ANTIGENS IN EGGS AND DEVELOPMENTAL STAGES OF THE SEA URCHIN : I. Immunological and Physicochemical Properties

A number of antigens in unfertilized eggs and embryos of the sea urchin Paracentrotus lividus were characterized with respect to both immunological and physicochemical properties. Experiments involved single diffusion in agar (Oudin technique) combined with mutual dilution, serial dilution, and heating of antigenic extracts, as well as immunoelectrophoresis with normal and heated extracts and agar electrophoresis followed by staining of the antigenic spots with protein specific dyes. The gradual transition in migration rates of bands of precipitates in Oudin tubes following mutual dilution of either extracts or antisera allowed the identification of 6 immunologically identical antigens in eggs and embryonic stages. Similarities with respect to diffusion coefficients, sensitivity to heat, electrophoretic mobility, and reaction to protein specific dyes indicated that the antigens in extracts of eggs and various developmental stages also had certain physicochemical properties in common. Such knowledge is of importance for an understanding of antigenic changes occurring during ontogenesis.     

Water-soluble proteins in early amphibian embryos. III. Immunoelectrophoretic analysis of the antigenic changes in the ectoderm of the early gastrula and neural plate during development

20 water-soluble antigen have been identified with the help of rabbit antisera to extracts of the early gastrula ectoderm and neural plate in Rana temporaria. All of them were also found in the early blastula embryos and unfertilized eggs. The identified antigens are characterized by a definite embryospecificity. As the development proceeds, the concentration of these antigens in the embryonic tissues decreases until the complete disappearance of corresponding immunoelectrophoretic reactions. By this characteristic all antigens under study are subdivided into four groups: I--five antigens identified at the early developmental stages only (until hatching, stage 29); II--nine antigens present up to stages 33--35; III--three antigens followed up to stages 39--40 (well formed tadpole); IV--three antigens were found at all developmental stages under study up to stages 45--47. 11 out of 20 identified antigens have antigenic similarity with the proteins of blood serum of adult amphibians. Besides, the early gastrula ectoderm contains antigens similar with those of the brain of adult amphibians.     

Characterization of antisera against mouse teratocarcinoma OTT6050: Molecular species recognized on embryoid bodies,preimplantation embryos,and sperm

Antisera raised in rabbits by hyperimmunization with small embryoid bodies of the transplantable teratocarcinoma OTT6050 recognize several distinct antigenic protein species on the surfaces of cells of the immunogen. Some of these antigens were found on the cells of preimplantation mouse embryos, on cells from parietal yolk sac carcinoma, and on mouse sperm. These antigens have been distinguished by polyacrylamide electrophoresis of the immune precipitates from detergent extracts of lactoperoxidase-iodinated cells. The intact embryoid bodies from the ascitic form of the OTT6050 teratocarcinoma exhibited five major protein bands (approximate MW 150K, 115K, 82K, 48K, and 12K), one band that ran at the dye front of the gels (R_f ≥1) and one minor band (approximate MW 22K). Two different rabbit antisera recognized an essentially identical pattern of antigens which, however, varied on the different cell types tested. Antisera were also elicited in syngeneic male mice using glutaraldehyde-fixed or irradiated OTT6050 embryoid bodies. The isoantisera had very poor titers in comparison to the absorbed xenoantisera, as assessed by complement-mediated cytotoxic activity against the immunizing cell types. Complement-mediated cytotoxicity could also be demonstrated using parietal yolk sac carcinoma cells, preimplantation mouse embryos from all cleavage stages, blastocysts, and immunosurgically isolated inner cell masses, as targets. The complexity of the antisera generated by intact embryoid bodies described here indicates that these structures bear multiple antigenic specificities not present on adult somatic cells, some of which are stage-specific embryonic polypeptides.     

Relationship of Mycoplasma pneumoniae to other human Mycoplasma species studied by gel diffusion

Taylor-Robinson, David (National Institute of Allergy and Infectious Diseases, Bethesda, Md.), Otakar Sobeslavsky, and Robert M. Chanock. Relationship of Mycoplasma pneumoniae to other human Mycoplasma species studied by gel diffusion. J. Bacteriol. 90:1432-1437. 1965.-Conditions are presented for the production of four lines of precipitate between Mycoplasma pneumoniae antigen and homologous hyperimmune rabbit serum in double diffusion in agar. The specificity of the reaction was shown by the fact that M. pneumoniae antigen did not react with antisera to the other human mycoplasma species, nor did M. pneumoniae antiserum produce lines with antigens prepared from the other human mycoplasmas. In addition, there was no reduction in the number or intensity of precipitation lines after absorption of M. pneumoniae antiserum with heterotypic mycoplasma antigens, or after absorption of heterotypic mycoplasma antisera with M. pneumoniae antigen. These findings indicate that, of the human mycoplasma species so far studied, M. pneumoniae is antigenically the most distinct.     

THE IMMUNOLOGICAL SPECIFICITY OF THE EUGLOBULIN AND PSEUDOGLOBULIN FRACTIONS OF HORSE AND H^MAN SERUM

That portion of horse and human serum globulin precipitated by 33 per cent saturation with ammonium sulfate and precipitated on subsequent dialysis was taken as euglobulin; and the fraction precipitated between 33 and 50 per cent saturation and remaining in solution on subsequent dialysis was taken as pseudoglobulin. The sera of rabbits injected with either of these antigens gave precipitation with both. However, two distinct and fraction-specific antibodies could be demonstrated by absorbing the sera with the one antigen, and testing the supernatant fluid with the other. The experimental results are adequately explained on the basis that there are at least two antigenically distinct globulins in serum which we may term globulin I and globulin II and which are largely associated with so called euglobulin and pseudoglobulin respectively. The ordinary methods of salting out and dialysis do not effect complete separation and each globulin preparation contains a trace of the other antigen. The antisera to these euglobulin and pseudoglobulin preparations therefore contain antibodies to both antigens. Each protein solution precipitates all the antibody specific for the one antigen and in addition, by virtue of the trace of contaminating protein, precipitates a portion, and only a portion of the antibody specific for the other antigen. The fact that antisera to whole serum contain these same fraction-specific antibodies suggests that this immunological specificity is an inherent property of two globulins present as such in serum and is not an artifact induced by their precipitation and purification. Lipoids extracted from the globulins by ether, petroleum ether, and alcohol give no demonstrable reaction with antisera to these globulins; antisera absorbed with a large excess of lipoid are not affected as regards their reactivity with the original protein; and globulins extracted with ether and petroleum ether at room temperature are not affected as regards their reactivity with antisera. It is concluded that the immunological specificity of the globulin fractions as evidenced by the precipitation reaction is not determined by lipoids associated with the protein.     

New approach to cell lineage analysis in mammals using the Cre-loxP system

The Cre-loxP site-specific recombination system was used for cell lineage analysis in mammals. We constructed an expression plasmid, pCETZ-17, which consists of cytomegalovirus enhancer/chicken beta-actin promoter (CAG), a portion of the rabbit beta-globin gene, loxP-flanked DNA sequence (containing enhanced green fluorescent protein (EGFP) cDNA), and lacZ gene encoding E. coli beta-galactosidase (beta-gal). When circular pCETZ-17 plasmid DNA was microinjected into the pronuclei of fertilized eggs and these eggs were allowed to develop to two-cell stage, 62.8% (59/94) of the two-cell embryos exhibited distinct fluorescence in one or both blastomeres, but never expressed lacZ protein, as evaluated by histochemical staining with X-Gal, a substrate for beta-gal. When both circular plasmids, pCETZ-17 and pCAG/NCre (containing CAG and DNA sequences encoding nuclear location signal and Cre), were co-injected into fertilized eggs, almost all (87.0%, 47/54) embryos exhibited low or no fluorescence, but 51.9% (27/52) exhibited positive staining for beta-gal activity. This indicates that transient expression of the Cre recombinase gene removed the loxP-flanked DNA sequence in pCETZ-17 and then caused expression of the downstream lacZ sequence. We next microinjected pCETZ-17 into the pronuclei of fertilized eggs, cultured these injected eggs for 1 day, and collected only two-cell embryos expressing EGFP in both blastomeres. One blastomere of the EGFP-expressing two-cell embryos was microinjected with pCAG/NCre, and these treated embryos were cultured for 1 day up to four-cell stage. When the developing four-cell embryos were subjected to staining with X-Gal, cell lineage-related staining pattern for beta-gal activity was observed in most (77.8%, 7/9) embryos. These findings were further confirmed using two-cell embryos derived from a transgenic mouse line carrying CETZ-17 transgene. Thus, our system, which is based on transient expression of the Cre recombinase gene directly introduced into nuclei of embryonic cells by microinjection, is a powerful means for cell lineage analysis in mammals.     

Detection of sperm antigens on mouse ova and early embryos.

Serum and colostrum antibodies against mouse sperm were developed in two rabbits after systemic and mammary gland immunizations. Indirect immunofluorescence utilizing fluorescein-labeled goat antisera against rabbit IgG and IgA, respectively, indicated that both immune serum (IS) and colostrum (IC) compared with control samples caused intensive staining of the acrosome and tail of sperm. Absorption of IS and IC with mouse serum and the spleen, kidney, liver, and brain of male mice did not reduce the strength or the pattern of staining reaction on sperm. The absorbed IS reacted with cell surfaces of oocytes, unfertilized ova, zygotes, two-cell and four- to eight-cell fertilized ova, and blastocysts. The absorbed IC, however, reacted only with the four- to eight-cell embryo and blastocyst. Further absorption of the IS with mouse ovary removed the reaction with unfertilized ova and the one- to two-cell fertilized ova, but the staining of later embryo stages was unaffected. Therefore, it appears that specific rabbit anti-sperm antibodies are detecting two cell-membrane antigens on mouse embryos: one originating from the ovary and the other arising after fertilization.     

Serogrouping of Halophilic Bdellovibrios from Chesapeake Bay and Environs by Immunodiffusion and Immunoelectrophoresis

Little has been reported on the serological relationship of halophilic bdellovibrios (Bd). Immunodiffusion analysis performed with rabbit or mouse Bd antisera developed against eight halophilic Bd isolates and one terrestrial Bd isolate, when reacted with soluble antigen preparations of 45 isolates of halophilic Bd, allowed separation into seven serogroups, which were distinct from the terrestrial isolate. Soluble antigen preparations of prey bacteria, Vibrio parahaemolyticus P-5 (P-5) and Escherichia coli ML 35 (ML 35), exhibited no reactivity with the antisera by immunodiffusion. Immunoelectrophoresis revealed the presence of three distinct antigens in homologous reactions and one shared antigen in heterologous Bd reactions. Shared antigens were noted between halophilic and terrestrial Bd, in addition to between halophilic Bd strains, indicating the possible existence of an antigen(s) which may be shared among all Bd. Again, no shared antigen was noted when P-5 or ML 35 was allowed by immunoelectrophoresis to react with the antisera. Prey susceptibility testing of the seven distinct groups of halophilic Bd, using 20 test prey, produced essentially identical spectra for each group, indicating that this was not a useful technique in delineating the Bd. While immunoelectrophoresis was able to demonstrate an antigen common to all Bd tested, immunodiffusion was able to delineate strains on the basis of a “serogroup specific” antigen. This suggests that immunological tools may serve as important means to study the taxonomy of halophilic Bd, as well as in the formation of a clearer taxonomic picture of the genus Bdellovibrio.     

Cross-reacting antigens of the mammalian ovular zona pellucida

Antigens of zona pellucida (ZP) of different mammalian species, including the man, were studied by means of various immunochemical methods. The analysis was carried out using rabbit antisera to the pig, mouse, guenon and Java macaque eggs. After immunoadsorption (by blood serum and tissue extracts) these anti-ZP-sera reacted with water-insoluble ZP components only (in immunofluorescence). The adsorbed anti-ZP-sera were specific not only to homologous ZP, but also to ZP of other mammalian species. The spectrum of antigens of the ape ZP was most closely related to that of human ZP. The pig eggs contained antigens common with the human ZP as well. The antiserum to the mouse ZP did not react with ZP of man, ape, pig, cow, rabbit and other species under study. The blood of sterile women contained the antibodies reacting (in immunofluorescence) with ZP of mouse, pig, guinea pig, sheep and man (very weakly). The data obtained suggest the complexity of antigenic mosaic of the mammalian ZP including both "cross-reacting" (common) antigens and species specific ones.     

A modification of the unlabeled antibody enzyme method using heterologous antisera for the light microscopic and ultrastructural localization of insulin, glucagon and growth hormone.

The requirement of using homologous antisera (primary antiserum and peroxidase-antiperoxidase (PAP) complex raised in the same species) in the unlabeled antibody enzyme method has been investigated at the light and electron microscopic level using the localization of insulin, glucagon and growth hormone as model systems. Optimum immunocytochemical staining for all three antigens was observed when sheep or goat antirabbit gamma-globulin (S-ARgammaG or G-ARgammaG) were used to couple rabbit peroxidase-antiperoxidase complex with either guinea pig antisera to insulin (GP-AIS) or glucagon (GP-AGS), or monkey antisera to rat growth hormone (M-ARGH). The cross-reactivity between S-ARgammaG or G-ARgammaG and immunoglobulins in these primary antisera were substantiated by immunoelectrophoresis and radioimmunoassay. S-ARgammaG was shown to produce precipitation arcs with GP-AIS and M-ARGH that were similar to those seen when the latter were reacted with rabbit antiguinea pig gamma-globulin antiserum and goat antimonkey gamma-globulin antiserum, respectively. Radioimmunoassay results revealed that immunoprecipitation of 6-10% as compared to homologous antisera controls yielded excellent staining localization when S-ARgammaG was used for immunocytochemistry. Thus, heterologous antisera (primary antiserum and PAP complex raised in different species) may be used in the unlabeled antibody enzyme method as long as the coupling antiserum shows cross-reactivity with immunoglobulins of the primary antiserum and the PAP complex.     

Isolation of Fasciola hepatica genus-specific antigens

Santiago de Weil N., Hillyer G. V. and Pacheco E. 1984. Isolation of Fasciola hepatica genus-specific antigens. International Journal for Parasitology14: 197–206. The Fasciola hepatica antigens which induce antibody formation in acute fascioliasis were isolated by acid elution after reacting an F. hepatica tegument antigen extract with a CNBr-Sepharose 4B column coupled with IgG obtained from the serum of rabbits infected with fascioliasis for 6–10 weeks. These isolated antigens were further separated by gel filtration using a column packed with Sephacryl S-200. In this manner three major peaks were obtained. The best serologic antigens were found in peak 2 which had a mol. wt range of 14,000–43,000. This peak contains genus-specific F. hepatica antigens which are highly reactive with fascioliasis serum. These antigens do not cross-react with either Schistosoma mansoni or with bovine serum albumin by gel diffusion. Monitoring by ELISA and gel diffusion with heterologous and homologous antisera showed that as purification by antibody affinity chromatography proceeded, cross reactivity with S. mansoni was eliminated. The rabbit antiserum obtained against peak 2, when tested by immunoelectrophoresis with a crude F. hepatica extract shows one main band identical to the main band observed with serum from acutely infected rabbits. Up to two other minor bands can be detected using concentrated homologous antisera. Fractions obtained from preparative iso-electric focusing of the F. hepatica tegument extract were reacted with sera from rabbits with acute fascioliasis. Two main bands were observed in immunodiffusion with antigens eluting in a pH range of 7.4–8.7. When these fractions were monitored with anti peak 2 antisera, two precipitin bands appeared with antigens eluting in a pH range of 7.4–7.9. The F. hepatica genus-specific antigen pool was applied to ELISA to evaluate its ability to detect antibody in a primary F. hepatica infection in rabbits. A rise in absorbance values could be detected by 2 weeks of infection, reached high levels by 6 weeks and remained high through 28 weeks of infection.     

Antigenic prenylated peptide conjugates and polyclonal antibodies to detect protein prenylation

Posttranslational modification of proteins with farnesyl and geranylgeranyl groups is a required modification for signaling proteins that includes the small GTPases in the Ras, Rho, and Rab families, heterotrimeric G proteins, and nuclear lamin proteins. To develop antibodies capable of detecting and distinguishing prenylated proteins, we synthesized two antigens, succinylglycine-(geranylgeranyl)cysteine methyl ester (SuccG-(gg)CMe, 1) and succinylglycine-(farnesyl)cysteine methyl ester (SuccG-(f)CMe, 2). These prenylated peptides were covalently coupled to bovine serum albumin (BSA) and to keyhole limpet hemocyanin (KLH) to produce polyvalent, immunogenic bioconjugates. Immunization of rabbits with these immunogens generated polyclonal antisera that contained significant titers of anti-geranylgeranyl and anti-farnesyl antibodies. The selectivity of the polyclonal antisera was examined using ELISA and dot blotting methods. The anti-farnesyl and anti-geranylgeranyl antisera crossreacted with both antigens. Attempts to purify the polyclonal antisera by either positive or negative immunoaffinity selection protocols failed to produce selective anti-geranylgeranyl and anti-farnesyl antibodies. Moreover, both crude antisera and purified antibodies also crossreacted with myristoylated and palmitoylated BSA conjugates. Immunofluorescence staining of EYFP-CVLL or EYFP-CVIM transfected CHO-K1 cells with rabbit polyclonal antisera showed colocalized membrane fluorescence. Thus, an important caveat for the use of antibodies raised against aliphatic antigens is that extensive controls must be performed to determine selectivity.     

Fc-mediated nonspecific staining of the porcine brain with rabbit antisera in immunocytochemistry is prevented by pre-incubation of the sera with proteins A and G.

Nonspecific staining was detected in immunocytochemical procedures on the porcine hypothalamus with rabbit antisera, irrespective of the antigen specificity of the sera, in magnocellular neurons of the paraventricular (PVN) and supraoptic nuclei (SON), and in the vasopressin- and oxytocin-containing nucleus (VON). The present study was designed to test the hypothesis that this staining is mediated by the Fc portion of rabbit immunoglobulins. Rabbit antisera against neuropeptides localized predominantly outside the PVN, SON, and VON were employed in combination with different detection methods. The intensity of the nonspecific staining varied depending on the antiserum and persisted after pre-absorption of the antisera with their homologous peptides. Nonspecific staining and antigen-specific staining were differentially affected by the method of tissue fixation. The nonspecific staining could be prevented by preincubation of the antisera with proteins A and G, which left the antigen-specific staining intact, whereas additional preabsorption with homologous peptide abolished all staining. These observations suggest that the Fc region of IgGs is indeed involved in the nonspecific staining. On press-blots of homogenates from SON tissue subjected to isoelectric focusing, one band in the low-pH region was found with all antisera. Pre-incubation of the antisera with protein A abolished the staining of this band but did not affect staining of antigen-specific bands. Pre-incubation with proteins A and G is proposed as a routine control to check for nonspecific staining mediated by the Fc region of IgGs in immunocytochemical procedures, particularly those that employ rabbit sera in porcine brain.     

Expression of epidermis-specific antigens during embryogenesis of the ascidian, Halocynthia roretzi

We have produced two monoclonal antibodies (Epi-1 and Epi-2) which specifically recognize epidermal cells and their derivative, the larval tunic, of developing embryos of the ascidian Halocynthia roretzi. The antigens, examined by indirect immunofluorescence staining, first appear at the early tailbud stage and are present until at least the swimming larval stage. There were distinct and separate puromycin and actinomycin D sensitivity periods for each antigen. Aphidicolin, a specific inhibitor of DNA synthesis, prevented the appearance of each antigen when embryos were exposed to the drug continuously from cleavage stages. These results suggest that the antigens are synthesized during embryogenesis by developing epidermal cells and that several rounds of DNA replication are required for the antigen expression. Early cleavage stage embryos, including fertilized but unsegmented eggs, in which cytokinesis had been blocked with cytochalasin B expressed the antigens, and blastomeres exhibiting the antigens were always of the epidermis lineage. In partial embryos produced by four separated blastomere pairs of the 8-cell embryos, the expression of antigens was seen only in those developed from the animal blastomere pairs, which are progenitors of epidermal cells. These observations indicate that differentiation of epidermal cells in ascidian embryos takes place in a typical "mosaic" fashion.     

Chaperon-like Activation of Serum-Inducible Tryptophanyl-tRNA Synthetase Phosphorylation through Refolding as a Tool for Analysis of Clinical Samples

Tryptophanyl-tRNA synthetase (TrpRS) expression alters in colorectal (CRC), pancreatic (PC), and cervical (CC) cancers. Here, phosphorylation of unfolded TrpRS and its fragments is stimulated by human cancer sera (CS; n = 13) and serum of rabbit tumor induced by Rous sarcoma virus, unaffected by donor sera (NS; 11/15) and abolished by alkaline phosphatase. At 20 years of follow-up, serum-inducible TrpRS phosphorylation found years before healthy donors (3/15) diagnosed with PC, CRC, or leukemia. I have examined a specificity of serum-inducible TrpRS phosphorylation and found, surprisingly, that serine phosphorylation of unfolded TrpRS is stimulated by anti-TrpRS rabbit antisera but is unaffected by rabbit nonimmune sera and antisera to other antigens. Anti-TrpRS immunoglobulin G (IgG) inhibits phosphorylation of full-length TrpRS and stimulates phosphorylation of its 20-kDa fragment. Phosphorylation of this fragment is stimulated also by CS but not NS. 2-Mercaptoethanol and cyclic AMP exerted synergistic inhibitory effect on TrpRS phosphorylation. Anti-TrpRS sera and casein act as chaperones increasing TrpRS phosphorylation through refolding. Histone-specific protein kinase activity in CS (n = 44) and anti-TrpRS sera was lower than that in NS (n = 11), rabbit nonimmune sera and antisera to other antigens. TrpRS inhibitors, tryptamine, and tryptophanol stimulate in vivo accumulation of enzymatically inactive, nonphosphorylated, aggregated and anti-TrpRS IgG refoldable TrpRS. Phosphorylation of postsurgical tissues (n = 18) reveals TrpRS in ovarian cancer (OVC) and CC but not in normal placenta and liver. In OVC, TrpRS phosphorylation increase correlates with elevated tryptophan-dependent ATP-inorganic pyrophosphate exchange. Although not inducing cancer, TrpRS triggers signaling concomitant with cancer.     

An embryo-specific protein of barley (Hordeum vulgare).

An immunological approach has been used to identify embryo-specific products that can be used as molecular markers of embryogenesis. Immunoadsorption of antisera to remove antigens common to embryos, meristematic cells and callus, revealed one major embryo-specific antigen, a polypeptide of 17 kDa. The antigen appeared at mid-stages of zygotic embryo formation and remained at similar levels up to six days post-germination of the seedling. The polypeptide could not be detected by protein staining, suggesting it is a non-abundant product. Appearance of the antigen could be induced by culture of zygotic embryos in vitro on abscisic acid (1 microM) or mannitol (9% mass/vol.). Cross-reactive products of near-identical molecular mass were observed in embryos of wheat, rye and oats but not distantly related cereals, nor embryos from dicotyledonous species. The timing of the appearance of the antigen was different in embryos formed from microspores during anther culture in vitro. In the cultured material, the 17-kDa polypeptide preceded the appearance of morphologically distinct embryonic structure.     

Nucleoprotein-based indirect enzyme-linked immunosorbent assay (indirect ELISA) for detecting antibodies specific to Ebola virus and Marbug virus

Full-length nucleoproteins from Ebola and Marburg viruses were expressed as His-tagged recombinant proteins in Escherichia coli and nucleoprotein-based enzyme-linked immunosorbent assays (ELISAs) were established for the detection of antibodies specific to Ebola and Marburg viruses. The ELISAs were evaluated by testing antisera collected from rabbit immunized with Ebola and Marburg virus nucleoproteins. Although little cross-reactivity of antibodies was observed in anti-Ebola virus nucleoprotein rabbit antisera, the highest reactions to immunoglobulin G (IgG) were uniformly detected against the nucleoprotein antigens of homologous viruses. We further evaluated the ELISA’s ability to detect antibodies to Ebola and Marburg viruses using human sera samples collected from individuals passing through the Guangdong port of entry. With a threshold set at the mean plus three standard deviations of average optical densities of sera tested, the ELISA systems using these two recombinant nucleoproteins have good sensitivity and specificity. These results demonstrate the usefulness of ELISA for diagnostics as well as ecological and serosurvey studies of Ebola and Marburg virus infection.     

黄鳝IgM的分离纯化及兔抗黄鳝IgM抗血清的制备

目的 分离纯化黄鳝血清免疫球蛋白,制备其兔抗血清,并检测抗血清的特异性。方法 用Protein A亲和层析的方法纯化黄鳝血清免疫球蛋白,通过SDS-聚丙烯酰胺凝胶电泳检测蛋白的纯度,免疫大耳白兔制备抗血清,利用免疫双扩散检测抗血清的效价,通过western blotting检测抗血清的特异性。结果 纯化了黄鳝血清免疫球蛋白,免疫双扩散法测定兔抗黄鳝免疫球蛋白血清效价为1∶32,western blotting结果显示抗血清具有很好的特异性。结论 成功纯化了黄鳝免疫球蛋白,制备了兔抗黄鳝IgM抗血清,为建立黄鳝的血清学检测系统奠定了基础。     

Some serological properties ofActinobacillus pleuropneumoniae strains of serotypes 1 through 5

Rabbits were hyperimmunized with live, formalin-killed, and heat-treated antigen preparations of the reference strains of serotypes 1 through 5 ofActinobacillus pleuropneumoniae in order to study the antibody response to both soluble and particulate antigens. The antibody response was studied by means of precipitation, agglutination, coagglutination, indirect hemagglutination, and complement fixation tests.Serotyping ofA. pleuropneumoniae strains was done by ring precipitation (RP) and coagglutination (CoA) tests with unheated and heated cell-saline extract as antigens and rabbit hyperimmune sera produced against either live cultures or formalin-killed whole-cell suspensions. The results showed that live cultures provoked more cross-reactive antibodies in rabbits, thus making the antisera unsuitable for use in serotyping by the RP test when unheated wholecell saline extract was used as antigen. Rabbit hyperimmune serum produced against formalinkilled bacterial suspension gave serotype-specific reactions in the RP test. Boiled or autoclaved cell-saline extracts gave serotype-specific reactions in the RP test even when rabbit anti-livecell sera were used. Serotype-specific reactions were obtained in the CoA test in both rabbit anti-live or anti-formalin-killed cell sera with either unheated or heated bacterial cell suspensions as antigens.Live and formalin-killed whole-cell suspensions as well as their saline extracts provoked a high antibody response in rabbits. Heating the cell suspension at 100°C for 1 h caused a significant reduction in their immunogenic potency, whereas autoclaving (121°C) of the cell suspension for 1 h almost completely destroyed their serotype-specific immunogenic properties, since the antibody response was either absent or very poor and not type-specific. However, neither boiling nor autoclaving of the cell suspensions caused significant reduction in their ability to react with preformed antibodies. Phenol-water-extracted antigens gave the highest degree of serotype specificity in the complement fixation test.     

Phosphatase active antigens in sea urchin eggs and embryos. I. Substrate specificity, pH-optima and inhibitors.

Phosphatase activity in sea urchin embryonic antigens was investigated by histochemical staining of immunoprecipitates separated by two-dimensional (crossed) immunoelectrophoresis. Unfertilized eggs were homogenized in a hypotonic medium which solubilized cytoplasmic antigens. Antigens integrated in membranes or enclosed in particles were solubilized by detergent treatment of the residual pellet. Two different phosphatase activities were discerned in the unfertilized eggs, nucleoside diphosphatase (EC 3.6.1.6.) and acid phosphatase (EC 3.1.3.2.). Nucleoside diphosphatase activity was obtained in both the water soluble and detergent extracted protein fractions. This activity was confined to one antigen. Acid phosphatase acitivity on the other hand was almost exclusively obtained in the detergent extracted fraction and about ten distinct antigens displayed this activity. The nucleoside diphosphatase active antigen preferentially hydrolyzed purine nucleoside diphosphates and to a lesser degree triphosphates of these nucleosides. The acid phosphatase active antigens had a broader substrate specificity and hydrolyzed equally well beta-glycerophosphate and nucleotides. Both activities were essentially inactive at neutral or alkaline pH values. The activities were inhibited by p-choloromercuribenzoate and accordingly stimulated by cysteine. Tartrate and sodium fluoride, however, inhibited the acid phosphatase activity while nucleoside diphosphatase activity was either stimulated or not affected at all by these agents.