Isolation of immunogenic and lethal peptides of alpha-toxin from Clostridium novyi type B.

The lethal alpha-toxin was isolated from the culture filtrate of Clostridium novyi type B using ammonium sulfate precipitation and ion exchange chromatography. The alpha-toxin has a mol. wt of 190,000 and does not contain any disulfide cross-linkages. It consists of a single polypeptide chain. The peptide fragments resulting from the cyanogen-bromide cleavage were isolated using reversed phase and gel filtration HPLC. The immunogenic actions of these peptides and peptide mixtures were studied in Balb/c mice. Three polyclonal antisera recognizing the uncleaved native toxin could be found using an ELISA test (Br3, Bro2, Bro5). One peptide mixture (Tx5), which was proved lethal in shell-less quail eggs (in vitro), was rechromatographed with gel filtration HPLC that resulted in one peptide with mol. wt 3000 (Txleth), which again proved lethal in the shell-less quail egg lethality test. The immunogenic peptides differ from the lethal one, therefore we assumed different locations on the polypeptide chain. The separation of the immunogenic, non-toxic fragment from the lethal one may allow the production of a highly specific non-toxic vaccine. By using synthetically produced immunogenic peptides, time-consuming purification methods and working with the whole toxin will become unnecessary.

Immunological identification of avian monomeric and polymeric immunoglobulin M and immunoglobulin A after fractionation on sodium dodecylsulfate pore gradient polyacrylamide gels.

Naturally existing variants of Immunoglobulin M (IgM) and Immunoglobulin A (IgA) in the serum of immunologically competent, as well as immunologically defective UM-B19 chickens can be detected without time consuming purification. The different molecular forms of IgM and IgA were separated by gel filtration. Pentameric IgM and dimeric IgA were well separated from the 7S-peak that contained monomeric IgM and IgA. A method for immunological identification of the protein components is described. Pore gradient gel electrophoresis was combined with antigen-antibody crossed electrophoresis in horizontal agarose slabs. The principle of the method is that IgM and IgA in their different molecular forms in the mixtures to be assayed after gel filtration are first separated with high resolution by sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis using a pore gradient of 3 to 15%. In the second step, a slab of polyacrylamide gel with the separated proteins in transferred onto agarose layers containing appropriate anti- mu- and anti-alpha antiserum and SDS in a concentration of .01%. The technique is easy to perform and gives reproducible results. In chicken serum, the monomeric state of IgM (184,000 d), along with the pentameric state (920,000 d) were identified. Serum IgA exists in dimeric (340,000 d) and in monomeric (170,000 d) states. No differences between the commercial Leghorn and dysgammaglobulinemic and normogammaglobulinemic lines were found.

Development of specific enzyme-linked immunosorbent antibody assay systems for the detection of chicken immunoglobulins G, M, and A using monoclonal antibodies.

The aim of the present study was the development of a sensitive and specific ELISA system for the quantitative and qualitative assay of chicken Ig Isotypes G, M, and A using monoclonal antibodies. Five hybridoma cell lines were developed that synthesized specific antibodies against chicken IgG and three lines each producing specific antibodies against IgM or IgA. Using an immunodiffusion test, the subclasses were determined. Isolation of monoclonal antibodies from ascites was carried out by way of affinity chromatography with protein G sepharose. The purity of the eluates were determined by both SDS-PAGE and HPLC. A Sandwich ELISA was found to be the most suitable technique for the assay. Specificity testing was carried out by Western blotting. An epitope analysis was also carried out. By variation of the single steps concerning incubation times, quantities, and concentrations of the substances to be applied, the whole procedure was optimized. Assay limits for individual Ig isotypes were determined. The limits were 20 ng/mL for IgG, 80 ng/mL for IgM, and 160 ng/mL for IgA.

Activity of putative pulmonary ornithokallikrein and angiotensin-converting enzyme during the onset of lung respiration in the chick embryo.

We have assayed a proteinase from chicken lungs, the properties of which were comparable to those of ornithokallikrein, the enzyme that catalyzes the formation of ornithokinin. The activity of this pulmonary proteinase showed an accelerated increase during the development of lung respiration (paranatal period). In contrast, the increase in the activity of angiotensin-converting enzyme (ACE), which degrades kinin, was not enhanced at this developmental stage. L-thyroxine administration accelerated the onset of lung respiration and the associated changes in the activities of ornithokallikrein and ACE. Thiourea, on the other hand, retarded the onset of lung respiration. Concomitantly, there was no pronounced increase in ornithokallikrein activity and no attenuation of the increase in ACE activity. Furthermore, the development of lung circulation, as measured by pulmonary hemoglobin concentration, paralleled the ornithokallikrein activity. The results suggest that the increase in ornithokallikrein activity and the suppression of ACE activation during the paranatal period help adapt the embryo to the neonatal period.

Distribution of immunoglobulins during embryogenesis in the chicken.

Whereas the yolk of freshly-laid eggs contains only IgG, apart from traces of IgA, we were able to measure, on average, 0.145 mg/ml IgM and 0.207 mg/ml IgA in the yolk sac contents of 21-day-old chicken embryos. Up to the 14th embryonic day, IgG is exclusively contained in the yolk sac contents. It was not possible to demonstrate an increase in the amount of immunoglobulins present by comparing the amounts of IgM and IgA in freshly-laid eggs and in the yolk sac contents of 21-day-old embryos. The offspring of hens with experimentally-induced agammaglobulinemia did not begin with the production of the immunoglobulin isotypes IgG, IgM and IgA until after hatching. From these results the conclusion can be drawn that the immunoglobulins IgM and IgA are transferred from the egg white to the yolk sac contents during the last third of embryonic development. Embryonic synthesis of these immunoglobulins can be discounted as a result of this study. Synthesis was found to be initiated between the 2nd and 7th day of life for IgG, between the 2nd and 4th day of life for IgM and between the 6th and 13th day of life for IgA.

The influence of various adjuvants on antibody synthesis following immunization with an hapten.

For the production of specific antibodies to the hapten MATP (4-Amino-1,2,2-trimethyl-phenylphosphonate) in Balb/c mice various non-toxic adjuvants were compared to Freund's complete adjuvant (FCA). For immunization the hapten MATP was coupled to the carrier human serum albumin (HSA). The immunostimulating effect of the synthetic lipopeptides Pam3Cys-OH, Pam3Cys-Ser-Ser-Asn-Ala and different concentrations of the lipohexapeptide Pam3Cys-Ser-(Lys)4 (Pam3Cys = S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-N- palmitoyl-(R)-cysteine as well as of aluminium hydroxide were tested. IgG antibody titers in serum were determined in ELISA. In dose-response studies 50 micrograms Pam3Cys-Ser-(Lys)4 per mouse was the most effective dose with a long period of high antibody levels after the second booster. Pam3Cys-Ser-Ser-Asn-Ala provoked only low antibody titers. Immunostimulation with Pam3Cys--OH did not result in an increased production of specific antibodies. Compared to the control group an enhanced antibody synthesis could be provoked with aluminium hydroxide. However, the increase was much smaller than by using FCA. The lipopeptide Pam3Cys-Ser-(Lys)4 turned out to be a very potent adjuvant. One week after booster injection into mice 50 micrograms of this substance helped to elicit a higher antibody titer than FCA. Hence, as far as the degree of antibody production is concerned, Pam3Cys-Ser--(Lys)4 represents an alternative adjuvant to FCA.

Characterization and functional properties of a novel monoclonal antibody which identifies a T cell receptor in chickens.

A monoclonal antibody, mAb6, was produced that specifically bound to chicken T lymphocytes. Immunofluorescence analysis using a fluorescence-activated cell sorter revealed that the antibody reacted with approximately 50% of blood lymphocytes and with approximately 40% of splenocytes and thymocytes. It did not react with bursal cells and erythrocytes. Among different types of hemopoietic cell lines, only a T cell line was reactive with mAb6. When coupled to Sepharose 4B beads, mAb6 was found to be highly mitogenic for chicken T cells. In soluble form, mAb6 inhibited concanavalin A-induced T cell proliferation and the cytotoxic activity of allosensitized T cells, the inhibition occurring in a dose-dependent manner for both assays. Thus, the tissue distribution and the effects of this antibody on T cell responses suggest that mAb6 recognizes a T cell receptor present on a large proportion of chicken T cells.

Quantification of the immunoglobulin classes IgG and IgA in the young and adult pigeon (Columba livia).

Pigeon immunoglobulin classes IgG and IgA were purified and specific isotype antisera were produced in rabbits. The antisera were used to develop a quantitative assay for both immunoglobulins. Serum IgG concentrations in relation to age showed a similar pattern in pigeons to that in chickens. The same applies to the transfer of maternal immunoglobulins via egg. Besides this transfer mechanism, an additional transfer of immunoglobulins exists in the pigeon via feeding of crop milk. Crop milk contains considerable amounts of IgA (1.45 mg/ml) and significantly less IgG (0.34 mg/ml). During day 1 intestinal absorption of IgA is possible to a very low extent. Most of the IgA, as well as IgG, remains in the intestine to provide local immunity.

The complete amino-acid sequence of the heavy chain of the human myeloma protein WIE, an immunoglobulin D.

The human myeloma protein WIE is a lambda-type immunoglobulin D; the amino-acid sequence of its Fc part and aminoethylated heavy chain was completely determined. The VH-part (subgroup III) begins N-terminally with 5-oxoproline, and it contains a long, unique CDR3 region. Since the constant part differs from known delta chains by one amino-acid substitution in the hinge region, IgD WIE probably represents an allotypic variant. As in other protein delta chains, O-glycosylations are confined to the hinge region. Furthermore, the ratios of N-glycosylations at the three positions are identical in IgD WAH [Takahashi, N. et al. (1984) J. Chromatogr. 317, 11-26.] and IgD WIE (100%, 50%, 100%). From the most conserved constant domain, C delta 3, a three-dimensional model was constructed to clarify the role of its delta-specific substitutions and glycosylation.

[Modification of avian humoral immunoreactions by Influex and Echinacea angustifolia extract]. / Beeinflussung der aviären humoralen Immunreaktionen durch Influex und Echinacea Angustifolia Extrakt.

Medicinal complex drugs as well as single ethanolic or aqueous extracts of several plants are commonly used to increase the natural resistance to various infections, though their efficacy and mechanism of action are not yet well elucidated. In the present study, we investigated two problems: firstly, whether the complex drug (Influex) and Echinacea angustifolia extract do stimulate the immunoglobulin and antibody synthesis in chickens immunized with human serum albumin; and secondly, whether a restoration of IgG-synthesis in immunodefective (dysgammaglobulinemic) UM-B 19 chickens is possible with these plant preparations, i.e. if the BG cells which may possibly be present can be polyclonally or antigen specifically stimulated. The preparations were administered orally in two doses, after which the complete immunoglobulin concentration was determined by rocket immunoelectrophoresis and the antibody production by ELISA. The effect of ethanolic solvent was taken into account. The administration of the complex drug to normal Leghorn chickens induced a rise in the serum immunoglobulin concentration, as well as an increase in the three classes of antibody. By the immunodeficient chickens (IgG concentration was below the level of test sensitivity at the start), the administration of the drug led to a slight production in IgG and antibody.