absence of BoLA class I antigens on bovine spermatozoa

Forty AI bulls were tested for BoLA class I antigens by means of eight specific polyclonal reagents. By means of immobilization and sperm penetration tests these antigens were not detected on sperm cells. Isoimmunization studies with the use of sperm as antigenic stimuli and insemination of frozen spermatozoa diluted in specific reagents did not prove the presence of BoLA class I antigens on bovine spermatozoa. The cytotoxic tests used in this investigation were not reliable.     

Absence of BoLA class I antigens on bovine spermatozoa

Summary. Forty AI bulls were tested for BoLA class I antigens by means of eight specific polyclonal reagents. By means of immobilization and sperm penetration tests these antigens were not detected on sperm cells. Isoimmunization studies with the use of sperm as antigenic stimuli and insemination of frozen spermatozoa diluted in specific reagents did not prove the presence of BoLA class I antigens on bovine spermatozoa. The cytotoxic tests used in this investigation were not reliable.     

Analysis of the HLA-linked SB gene system with cloned and uncloned alloreactive T-cell lines

Further enhancement of cellular typing for antigens of the new HLA-linked "SB" gene was undertaken by T-cell expansion and cloning. The PLT reagents which define these antigens could be expanded over 100-fold with interleukin 2 (IL-2), without loss of specificity. Cloning efficiencies in limiting dilution of over 50 percent could be achieved, although only 5 percent of these could be expanded extensively. Detailed analysis was performed with clones from a highly restricted PLT reagent raised between an HLA recombinant donor and his sibling whose only known HLA difference was SB4. The antigens recognized by the 11 independent clones analyzed appeared to segregate in this family with HLA. Although the two SB4-positive haplotypes in the family were essentially indistinguishable by the clones, they detected heterogeneity among unrelated donors matched for SB4 (as defined by bulk PLT reagents). Such heterogeneity did not appear to be due to differences between clones in the kinetics of their responses, but could be explainable by complexity of the SB molecule or by new HLA antigens.     

Experimental basis for using Soviet bentonite clays as immunosorbents for serological studies

The methodological principles of using Soviet bentonite clays as immunosorbents necessary for serological studies in bacterial infections were worked out. The method for the preparation of antigenic bentonite diagnostic reagents was experimentally substantiated. Bentonite diagnostic reagents were first used in a new method for the rapid indication of bacterial antigens in environmental objects with a view of solving epidemiological problems.     

Monoclonal antibodies to parasite antigens found in the serum of Dirofilaria immitis-infected dogs

We recently reported that parasite antigens are detectable in the serum of Dirofilaria immitis-infected dogs by counterimmunoelectrophoresis (CIE). Hybridoma cell lines that produce monoclonal antibodies specific for these antigens were obtained by immunizing mice with a partially purified antigen preparation, fusing spleen cells with SP-2 myeloma cells, and screening cell culture supernatants for antibody by ELISA and CIE inhibition. Antibodies specific for two epitopes shared by the two major circulating parasite antigens were identified. Immunoperoxidase studies showed that the epitopes recognized by the monoclonals were widely distributed in D. immitis, but the female uterus and eggs were particularly strongly labeled. A monoclonal antibody-based ELISA was developed to measure parasite antigens in dog sera. Parasite antigens were detected in 45 of 46 sera from infected dogs but were absent in sera from uninfected dogs and sera from dogs infected with Dipetalonema reconditum. Serum antigen content was significantly correlated with the number of female worms recovered from infected dogs (r = 0.82, p less than 0.001). Antigenemia was first detected 6 mo after infection, and antigen levels remained fairly stable between 9 and 21 mo after infection. Parasite antigen detection with this monoclonal antibody-based ELISA appears to be superior to previously described diagnostic methods for canine dirofilariasis in terms of sensitivity, specificity, and relation to infection intensity.     

Nonhistone nuclear antigens reactive with autoantibodies. Immunofluorescence studies on distribution in synchronized cells

Sera from patients with certain autoimmune diseases tht contained autoantibodies to nonhistone nuclear antigens were used as reagents in an indirect immunofluorescent study. The distribution of these nuclear antigens was determined in synchronized human B lymphoid cells. Autoantibodies to Sm antigen, nuclear ribonucleoprotein complex and SS- B antigen were used. Although all three nonhistone antigens appeared to show speckled nuclear straining patterns in the Go phase, different patterns of staining were present at other periods of the cell cycle. The SS-B antigen showed a distinctly nucleolar localization during the G1/early S phase. These studies demonstrate that autoantibodies occurring in certain human diseases can be useful reagents for the immunohistological localization of nuclear macromolecules and for tracing their pathways during different phases of cell growth and differentiation.     

Immunity of dogs against Babesia canis, its vector tick Dermacentor reticulatus, and Ixodes ricinus in endemic area

Previous epidemiological studies allowed us to accurately define endemic areas of canine babesiosis and tick distribution in southeastern France (Martinod, 1983). Using a micro-ELISA test 100 dogs sera were tested with 3 antigens: Babesia canis, Dermacentor reticulatus and Ixodes ricinus. Antibodies against B. canis and its vector D. reticulatus were detected in an endemic area, sometimes with high levels (optical density 1.38 and 0.80 respectively). A correlation factor and regression lines were found between ELISA activity of B. canis and vector tick antigens, even for dogs which never showed any babesiosis symptoms. These results were compared with those of an area without any babesiosis. Furthermore I. ricinus antigens detected ELISA activity in sera of dogs; some cross reactions were observed between I. ricinus and D. reticulatus antigen.     

In vitro-isolated human cytotoxic T-lymphocyte clones detect variations in serologically defined HLA antigens

T cells of two donors, JR (HLA-A23, 29; B7,7; G; DRw5) and HG (HLA-A2, 23; B40, w44; Cw4), were stimulated with cells from an HLA homozygous lymphoblastoid cell line JY (HLA-A2, 2; B7,7, C-, DRw4, 6) and cloned by limiting dilution after the third stimulation. Two cytotoxic T-cell (CTL) clones, JR-2-16 (from donor JR) and HG-31 (from donor HG), were used for detailed studies. The results of a panel study using lymphocytes from HLA-typed individuals and a study with two HLA recombinant families indicate that the antigens recognized by the CTL clones JR-2-16 and HG-31 were highly associated with HLA-A2 and HLA-B7, respectively. Blocking studies with a monoclonal antibody recognizing a framework determinant on HLA-A, -B and-C antigens and a monoclonal antibody reacting with HLA-A2 support the notion that JR-2-16 and HG-31 interact with the HLA-A2 and the HLA-B7 antigens per se. However, these clones did not recognize the HLA-A2 and HLA-B7 of all donors typed for these antigens, suggesting that the HLA-A2 and HLA-B7 antigens of these particular donors are variants of the serologically defined HLA antigens. These results indicate that in vitro-derived human CTL clones detect variations in the serologically defined allospecificities and can be used as reagents to elucidate the polymorphism of HLA antigens further.Abbreviations used in this paper: CTL cytotoxic - T lymphocytes - BSA bovine serum albumin - PHA phytohemagglutinin - Con A concanavalin A.     

Erythrocyte antigens in Norwegian goats: serological and genetic studies.

Goat alloantisera and bovine blood typing reagents were used to characterize eight erythrocyte antigen specificities in Norwegian goats by cluster analysis, absorption and family studies. Most of the goat sera were produced by injecting dams once or twice with blood cells or blood from their own kids. The characterized specificities were designated N1-N8. The two specificities N5 and N8 were recognized both by goat alloantisera and by reagents against the bovine factors E'1 and E'2 (N5) and I (N8), which are allelic factors in the bovine B-system. In goat families, the two specificities also behaved as alleles. Consequently, the locus or gene system coding for these specificities was called the B-system of goats. The six other erythrocyte antigens were provisionally assigned to six separate loci. In addition, a bovine anti-sheep R factor reagent reacted with cells from 3.3% of the goats tested, whereas a monoclonal antibody against the Forssman antigen reacted with all the goats tested.     

Evaluation of ready-to-use liquid reagents for clinical chemistry in laboratory animals.

We evaluated the ready-to-use liquid reagents for clinical chemistry (6 tests), to assess their suitability for use in the toxicology laboratory setting. Hitachi 736 automated analyzer was used for the analyses. The evaluation included the following studies: Precision, Linearity, Effects of interference substances such as hemolytic hemoglobin, bilirubin, turbidity to the analytical values and correlation to the solid reagents, which are prepared each time they are needed. The precision and linearity data were within the reagents' specifications. Results of comparison of the liquid reagents and the solid reagents in analyzing plasma samples of rats, dogs and monkeys were generally good except for a bias in results for GOT and GPT, regardless of the animal species tested. It is concluded that these types of liquid reagents can be used in clinical pathology examinations in animal studies.     

Synthesis of bis- and tris-branched COOH-terminal pegylating reagents: conjugation to NH-terminal peptides.

In previous studies we reported an orthogonal protection scheme that was developed for the solution-phase synthesis of a family of bis- and tris-pegylating reagents which contain a free NH(2)-terminus. These pegylating reagents were coupled to the COOH-terminus of a model peptide. In the present study we report on the solution synthesis of a novel family of bis- and tris-pegylating reagents which contain a free COOH-terminus. To illustrate their general utility, conditions were developed for the coupling of these novel pegylating reagents to the NH(2)-function of a model pentapeptide. Taken together, our studies demonstrate that these pegylating reagents are well suited for conjugation to peptides and proteins that contain either free COOH- or NH(2)-functions. These reagents may have general utility in therapeutic development as branched pegylation has been shown to provide more effective protection of proteins from proteolysis by shielding the protein surface from approaching macromolecules.     

Monoclonal antibodies as a tool for phylogenetic studies of major histocompatibility antigens and β 2-microglobulin

The cross-reactivity of several monoclonal antibodies recognizing monomorphic determinants of human HLA-A, B, C, and DR antigens and human 2-microglobulin (2m) has been studied on peripheral blood leukocytes in 24 different species. An monoclonal HLA-A-, B-, and C-specific antibody and four monoclonal HLA-DR-specific antibodies cross-reacted with cells from all the primate species tested. Furthermore, antibodies HLA-DR-specific were positive with peripheral blood leukocytes (PBL) from cows, goats, sheep, horses, and dogs. Two monoclonal 2m-specific antibodies, which were positive with PBL from certain primates, also reacted with cells from cows, goats, sheep, horses, and dogs. Two other #2m-specific antibodies reacted only with PBL from chimpanzees. No reaction could be detected with all our reagents in other classes tested (birds, reptiles, amphibians, and Teleostei).     

New immunolatex spheres: visual markers of antigens on lymphocytes for scanning electron microscopy

New immunochemical reagents consisting of antibodies bound to small latex spheres were used as visual markers for the detection and localization of cell surface antigens by scanning electron microscopy. Cross-linked latex spheres of various sizes from 300 to 3,4000 A in diameter were synthesized by aqueous emulsion copolymerization of methacrylate derivatives containing hydroxyl and carboxyl functional groups. Proteins and other molecules containing primary amino groups were covalently bonded to the acrylic spheres under a variety of mild conditions by the aqueous carbodiimide, cyanogen bromide, and glutaraldehyde methods. For use in the indirect immunochemical-labeling technique, goat antibodies directed against rabbit immunoglobulins were bonded to the spheres. These immunolatex reagents were shown to bind only to cells (red blood and lymphocytes) which had previously been sensitized with rabbit antibodies against cell surface antigens. Mouse spleen lymphocytes with exposed immunoglobulins on their surface (B cells) were labeled with these spheres and distinguished from unlabeled or T lymphocytes by scanning electron microscopy. The distribution of Ig receptors on lymphocytes was also studied using the spheres as visual markers. When lymphocytes were fixed with glutaraldehyde and subsequently labeled with the immunolatex reagents, a random distribution was observed by scanning electron microscopy; a patchy distribution was observed when unfixed lymphocytes were used. These results are consistent with studies using ferritin-labeled antibodies (S. De Petris and M. Raff. 1973. Nature [Lond.]. 241:257.) and support the view that Ig receptors on lymphocytes undergo translational diffusion. In addition to serving as visual markers for scanning electron microscopy, these latex spheres tagged with fluorescent or radioactive molecules have applications as highly sensitive markers for fluorescent microscopy and as reagents for quantitative studies of cell surface antigens and other receptors.     

Passive transfer of protective immunity to larval Dirofilaria immitis from dogs to BALB/c mice

Protective immunity to larval Dirofilaria immitis has been demonstrated in both the natural host, the dog, and in an experimental host, the mouse. In the present study, sera were collected and pooled from dogs that had been shown to have protective immunity to larval D. immitis. The pooled serum was inoculated into normal BALB/cByJ mice that then were challenged with third-stage larvae (L3) implanted in diffusion chambers. Two weeks postchallenge no significant difference was seen in either parasite survival or growth. Three weeks postchallenge, there was a significant decrease in parasite survival in mice receiving serum from immune dogs. Living larvae recovered at 3 wk postchallenge were significantly shorter than cohorts recovered from control mice. Antibody responses to L3 and forth-stage larvae (L4) surface antigens, to L3 and L4 aqueous soluble antigens, and to an excretory-secretory antigen fraction were measured. Only antibody responses to L3 surface antigens were elevated in the immune serum as compared to controls, thus suggesting a possible role for antibodies with specificity for surface antigens in protective immunity.     

Serology of Neisseria gonorrhoeae. Classification by co-agglutination

The co-agglutination (COA) method has been adapted for serological classification of Neisseria gonorrhoeae. COA reagents were prepared with selectively absorbed rabbit hyperimmune antibodies against gonoccal (GC) major outer membrane protein (MOMP) serotype strains. Using these reagents, the 16 MOMP reference strains could be referred to at least three antigen classes, tentatively named W, J and M. The GC antigens of class W were divided into three groups I, II and III, and they were in part sensitive to pronase. The antigens of class J reflected strain specific or serotype reactions, some sensitive and others resistant to proteolytic enzymes. The antigens of class M were sensitive to periodate and resistant to pronase. Strains used in serological studies by other authors were tested. The properties of class W correlated well with those of the so-called micro-immunofluorescence and immunotype systems, and class M with those of the so-called endotoxin and acid polysaccharide systems. Strains from three different laboratories could all be grouped by class W and M reagents. Identical strains obtained independently from different laboratories gave very similar reaction patterns with the reagents available. Repeated GC-isolates from patients infected with beta-lactamase producing strains showed stable reactions with class W and J reagents, while there was a time-related variation of the class M pattern. We have found that the COA method is rapid, easy and reproducible in the serological classification of Neisseria gonorrhoeae and all the 117 GC-strains tested could be classified.     

Resistance of dogs to Echinococcus granulosus

Further studies on the immunization of dogs against E. granulosus indicated that certain dogs have a natural resistance, which is not mediated by specific antibodies or sensitized lymphocytes to tapeworm secretory antigens. In a pilot experiment with two pups, E. granulosus recovered from an immunized pup were arrested in their development and exhibited 100% inhibition of egg production compared to 100% egg production in mature worms recovered from a control pup. When the experiment was repeated with 10 pups, three of five control pups given Freud's adjuvant and Bordetella pertussis carried E. granulosus which were arrested in their development and showed 100% inhibition of egg production. Two of five immunized pups also exhibited signs of resistance. Arrested worms recovered 40 days after infection appeared to be at a stage equivalent to about 18 days of optimal development. Although immunized pups exhibiting resistance showed strong positive in vitro and in vivo immune responses, all control pups gave negative reactions to tapeworm secretory antigens.     

Uncinaria stenocephala: assessment of antigens for the immunodiagnosis of canine uncinariosis

Although Uncinaria stenocephala is the most frequent hookworm in the intestine of dogs from Northern, Central and Southern Europe, little is known about its host-parasite relationship. Three groups of sera from dogs (Group 1: dogs naturally infected only by U. stenocephala; Group 2: helminth-free dogs at necropsy, and Group 3: dogs parasitized by other helminths) were analyzed by ELISA using U. stenocephala antigens from adult worms (somatic and excretory-secretory antigens) and from L3 larvae (somatic antigens). All three sources of antigens were found to be suitable for immunodiagnosis of canine uncinariosis with up to 90% efficacy. However, an analysis to assess the diagnostic value of the different antigens demonstrated that the adult excretory-secretory antigens had a higher diagnostic efficacy (96.7%), indicating that this is the best antigen source for the diagnosis of Uncinaria infection.     

Detection of circulating Dirofilaria immitis antigens in random source laboratory dogs: evaluation of two commercial serodiagnostic tests

Two commercially available serodiagnostic tests for Dirofilaria immitis antigens were evaluated for sensitivity, specificity, predictive values, and reliability using serum from 110 random source dogs. Both tests were performed in two separate laboratories on serum samples randomized in five blocks of 22 samples each. Dogs were examined for microfilariae using the modified Knott's technique, and for adult parasites by necropsy. Forty-eight of the 110 dogs (43.6%) had either adult or juvenile parasites within the cardiopulmonary vasculature or microfilariae in the peripheral blood. Of those 48, 26 (54.2%) were amicrofilaremic and had cardiopulmonary parasite populations ranging from one to greater than 50. In both laboratories, both commercial tests failed to detect infection in eight of the 26 amicrofilaremic dogs. Three amicrofilaremic dogs were positive by both tests in both laboratories. Four dogs (3.6%) had microfilariae without adults. Two of those four dogs were negative by both commercial tests in both laboratories. One commercial test had 38 false negatives in one laboratory, 13 of which were also negative in the second laboratory. The other test had 21 false negatives in one laboratory and 20 in the other laboratory. Fourteen of these samples were falsely negative in both laboratories. False positives were low in both laboratories for both tests.