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。     

Characterization of sialyltransferases from serum of normal and hepatoma Mc-29 bearing chickens

Sialyltransferase was measured in serum of normal and hepatoma Mc-29 bearing chickens. By preparative isoelectric focusing the multiple forms of sialyltransferase from both kind of serums was studied as well. By using influenza virus neuraminidase an attempt was made for partial structural characterization of the sialylation sites in asialofetuin applied as exogenous acceptor for sialyltransferase determination. It was established an elevated serum sialyltransferase activity in tumor bearing chickens with tumor an enzyme form was detected with pI-4.99 identical with an enzyme form described previously in solubilized plasma membrane preparations from hepatoma Mc-29. Monitoring of multiple forms of serum glycosyltransferases may be of value in answering the problem concerning the tissue origin of serum enzymes.     

A simplified and efficient procedure for the purification of apolipoprotein AI from human serum high-density lipoprotein-3 by preparative isoelectric focussing on polyacrylamide gel beads

An improved method is described for the purification of milligram amounts of apolipoprotein AI from serum apo-HDL₃ by isoelectric focussing on polyacrylamide gel beads. The procedure involves a single focussing over a narrow (1.3 unit) pH gradient, and permits isolation of apo-AI of exceptional purity and in high yield (75% recovery of HDL₃ protein, ca. 50% corresponding to pure apo-AI). The electrophoretic mobility, pI values, molecular weight, antigenicity and amino acid composition of such apo-AI were indistinguishable from those reported in the literature. A rabbit antiserum to apo-AI isolated by focusing exhibited similar immunological reactivity to one prepared from an antigen isolated by gel filtration chromatography; moreover, apo-AI purified by the respective procedures reacted identically with both antisera. We conclude that isoelectric focussing on a support of polyacrylamide gel beads (as Bio-Gel P60) presents certain advantages for the isolation of highly purified apo-AI over both conventional chromatographic procedures and isoelectric focussing on a Sephadex support.     

The passive cutaneous anaphylaxis (PCA) test in Cryptococcosis

Summary The PCA test appeared to be a useful indicator of past infection due toCryptococcus neoformans when the extracellular starch antigen was used. The whole cell antigen, crude cell wall antigen, ruptured cell antigen, crude carbohydrate antigen and the crude capsular polysaccharide antigen were not found to be effective.Serums from the nine rabbits injected withC. neoformans showed some degree of reactivity in the PCA test with the homologous extracellular starch antigen. Serums from rabbits injected withC. diffluens andC. laurentii gave no response.None of the rabbits exhibited evidence of previous sensitization and none were obviously infected. Six rabbits injected withC. neoformans were skin tested 4 weeks later with all of the antigens. Positive tests were obtained in four rabbits but only with the cell wall antigen or the ruptured cell antigen. PCA activity was not observed in the serums of mice injected with strains ofC. neoformans although some came from mice showing lesions and some came from fatally infected mice.Of the 500 human serums tested for PCA activity using the extracellular starch antigen, only one reacted strongly and with a sufficiently high titer to suggest previous infection. Serums from three cases of cryptococcosis failed to react.This investigation was supported by Communicable Disease Center grant CC 00151-01.The material in this paper is taken in part from a dissertation presented to the Graduate School of The University of Arizona in partial fullfilment of the requirements for the Ph.D. degree.     

Entamoeba histolytica: cytopathogenicity, including serum effects on contact-dependent and toxin-induced lysis of hamster kidney cell monolayers

Some properties of toxin, isolated from extracts of axenically cultivated Entamoeba histolytica or excreted by intact amoebae, were investigated using a toxicity assay in microplates with monolayers of baby hamster kidney cells. Preparative isoelectric focusing showed that the highest cytotoxic activity was present in a fraction of antigen containing protein bands with an isoelectric point between 4.5 and 5. Activity of the toxin was stable between pH 4 and 10. Nonimmune rabbit serum and concanavalin A, coupled to Sepharose beads, were able to bind a large amount of toxin. Cytotoxicity of antigen was inhibited by specific immune IgG and by unknown factors in nonimmune serum with a molecular weight between 50,000 and 100,000. The toxin was inactivated by trypsin, but not by trypsin inhibitor. Its activity was thiol dependent. Serum also had a marked inhibitory effect on contact lysis of BHK cells induced by intact trophozoites. A considerable reduction of both contactdependent and toxin-induced Cytopathogenicity was observed when Diamond's TP-S-1 medium was used in the assay, in which the TP broth had been autoclaved. It is suggested that Entamoeba histolytica exocytozes toxin, which acts on adjacent cells during close contact.     

Marke''s disease herpesviruses. III. Purification and characterization of Marek''s disease herpesvirus B antigen.

Sera from chickens naturally infected with Marek's disease herpesvirus (MDHV) form preciptin lines with at least two immunologically distinct soluble antigens designated MDHV-A and MDHV-B. Partial purification and characterization of the glycoprotein MDHV-A antigen was previously reported. MDHV-B was found predominantly in the sonically treated extracts of infected cells, in contrast to the predominantly extracellular MDHV-A. Analysis of these extracts from [14C]glucosamine-labeled cells by immunodiffusion with chicken anti MDHV-B serum negative for MDHV-A followed by autoradiography confirmed that MDHV-B was a common antigen between MDHV and herpesvirus of turkeys and revealed that it was also a glycoprotein. Because of their glycoprotein nature, both MDHV-A and MDHV-B bound to concanavalin A affinity chromatography columns and could then be eluted by alpha-methyl-D-mannoside and recovered for further analysis. Concanavalin A affinity chromatography was an excellent technique for initial purification of MDHV-A and MDHV-B, since approximately 5- and 15- fold purification, respectively, was achieved in a single simple step. MDHV-B was resistant to trypsin under conditions where MDHV-A was sensitive, but was similar to MDHV-A in resistance to pH 2.0 and to 1.0 or 2.0 M urea and 0.05% Brij 35. Partially purified MDHV-B was analyzed by sucrose gradient sedimentation, isoelectric focusing, and gel filtration on Sephadex G-200 in the presence of 1.0 or 2.0 M urea and 0.05% Brij 35 to purify the antigen and to determine its physical and chemical properties in comparison with those already reported for MDHV-A. MDHV-B had a much lower isoelectric point in pH 4,54, a higher sedimentation coefficient of 4.4S, and a greater molecular weight of 58,250. These data indicate that MDHV-B is physically distinct from MDHV-A antigen, although the size difference is not sufficient to allow for effective separation. In contrast, the isoelectric point difference of greater than 2 pH units makes isoelectric focusing an effective means of purifying the antigens free of one another. The four-step purification procedure achieved greater than 200-fold purification of MDHV-B. Immunization of rabbits with this highly purified antigen results in the preparation of antisera that appeared monospecific for MDHV-B in immunodiffusion.     

Effect of HIV antigen glycoproteins and morphine on accompanying reactions of liberating Ag+-sensitive SH-containing nonprotein compounds in the "antigen-antibody" interaction

The interaction of blood serum immunoglobulins of M, G, and A classes of the donors with monospecific serums (MSS)-anti-IgM, anti-IgG and anti-IgG was established to be associated by Ag(+)-sensitive-SH containing non protein compounds release. This phenomenon formation should be related to a parallel running associated reaction mediated by conformational and/or some other changes of immunoglobulins macrostructure under highly specific intermolecular interaction with adequate MSS in the reactive mixtures. As a rule these processes are associated by the break and reduction of mixed disulphide bounds between thiol containing nonprotein compounds and proteins. HIV antigen glycoproteins and morphine preliminary introduced into the analogic reactive mixtures were found to block this phenomenon. If in these reactive mixtures the serums including three serotypes hepatitis B virus antigen is introduced this phenomenon is preserved. This effect of HIV antigen glycoproteins and morphine could be explained by their direct and/or mediated influence on the immunoglobulins macrostructure. As a result of the latter the immunoglobulins structure-functional status is infringed, being indirectly evidenced by absence of the associated reaction of release Ag(+)-sensitive-SH containing non protein compounds in the reactive mixtures. The processes presented are capable to play an essential role in formation of polyclonal gammapathy under HIV-infection.     

Marek's Disease Herpesviruses II. Purification and Further Characterization of Marek's Disease Herpesvirus A Antigen

Marek's disease herpesvirus A antigen was purified greater than 200-fold with a 24% recovery by ion exchange column chromatography, isoelectric focusing, and preparative polyacrylamide gel electrophoresis. The antigen had an isoelectric point of 6.68 ± 0.03 in the presence of 1 M urea and 0.05% Brij 35, a nonionic detergent, and approximately 6.5 in the absence of dissociating agents. When analyzed by electrophoresis on analytical polyacrylamide gels, the purified antigen migrated as a single broad band which stained for both protein and carbohydrate, suggesting that it was a highly purified heterogeneous glycoprotein. However, the antigen was not purified to homogeneity as determined by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate and by immunodiffusion analysis. Antibody to Marek's disease herpesvirus A antigen was prepared in a rabbit, and antibody to two contaminating antigens was removed by adsorption to yield monospecific antisera.     

Characterization of male meiotic germ cell-specific antigen (Meg 1) by monoclonal antibody TRA 369 in mice.

We have identified a male meiotic germ cell-specific antigen (Meg 1) with monoclonal antibody (mAb) TRA 369 in mice. The Meg 1 antigen was strongly expressed in specific steps of meiotic germ cells from pachytene spermatocyte to early spermatid, and not in other germ cells or somatic cells. Immunohistochemical examination revealed that the antigen was localized to the cytoplasm and was not distributed in the nucleus or on the cell surface. This antigen was demonstrated to have a molecular weight of 93 kDa and an isoelectric point of 5.2 by Western blotting. This molecule was first detected in the testis of 13-day-old mouse when pachytene spermatocytes first appeared. Thus this is a differentiation-specific antigen in male meiotic germ cells, and mAb TRA 369 is a useful tool to study the regulation of germ cell differentiation and to define germ cell development in a molecular level.     

Antigenic determinants of blood serum proteins detected in the structure of hepatitis B surface antigen by the method of affinity chromatography]

Tropism of the antigen of hepatitis B to the antibodies against normal serum proteins was revealed by the method of affine chromatography; this pointed to the possibility of the presence in the HBs-antigen structure of some antigenic determinants of the serum proteins or to the association of serum proteins with the HBs-antigen. A change of the isoelectric spectrum of the HBs-antigen and its tropism in affine chromatography to the antibodies against serum proteins possibly depended both on the nonhomogeneity of the antigenic determinants included into the composition of the antigen, and on the presence of the HBs-antigen--HBs-antibody or HBs-antigen--serum proteins complexes.     

Restricted dietary protein in pregnant beef cows: II. Effect on the immune response

Cows fed 0.37 Kg crude protein per day for four months before calving showed no decrease in colostral IgG, IgA, or IgM from cows fed a normal ration. Similarly, no difference in serum immunoglobulin concentration was detected in their calves. Cows in the low protein group developed significantly lower (P < 0.01) titers to Salmonella pullorum antigen than did the normal group. The data show significant positive linear regressions of calf agglutination titer to S. pullorum on the dam's colostral titers. Serum levels of IgG measured at 24 hours were significantly lower in calves from second-calf heifers than from these of other age groups. The IgA levels of serums from calves from the six-year-old cows were significantly higher than the serum levels of calves from younger dams (P < 0.01).     

盐城地区未成年过敏性疾病患者吸入性过敏原IgE检测结果分析

摘要 目的：通过研究分析盐城地区未成年过敏性疾病患者吸入性过敏原特异性IgE检测结果分布变化特点,为过敏性疾病预防和临床诊疗提供科学依据。方法：自2020年1月至2021年3月期间,选择430例未成年(<18岁)过敏性疾病患者,血清检测方法采用欧蒙公司生产的过敏原特异性IgE检测试剂盒。结果：430例患者中,血清过敏原IgE阳性166例(38.60%),其中尘螨和屋尘是主要的吸入性过敏原。血清IgE阳性率男性39%,女性36% (P>0.05),中学组女性阳性率（61.11 %）高于男性（45.83 %）（P<0.05）。不同年龄组间IgE阳性率有统计学差异（P<0.05）,蟑螂、尘螨、霉菌、豚草、屋尘和年龄组间有统计学差异（P<0.05）。不同临床症状与IgE阳性率有统计学差异(P<0.05),其中呼吸道过敏症状组IgE阳性率最高（48.30 %）,不同症状组间主要过敏原都是尘螨。屋尘阳性患者均合并尘螨阳性,其中70.93 %的患者表现出了呼吸道过敏症状。结论：尘螨、屋尘是盐城地区未成年过敏性疾病患者最主要的吸入性过敏原,研究血清过敏原分布,对未成年人过敏性疾病的预防、诊疗具有重要意义。     

Taenia solium: characterization of a small heat shock protein (Tsol-sHSP35.6) and its possible relevance to the diagnosis and pathogenesis of neurocysticercosis

A cDNA encoding for a predicted small heat shock protein (sHSP), Tsol-sfISP35.6, has been isolated by antibody screening of a Taenia solium c-DNA library. The clone was a full-length sequence (1172 bp) with an open reading frame of 945 bp and encoded for a 314 amino acid protein with deduced molecular mass of 35.6 kDa, isoelectric point of 5.6 arid the characteristic HSP20/alpha-crystallin domain duplicated. It was highly conserved, with a high sequence similarity with other platyhelminth sHSPs. Western blot analysis, using serum from neurocysticercosis patients (NCC), indicated that the purified Tsol-sHSP35.6 expression product was immunogenic, while in indirect ELISA, using the purified Tsol-sHSP35.6 expression product as antigen and serum samples from pigs and humans, 80% of T. solium infected pigs and 84% of patients with active, or 71% of patients with inactive NCC were sero-positive. The possible relevance of Tsol-sHSP35.6 in the diagnosis and pathogenesis of NCC is discussed.     

A new lymphocyte surface protein present in normal urine. I. Isolation and physicochemical properties

A lymphocyte surface glycoprotein designated urinary acidic antigen (UA) has been isolated from normal urine by a combination of preparative isoelectric focusing and ammonium sulfate precipitation. It has an m.w. of 14,000 to 17,500 daltons, and is approximately 60% carbohydrate and 40% amino acid in content. The protein exhibited the following physical properties: S20,omega = 1.24; v = 0.693 ml/g; E1%1 cm, 278 nm = 2.08; and pI-2.5. It appears to be unrelated to beta 2-microglobulin, protein HC, urinary proteose, microglobulin, or any previously described normal urine or human serum protein.     

Schistosoma mansoni: Characterization of antigens in excretions and secretions

Excretory and secretory antigens of Schistosoma mansoni were obtained by in vitro cultivation of worms in Medium H-199, under sterile conditions at 37 C, in the dark, in an atmosphere of 92% air and 8% CO₂. This procedure yielded about 1 μg soluble excretion-secretion products per worm per 24 hr. The composition of the “excretory and secretory antigen” (ESA) preparation is complex. Analysis by isoelectric focusing revealed the presence of about 10 major and about 30 minor protein components. Immunological analysis of the ESA preparation was performed by immunoelectrophoresis. At least five precipitin arcs were seen with infected mouse serum, and seven with rabbit anti-ESA serum. Immunoelectrophoresis of molecular-weight fractions of ESA showed a total of 17 different antigens. One of these antigens was excreted exclusively by female worms. The antibody response in rabbits to preparations obtained by homogenization of adult worms, or by extraction of the tegument, was very different from the response to excretory and secretory antigens. Considerable cross-reactivity between these preparations did, however, occur.     

Evidence for the phosphorylation of a proenkephalin-derived peptide, peptide B

The potential phosphorylation of a proenkephalin-derived peptide, Peptide B, was investigated in primary cultures of bovine adrenal chromaffin cells and fresh adrenal medullary tissue. Cultures were labeled with [32P]phosphate for 24 h and extracts subjected to immunoprecipitation using affinity-purified anti-serum directed against the carboxyl terminus of Peptide B. A 4.6-kDa-labeled peptide was observed in autoradiograms of immunoprecipitates separated by sodium dodecyl sulfate-polyacrylamide electrophoresis; this peptide was not observed when excess antigen was present during the immunoprecipitation. Radioimmunoassay of extracts prepared from adrenal medullary tissue and separated by isoelectric focusing revealed the presence of four isoelectric forms of Peptide B-immunoreactive peptides; these peptides also exhibited Met-enkephalin-Arg-Phe immunoreactivity. The isoelectric points of these peptides (4.5, 4.3, 4.1, and 3.9) were consistent with the predicted pI values for phosphorylated derivatives of Peptide B. Treatment of samples with alkaline phosphatase prior to isoelectric focusing resulted in the conversion of the more acidic forms to the least acidic form. The presence of phosphate in the more acidic peaks was additionally verified by isoelectric focusing of 32P-labeled immunoprecipitates; the pI values of the radioactive peptides corresponded precisely to the peaks of immunoreactivity. In adrenal medullary tissue, the relative contributions of the various phosphorylated species to the total Peptide B immunoreactivity were as follows: unphosphorylated form, 13%; singly phosphorylated, 31%; doubly phosphorylated, 37%; and triply phosphorylated, 17%. Thus more than 85% of the Peptide B molecules present in the bovine adrenal medulla are phosphorylated.     

Purification and characterization of a high molecular weight eosinophil chemotactic factor from Schistosoma japonicum eggs

A high m.w. eosinophil chemotactic factor (ECF-SjE) was isolated and purified from a soluble egg antigen preparation (SEA) of Schistosoma japonicum by gel filtration on Sephacryl S-200, anion-exchange chromatography on DE52, and isoelectric focusing. ECF-SjE had a m.w. of more than 900,000 and an isoelectric point of 4.1. It contained 40% (w/w) sugar residues and bound to concanavalin A (Con A). The chemotactic activity of ECF-SjE was heat stable (100 degrees C, 60 min) and resistant to pronase digestion, but was destroyed by periodate oxidation. IgG antibody to ECF-SjE was detected in the serum of a rabbit infected with S. japonicum, demonstrating the antigenic nature of ECF-SjE. The antigenicity of ECF-SjE was also sensitive to periodate oxidation. Thus, ECF-SjE is a glycoprotein or proteoglycan from the eggs of S. japonicum, and the sugar chain is important for the expression of chemotactic and antigenic activities. However ECF-SjE differs from the major allergenic components of S. japonicum (JEAL) in m.w. and isoelectric point. A low m.w. eosinophil chemotactic factor was also detected in SEA. Together they are proposed to have a role in the direct accumulation of eosinophils in the egg-induced granulomas in S. japonicum infection.     

Glutamate dehydrogenase in the first leaf of wheat

Glutamate dehydrogenase extracted from wheat leaves ( Triticum aestivum L. cv. Capitole) taken at two different physiological stages was analysed by electrophoretic and immune-chemical techniques. Two NAD-dependent antigens were identified which bear the balk of the glutamate dehydrogenase activity in the two extracts. The first enzyme was found in much larger amounts in young than in senescent leaves and the reverse situation was observed for the second antigen. The possible relationships between this antigenic polymorphism and the heterogeneity detected by isoelectric focusing from the two extracts were investigated. A charge heterogeneity (isoelectric points about 5.7 and 4.8) was found for the first antigen in both extracts. The second antigen appeared homogeneous (isoelectric point about 5.7) at least in senescent leaves. The last result indicates that two quite different antigens appear in the same isoelectric focusing zone.     

Radioimmunoassay of a glycoprotein associated with malignancy

A glycoprotein associated with malignancy was purified from the 0.6M perchloric acid-soluble fraction of serum obtained from cancer patients. The purified glycoprotein contained sialic acid, which was responsible for binding to wheat-germ agglutinin-Sepharose. Gel electrophoresis showed one band with an apparent Mr of 50 000-55 000, and the isoelectric point was 4.4 +/- 0.1. The glycoprotein could be distinguished from carcinoembryonic antigen and alpha-fetoprotein. Iodination of this material with chloramine-T permitted development of a radioimmunoassay.     

Changes in the isoelectric focusing profile of pituitary follicle-stimulating hormone in the developing male rat

Pituitary glands, hypothalami, and trunk blood were obtained from male rats at 5, 15, 18, 21, and 29 days of age, on the day of balanopreputial separation (Days 42-45), and during adulthood. The forms of follicle-stimulating hormone (FSH) present within each pituitary were separated by polyacrylamide gel isoelectric focusing. Serum and pituitary gonadotropins, hypothalamic luteinizing hormone-releasing hormone (LHRH), and the profile of FSH forms across the isoelectric focusing gel were determined by radioimmunoassay. No change in the relative proportions of FSH forms were observed between 5 and 21 days of age. Likewise, only slight changes in serum and pituitary gonadotropin levels and hypothalamic LHRH content were observed at these times. After 21 days of age, dramatic increases in serum and pituitary gonadotropin levels were observed. Similarly, a shift in FSH forms within the pituitary to more basic and bioactive forms was observed at this time. These results demonstrate that, during the transition through puberty in the male rat, not only the absolute amount, but also the isoelectric focusing profile, of FSH change.