The apparent inability of cyprinid fish to produce skin-sensitizing antibody

Both egg albumin and horse serum failed to induce systemic anaphylactic shock in cyprinid fish. The production of skin-sensitizing antibody by these fish to either horse serum, egg albumin or antigenic extracts of the cestode, Caryophyllaeus laticeps or the acantho-cephalan, Pomphorhynchus laevis , could not be demonstrated by homologous passive cutaneous anaphylaxis (PCA) or by heterologous PCA reaction in guinea-pigs. It was considered therefore, that fish are unable to produce such antibody, at least in response to these antigens, and that it is unlikely that skin-sensitizing antibody is involved in the rejection of C. laticeps by dace Leuciscus lueciscus (L.). The phylogenetic origins of skin-sensitizing antibody are discussed.     

Induction of immunosuppression to bacterial antigens in carp, Cyprinus carpio

Typical primary antibody responses were found after injecting carp maintained at 25°C with formalin-killed (Fk) Vibrio anguillarum bacteria, but not in fish injected with the same antigen held at 12° C. There were no significant increases over the primary responses in fish receiving a second injection 60 days after the first injection.
The differences in the results between fish groups receiving three V. anguillarum antigens at two dosages were found to be insignificant in most of the experiments. A comparison of the antibody titres of fish groups after challenge with Fk bacteria showed no difference from control fish receiving a single injection similar to the challenge dose or fish receiving a previous injection with the antigens emulsified in complete Freund's adjuvant (CFA) at 25° C. However, significant immunosuppression was found in fish that were given the primary injection intracardially at 25° C or 12° C, or with CFA emulsion at 12° C.     

Survival of Vibrio cholerae 01 in common foodstuffs during storage at different temperatures

Survival of Vibrio cholerae El Tor serotype Inaba was examined in pasteurized milk, freshwater fish, raw beef and raw chicken at a variety of temperatures. Both food type and incubation temperature affected survival. At the lowest temperatures, V. cholerae remained viable in meats for up to 90 d at—5°C and 300 d at —25°C. In milk, however, it was not detectable after 34 d at —5°C and 150 d at —25°C. At 7°C it survived 32 d, on average, in milk and only 18–20 d in the other foods. At room temperatures survival periods were shorter, never exceeding 10 d, and it was not detected after 2 d incubation at 35°C in chicken and fish.     

At least two loci encode polymorphic class I MHC antigens in the horse

Summary. Six monoclonal antibodies and ten alloantisera were used to precipitate cell surface molecules of approximately 44kDa (class I MHC antigens) from radiolabelled equine peripheral blood lymphocytes. All ten antisera were raised against antigens of a single donor horse (horse 0834, ELA-A2,-A2). Four methods of producing antisera were compared: one or two pregnancies, skin allografting, and skin grafting followed by pregnancy. Immunization by pregnancy appeared to produce antibodies against class I products only, while skin grafting raised antibodies to class II antigens as well. Nine of the antisera were raised across an entire MHC haplotype barrier, while one recipient carried the ELA-A2 antigen of the donor. The pregnancy antiserum raised across this barrier probably identifies a second polymorphic class I locus in the horse. Sequential immunoprecipitation using this antiserum in the first stage and an anti-MHC haplotype antiserum or monoclonal antibody reagent in the second stage supported this hypothesis. Gene products of this second ELA class I locus are immunogenic in pregnancy.     

免疫磁珠技术分离唾液A/B血型抗原并用于吸附血清A/B抗体

目的:应用免疫磁珠分离技术获得具有良好抗原性的A/B血型抗原,并探究其作为ABO血型抗体吸附剂去除A/B抗体的可行性。方法:将含有血型物质的唾液进行预处理,再与包被了抗体的磁珠混合,分离出纯度较高的A/B抗原,运用酶联免疫及凝集抑制试验验证所得抗原的抗原性及是否存在交叉反应。用未纯化A/B抗原和纯化A/B抗原包被磁珠,对含有抗A/B IgM、IgG的血清进行抗体吸附,用纯化A/B抗原对100份来自O型血孕妇的临床血清样本进行抗体吸附,分别评价其吸附效果。结果:纯化抗原与对应抗体反应后,其吸光度显著高于对照组(A抗原与A抗体0.85±0.12 vs.0.27±0.03,P0.01;B抗原与B抗体0.86±0.09 vs.0.24±0.06,P0.01),与其它类型抗体反应后的吸光度值与对照组比较差异无统计学意义(P0.05)。进行红细胞凝集抑制试验时,纯化抗原可显著抑制相应抗体与红细胞的凝集反应,对其它类型抗体与红细胞的凝集没有抑制作用。血清抗体吸附实验表明纯化抗原的吸附效率比未纯化抗原的高(97.00%vs.88.00%,P0.001)。临床样本抗体吸附实验显示,纯化A抗原对抗A IgM/IgG的吸附效率分别为96.88%、98.44%;纯化B抗原对抗B IgM/IgG的吸附效率分别为96.88%、98.44%。结论:磁珠纯化抗原能特异性地与对应抗体结合,有效吸附血清中的血型抗体,有望作为合成A/B抗原的替代品。     

Respiratory metabolism of Utah chub, Gila atraria (Girard) and speckled dace, Rhinichthys osculus (Girard)

Measurements of active, standard and routine oxygen consumption were made for 207 Utah chub at 6°, 9°, 12°, 18° and 22° C; and 123 speckled dace at 4°, 8°, 12° and 18° C. These were expressed as mathematical functions of water temperature and size of the fish. The mean slopes of log_e oxygen consumption (mg O₂/h) and log_e wet weight (g) were 0.771, 0.526, and 0.619 for active, standard, and routine rates respectively for Utah chub, and 0.943, 0.486, and 0.683 respectively for speckled dace.     

Application of chemically desialylated and degalactosylated human glycophorin for induction and characterization of anti-Tn monoclonal antibodies

Human erythrocyte glycophorin was desialylated by mild acid hydrolysis and degalactosylated by Smith degradation. Two monoclonal antibodies (Tn5 and Tn56) obtained by immunization of mice with this artificial Tn antigen were characterized and compared in some experiments with two antibodies (BRIC111 and LM225) obtained in other laboratories by immunization with Tn erythrocytes. The specific binding of the antibodies to glycophorins desialylated and degalactosylated on the nitrocellulose blot and to asialo-agalactoglycophorin-coated ELISA plates, and reactions with authentic Tn antigen served for identification of their anti-Tn specificity. The antibodies were further characterized in inhibition assay with various glycoproteins. The antibody Tn5 (similar to BRIC111) was shown to be specific for human erythrocyte Tn antigen, whereas Tn56 reacted strongly with different glycoproteins carrying O-linked GalNAc- residues, and was strongly bound to the murine adenocarcinoma cell line Ta3-Ha. The antibodies Tn5, Tn56 and BRIC111 were similarly inhibited by ovine submaxillary mucin (OSM) and asialoOSM, but the antibody LM225 showed a distinct preference in reaction with OSM (sialosyl-Tn antigen). The results show that Tn antigen, obtained by chemical modifications of human glycophorin, enables the preparation and characterization of anti-Tn monoclonal antibodies, without using rare Tn erythrocytes.Abbreviations HuGph human erythrocyte glycophorin - HoGph horse erythrocyte glycophorin - OSM ovine submaxillary mucin - mAb monoclonal antibody - ELISA enzyme-linked immunosorbent assay - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis - PBS phosphate buffered saline (0.01m Na₂HPO₄/0.15m NaCl, pH 7.2) - BSA bovine serum albumin - TBS 0.05m Tris-HCl/0.15m NaCl, pH 7.2 - TGr transformation grade     

Thermoregulatory behaviour, diel activity and the relationship of spontaneous locomotor activity to temperature in the sea raven, Hemitripterus americanus (Gmelin)

Twelve sea ravens, Hemitripterus americanus , were tested individually for three day periods in electronic shuttleboxes to measure their preferred and avoided temperatures, diel activity pattern and relationship of activity to temperature. The fish avoided temperatures below 9°C or above 25°C, preferring temperatures with a central tendency of 15–17°C. The fish were primarily nocturnal, being more active at night than by day. Locomotor activity increased eightfold between 10 and 20°C, but exhibited an anomalous decrease between 14 and 18°C, corresponding to the preferred-temperature zone, characteristic of maximal summer temperatures in the natural habitat.     

用Vero细胞制备马抗狂犬病血清抗原

以Vero细胞为基质制备马抗狂犬病血清用抗原,以期建立有效、经济、简便的抗原制备方法。用含10％马血清营养液对Vero细胞作适应性培养,接种狂犬病毒,以含1％～3％马血清营养液作维持液培养病毒,于第5,8天收获病毒液,经灭活、浓缩、离心等制成抗原。第5,8天收获病毒滴度可稳定在7．010gLD50／mL以上,灭活抗原具良好的抗原性,用NIH法测定效价达6．0IU／mL以上,可用作抗原生产抗狂犬病血清。     

Response and function of cutaneous mucosal and serum antibodies in barramundi (Lates calcarifer) acclimated in seawater and freshwater

Mucosal and serum antibody responses were studied in sibling barramundi (Lates calcarifer) acclimated in either seawater or freshwater following vaccination by intraperitoneal injection or direct immersion in an inactivated Streptococcus iniae vaccine. As expected, route of vaccination had a marked effect on immune response, with direct immersion resulting in low serum antibody levels against S. iniae by ELISA detected 21 days post vaccination at 26 degrees C, whilst a significant response was detected in mucus. A strong specific antibody response was detected in both mucus and serum 21 days following intraperitoneal injection. Fish acclimated in seawater prior to vaccination showed a markedly higher specific mucosal antibody response than sibling fish acclimated in freshwater, regardless of the route of vaccination, whilst the serum antibody response was not affected by salinity. Both mucosal and serum antibodies from fish in seawater and freshwater were capable of binding antigen at salinities similar to full strength seawater in a modified ELISA assay. These results indicate that this euryhaline fish species is not only able to mount significant specific antibody response in cutaneous mucus, but that these antibodies will function in the marine environment.     

The Effects of Temperature on the Incubation and Latent Periods of Powdery Mildew (Erysiphe polygoni) on Clematis

The effects of temperature on the length of the incubation and latent periods of clematis powdery mildew, caused by Erysiphe polygoni , were studied. At constant temperatures over the range of 10–29°C, the length of the incubation period ranged from 4 to 11 days, and the length of the latent period ranged from 4 to 13 days; no visible colonies developed at 30°C after 19 days. The relationship between temperature and the rates of fungal development within the incubation and latent periods (expressed as the reciprocal of the lengths of the incubation and latent periods) under constant temperature were described well by a non-linear model. The resulting curves were not the usual form of an asymmetrically bell-shaped type; instead, they were of exponential type with the development rate increasing with increasing temperatures. The relationship between temperature and the development rate during the post-incubation (before sporulation) period was non-linear with an optimum temperature of approximately 20°C.     

Isolation of a Pseudomonas species from fish intestine that produces a protease active at low temperature

A psychrotrophic bacterium producing a protease active at low temperatures was isolated from fish intestine and identified as a Pseudomonas species. Optimum growth and protease-producing temperatures of this strain were 15°C and 10°C, respectively. The maximum temperature for proteolytic activity was 25°C, an unusually low temperature.     

Influence of environmental conditions on the ovarian cycle and serum chemistry of Cyprinus carpio in the Dal lake, Kashmir (India)

SUMMARY 1. The study presents adaptive and acclimatory changes observed in Cyprinus carpio communis L with respect to ovarian cycle and serum levels of electrolytes, calcium, total proteins and albumin as a consequence of wide temperature and sunshine variation in the natural environment of Dal lake in Srinagar, Kashmir (India).
2. The atmospheric temperatures in Srinagar show well-defined seasonal and diurnal variations. During the period of study, the mean monthly average temperature in summer (June-August) was 24°C whereas in winter (December-February) it fell to 3.6°C. The total hours of sunshine during the winter months were about half of those found in summer.
3. Under local thermal and photo conditions the fish was found to spawn once a year compared to biannual spawning in tropical chmates.
4. A significant decrease was found in serum sodium, potassium and chloride concentrations during the months of January and February when the mean minimum atmospheric temperatures reached −2.5°C and −l.2°C respectively.
5. The serum calcium levels showed a marked increase during the period corresponding to ovarian maturity and spawning of fish.
6. A significant rise in total serum protein concentration was induced by decreased environmental temperatures. Serum albumin levels were found to exhibit a sharp fall coinciding with the spawning period.     

On-chip complement activation adds an extra dimension to antigen microarrays

Antibody profiling on antigen microarrays helps us in understanding the complexity of responses of the adaptive immune system. The technique, however, neglects another, evolutionarily more ancient apparatus, the complement system, which is capable of both recognizing and eliminating antigen and serves to provide innate defense for the organism while cooperating with antibodies on multiple levels. Complement components interact with both foreign substances and self molecules, including antibodies, and initiate a cascade of proteolytic cleavages that lead to the covalent attachment of complement components to molecules in nanometer proximity. By refining the conditions of antibody profiling on antigen arrays we made use of this molecular tagging to identify antigens that activate the complement system. Antigen arrays were incubated with serum under conditions that favor complement activation, and the deposited complement C3 fragments were detected by fluorescently labeled antibodies. We used genetically C3-deficient mice or inhibition of the complement cascade to prove that the technique requires complement activation for the binding of C3 to features of the array. We demonstrate that antigens on the array can initiate complement activation both by antibody-dependent or -independent ways. Using two-color detection, antibody and complement binding to the relevant spots was measured simultaneously. The effect of adjuvants on the quality of the immune response and binding of autoantibodies to DNA with concomitant complement activation in the serum of mice suffering from systemic autoimmune disease was readily measurable by this new method. We propose that measurement of complement deposition on antigen microarrays supplements information from antibody binding measurements and provides an extra, immune function-related fingerprint of the tested serum.     

Upper thermal limits for feeding and growth of 0+ Arctic charr

When Arctic charr Salvelinus alpinus from two diVerent stocks were fed live Neomysis integer , the upper thermal limits for feeding and growth were established in the range 21·5–21·8° C. These critical temperatures might have been underestimates, because fish tend to show increased sensitivity to handling at high experimental temperatures. In the second experiment, the proportion of feeding undisturbed charr from four stocks decreased initially as temperature was raised in steps from 18 to 22° C. At the lower temperatures, 18 and 20) C, almost all fish resumed feeding, but the recovery time was longer and more fish ceased to feed at 20) C than at 18° C. When the temperature was increased to 21° C, 50% of the fish ceased feeding permanently, and all fish ceased feeding within 2 days at 22° C. It is concluded that 0+ charr cease to feed and grow at c .21·5) C and that the critical temperatures for feeding and growth coincide.     

Antibodies in cold stressed mice recognize a surface protein in Toxoplasma gondii tachyzoites

Physical or psychological stressors are known to have significant consequences for immune function and the outcome of disease in human and animal models. In mice, cold water stress (CWS) has been shown to delay control of acute infection and reactivation of latent infections. Increased levels of parasite-specific IgG and IgM antibodies are observed when CWS is applied in the chronic phase. The present study examined the effects of a physical stressor, CWS, on tachyzoites antigens of Toxoplasma gondii, with particular emphasis on a low molecular weight antigen, 5 kDa, which seems to be recognized by antibodies from mice subjected to CWS in the chronic phase. This antigen is not recognized by antibodies from infected mice not subjected to CWS. Sera obtained from stressed and infected (CWS + INF) mice subjected to CWS during the chronic phase (CWS + INF + CWS) were used to harvest anti-5-kDa antibodies for immunolocalization studies. Tachyzoite lysate preparations were electrophoretically separated and transferred to nitrocellulose membranes. Strips of nitrocellulose containing tachyzoite antigens in the 4-10-kDa range were used to select for anti-5-kDa antibodies. Harvested anti-5-kDa localized this antigen on the surface of tachyzoites. This antigen was not present in bradyzoite preparations. Treatment with phosphatidylinositol-specific phospholipase C showed this antigen was not anchored to the cell membrane through glycosyl-phosphatidylinositol. Strong antibody responses in stressed animals during the chronic phase are associated with parasite reactivation. The 5-kDa antigen constitutes a unique immunogenic component of T. gondii, with significant diagnostic potential for identifying reactivation of latent infections.     

Cross-subtype neutralizing antibodies induced in baboons by a subtype E gp120 immunogen based on an R5 primary human immunodeficiency virus type 1 envelope

Global human immunodeficiency virus type 1 (HIV-1) diversity may require engineering vaccines to express antigens representing strains prevalent in the target population of vaccine testing. The majority (90%) of incident infections in Thailand are genetic subtype E, with a small percentage of subtype B infections in the intravenous drug user populations. We have evaluated and compared the binding and HIV-1 neutralizing properties of serum antibodies induced in baboons by CHO cell-expressed monomeric gp120 derived from a CCR5-using (R5) subtype E primary HIV-1CM235 or a CXCR4-using (X4) subtype B T-cell line-adapted (TCLA) HIV-1SF2 isolate. In contrast to the subtype-specific HIV-1 neutralizing antibodies induced with recombinant HIV-1SF2 gp120 (rgp120SF2), rgp120CM235 immunization induced antibodies capable of neutralizing both subtype E and subtype B TCLA HIV-1 isolates. However, neither immunogen induced antibodies capable of neutralizing primary HIV-1 isolates. Antibody induced by rgp120CM235 preferentially bound natively folded gp120 and retained strong cross-reactivity against multiple gp120 strains within subtype E as well as subtype B. In contrast, antibody responses to rgp120SF2 were directed predominantly to linear epitopes poorly exposed on native gp120 and had more limited cross-recognition of divergent gp120. Fine epitope mapping revealed differences in antibody specificities. While both rgp120CM235 and rgp120SF2 induced antibodies to regions within C1, V1/V2, V3, and C5, unique responses were induced by rgp120CM235 to multiple epitopes within C2 and by rgp120SF2 to multiple epitopes within C3, V4, and C4. These data demonstrate that strain and/or phenotypic differences of HIV-1 subunit gp120 immunogens can substantially alter antibody binding specificities and subsequent HIV-1 neutralizing capacity.     

Primary antibody-Fab fragment complexes: a flexible alternative to traditional direct and indirect immunolabeling techniques.

Immunolabeling with immune complexes of primary and secondary antibodies offers an attractive method for detecting and quantifying specific antigen. Primary antibodies maintain their affinity for specific antigen after labeling with Fab fragments in vitro. Incubation of these immune complexes with excess normal serum from the same species as the primary antibody prevents free Fab fragments from recognizing immunoglobulin. Effectively a hybrid between traditional direct and indirect immunolabeling techniques, this simple technique allows primary antibodies to be non-covalently labeled with a variety of reporter molecules as and when required. Using complexes containing Fab fragments that recognize both the Fc and F(ab')2 regions of IgG, we show that this approach prevents nonspecific labeling of endogenous immunoglobulin, can be used to simultaneously detect multiple antigens with primary antibodies derived from the same species, and allows the same polyclonal antibody to be used for both antigen capture and detection in ELISA.     

Human Germline Antibody Gene Segments Encode Polyspecific Antibodies

Structural flexibility in germline gene-encoded antibodies allows promiscuous binding to diverse antigens. The binding affinity and specificity for a particular epitope typically increase as antibody genes acquire somatic mutations in antigen-stimulated B cells. In this work, we investigated whether germline gene-encoded antibodies are optimal for polyspecificity by determining the basis for recognition of diverse antigens by antibodies encoded by three V_H gene segments. Panels of somatically mutated antibodies encoded by a common V_H gene, but each binding to a different antigen, were computationally redesigned to predict antibodies that could engage multiple antigens at once. The Rosetta multi-state design process predicted antibody sequences for the entire heavy chain variable region, including framework, CDR1, and CDR2 mutations. The predicted sequences matched the germline gene sequences to a remarkable degree, revealing by computational design the residues that are predicted to enable polyspecificity, i.e., binding of many unrelated antigens with a common sequence. The process thereby reverses antibody maturation in silico. In contrast, when designing antibodies to bind a single antigen, a sequence similar to that of the mature antibody sequence was returned, mimicking natural antibody maturation in silico. We demonstrated that the Rosetta computational design algorithm captures important aspects of antibody/antigen recognition. While the hypervariable region CDR3 often mediates much of the specificity of mature antibodies, we identified key positions in the V_H gene encoding CDR1, CDR2, and the immunoglobulin framework that are critical contributors for polyspecificity in germline antibodies. Computational design of antibodies capable of binding multiple antigens may allow the rational design of antibodies that retain polyspecificity for diverse epitope binding.     

Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections.

We describe a new approach for retrieval of antigens from formalin-fixed, paraffin-embedded tissues and their subsequent staining by immunohistochemical techniques. This method of antigen retrieval is based on microwave heating of tissue sections attached to microscope slides to temperatures up to 100 degrees C in the presence of metal solutions. Among 52 monoclonal and polyclonal antibodies tested by this method, 39 antibodies demonstrated a significant increase in immunostaining, nine antibodies showed no change, and four antibodies showed reduced immunostaining. In particular, excellent immunostaining results were obtained with a monoclonal antibody to vimentin as well as several different keratin antibodies on routine formalin-fixed tissue sections after pre-treatment of the slides with this method. These results showed that after antigen retrieval: (a) enzyme predigestion of tissues could be omitted; (b) incubation times of primary antibodies could be significantly reduced, or dilutions of primary antibodies could be increased; (c) adequate staining could be achieved in long-term formalin-fixed tissues that failed to stain by conventional methods; and (d) certain antibodies which were typically unreactive with formalin-fixed tissues gave excellent staining.