Chromatographic Studies of Sera from Calves Vaccinated with Brucella Abortus, Strain 19

The presence of antibody was detected by agglutination tests in the serum of calves four days after vaccination with Brucella abortus strain 19. Titres had reached a maximum by seven to ten days post-vaccination. Sucrose density-gradient ultracentrifugation demonstrated that the earliest antibodies were macroglobulins, IgM (19Sγ; γM)-globulins. Lighter antibodies, IgG (7Sγ₂; γG)-globulins, appeared a few days later. With time, antibody titres fell, IgM declining somewhat more quickly than IgG. After revaccination some seven months later, there was a rapid rise in both IgM and IgG.

Anion-exchange column chromatography (DEAE-cellulose) and gel filtration (Sephadex G-200) were applied in separating the two forms of antibody. The former method, in which a gradient buffer system was used, proved to be the more efficient; the IgG antibodies apeared in early eluates at pH 7.8 to 8.0 and low ionic strength, 0.03M, whereas IgM was eluted late when the pH had fallen below 6.0 and the molarity had increased to beyond 0.2. DEAE cellulose chromatography detected IgG as well as IgM sera collected as early as five days after vaccination.

     

The Effect of Unheated Bovine Serum on the Complement-Fixing Activity of Heat-Inactivated Bovine Antiserum with Homologous Antigen. IV. Chromatographic Studies

Unheated, non-dialyzed, normal bovine sera were fractionated by column chromatography on the cross-linked dextran, Sephadex G-25, and the fraction tested for “supplementing” properties, that is for complement-fixation augmenting activities when added to mixtures of heated bovine antiserum and homologous antigen. Supplementing activity was shown by precipitated fractions from earlier eluates with pH values below 7.2 and also by both supernatant and precipitated fractions of the later eluates with pH values from 7.6 to 8.1. The possibility is briefly discussed that certain alkaline protein substances of relatively lower molecular weight may be involved in the supplementing activities of the later fractions. Heating at 56°C. for 30 min. destroyed the supplementing activity of each of these fractions.

Some of the supplementing fractions proved to be anti-complementary, others were not or only slightly so. First component of complement, C¹1, was detected in the precipitated fractions of certain of the earlier eluates with pH values below 6.5; second component of complement, C¹2, was found exclusively in supernatant fractions of earlier eluates with pH values less than 6.2. Conglutinin was not separated from C¹1 by this method.

     

Fractionation on Cross-Linked Dextran, Sephadex G-25, of Sera from Sheep Infected with Johne's Bacilli. Preliminary Report

Ten sera collected during the winter months from six sheep infected with Johne's bacilli were fractionated on Sephadex G-25 columns, and all fractions tested for complement-fixing antibody, anticomplementary properties and for supplementing and inhibitory activities when added to complement-fixation tests of a heterologous antigen-antibody system: bovine or ovine antiserum with Brucella abortus antigen. Serum from a normal sheep was similarly fractionated and examined.

The complement-fixing activity with a carbohydrate fraction of Johne's bacilli was confined principally to supernatant fractions of the earlier eluates containing the greater part of the serum proteins. Some of the unheated earlier and later fractions displayed a limited degree of supplementing effect. Inhibitory activity was demonstrated by certain of the earlier and later eluates after they had been heated, particularly those of antisera with initially low complement-fixing antibody titer.

     

Protein constituency of unbound fraction of bovine serum applied to anion exchange columns at different pHs and molarities with or without ethylenediamine tetraacetic acid. Application to preparation of bovine IgG1

Ethylenediamine tetraacetic acid disodium salt (EDTA) was found to be an important addition to anion exchange buffers in terms of the profile and amounts of eluted bovine serum proteins. Hydrogen ion concentration and buffer composition were important when different types of anion exchanger (DEAE 52 cellulose and QAE Sephadex A 50) were used for separation of bovine serum proteins as functional groups of the different anion exchangers did not behave similarly and were therefore not interchangeable. These findings applied to the purification of bovine serum IgG1 amounting to 15 to 25% of the total IgG1 was accomplished using QAE Sephadex A 50 anion exchange chromatography. This was followed by absorption of the IgG1 fraction with Staphylococcus aureus containing protein A to remove minor IgG2 contaminants and gel filtration to exclude traces of the third component of complement (C3).     

The antigenic activity of chromatographic fractions of a saline extract of Taenia saginata (Goeze, 1782) proglottides

A saline extract of a homogenate of Taenia saginata proglottides was partially purified by gel filtration chromatography on Sephadex G200 or Sepharose 4B and by ion exchange chromatography on DEAE cellulose. Gel filtration produced two distinct fractions with different antigenic properties. The first was of molecular weight of approximately 1,000,000 and contained a high level of activity in the haemagglutination inhibition test. The second fraction of molecular weigh of approximately 100,000 contained most of the immuno-precipitin activity. Other fractions had little or no antigenic activity. Eight fractions were obtained by DEAE cellulose chromatography, of which 4 had detectable antigenic activity. Subsequent rechromatography of fractions obtained by gel filtration on DEAE cellulose produced relatively pure fractions of high antigenic activity, from which small molecular weight contaminants had been removed.     

Preparation of porcine IgA and its detection using the fluorescent antibody technic]

The preparation of immunoglobulin A (IgA) from porcine colostrum, intestinal content and serum is described. The best results were achieved with colostrum, from which an antigen of satisfactory purity was prepared by purification on Sephadex G-200, on DEAE cellulose and subsequent filtration on Sephadex G-200. The serum to this antigen raised in rabbits was adsorbed to an immunoadsorbent from porcine serum (PS) or porcine IgG. The adsorbtion of the serum against secretory IgA (SIgA) to PS removed its undesirable heterologous and nonspecific reactivity. The anti-SIgA serum adsorbed in this way still reacted with IgA from porcine serum. In the direct and indirect immunofluorescent staining we detected the main antigenic determinants of the SIgA molecule, i. e. the heavy chains and the secretory component.     

Purification and properties of porcine allantoic fluid retinol-binding protein

Retinol-binding protein (RBP) was purified from Day 60 porcine allantoic fluid by a combination of diethylaminoethyl cellulose, G-100 Sephadex, G-50 Sephadex, Phenyl-Sepharose, and Reactive Green 19-dye-agarose chromatography. The yield was 1 to 2 mg of RBP, which generated a single M_r 20,000 band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and up to four isoelectric variants (isoelectric variants 1 to 4) after two-dimensional PAGE (2D-PAGE). The protein cross-reacted with antiserum raised against human RBP. When incubated with [³H]retinol and subjected to G-100 Sephadex chromatography, [³H]retinol coeluted with the protein. These results indicate that the purified protein is an RBP. When purified RBP was subjected to native 2D-PAGE, six forms of RBP were observed. Three native forms were fluorescent, and three were not fluorescent, suggesting that these forms were RBP with and without retinol, respectively. Denaturing 2D-PAGE analysis of each native form of RBP suggested that two of the nonfluorescent and two of the fluorescent native forms of RBP corresponded to isoelectric variant 1 on denaturing 2D-PAGE, whereas the other fluorescent and nonfluorescent forms corresponded to isoelectric variant 2. The incubation of RBP with 50 μM retinol enhanced the amount of both isoelectric variants present as fluorescent RBP, but uptake by isoelectric variant 1 was greater than that by isoelectric variant 2. These data indicate that RBP can be purified from porcine allantoic fluid and suggest that the isoelectric variants may differ in their affinity for retinol.     

Retinol transport system in cattle and purification of retinol-binding protein from bovine serum

Retinol transport system in cattle was investigated, followed by the purification and characterization of bovine serum retinol-binding protein (RBP). Gel filtration of serum from cow produced two retinol peaks, peak 1 and 2. The major, peak 1 having higher molecular weight corresponded to the retinol peak from human serum which consisted of RBP and prealbumin (PA). The peak 2 which was not presented in the human serum had lower molecular weight (about 20,000). In the presence of 3.0 M urea, the peak 1 was almost disappeared and peak 2 was increased. On the other hand, in the serum from calf, major retinol peak was corresponded to the peak 2 from cow. These results suggested that, in cow, retinol was transported by the complex of RBP and another protein, presumably PA, but in calf, mainly by RBP alone. Purification of bovine RBP was carried out by using four chromatographic steps as follows; 1. DEAE-cellulose (pH 6.0), 2. Sephadex G-100 (using the buffer containing 3.0 M urea), 3. DEAE-cellulose (pH 8.3), 4. Sephadex G-100. From 1,100 ml of serum, 14.1 mg of bovine RBP was finally obtained and the overall recovery was estimated to be about 32%. Its molecular weight, ultraviolet absorption and fluorescence spectra, electrophoretic mobility, and amino acid composition were similar to those of other species.     

A Preliminary Study of the Development of 19s and 7s Immunoglobulins in Sera of Two Sheep Vaccinated With Brucella Abortus, Strain 19

The pattern of antibody response to vaccination with Brucella abortus, strain 19, was studied in two sheep. Agglutinative activity was detected by the third and fifth days and complement - fixing activity by the fifth and seventh days post-vaccination.

Density gradient centrifugation and DEAE cellulose column chromatography showed the 19S antibody developed first, followed soon after by 7S antibody. The former had disappeared by the 25th day but the latter persisted longer in both sheep. A small amount of 19S antibody was detected in sheep 1 following a booster dose of vaccine but 7S antibody constituted the major secondary response.

The standard tube agglutination test was found to be more efficient than the complement-fixation test for titration of 19S antibody. An increase in the salt concentration to 10% in tube agglutination test rendered it more sensitive in demonstrating 7S antibody.

     

Preparation of Mycoplasma hyopneumoniae antigen for the enzyme-linked immunosorbent assay.

Methods of preparation of Mycoplasma hyopneumoniae antigens for the enzyme-linked immunosorbent assay to detect specific antibody, and properties of the antigens, are described. The reactivity and specificity of antigen prepared by Sephacryl S-300 column chromatography after treatment of M. hyopneumoniae cells with Tween 20 (S-300 antigen) were superior to those of antigen prepared by Sephadex G-25 column chromatography after treatment with Tween 20, or to lipid antigen. There were no differences among strains MI-3, J and VPP11 of M. hyopneumoniae. The S-300 antigen did not show cross-reactivity against porcine hyperimmune sera produced by M. hyorhinis, M. hyosynoviae, M. hyopharyngis, M. flocculare and Acholeplasma granularum. Antibody was first detected in sera of pigs inoculated intranasally with M. hyopneumoniae at two to four weeks after inoculation and seven to eight weeks after pigs were contact-exposed to the same mycoplasma.     

The partial purification of Clostridium perfringens beta toxin.

An attempt was made to purify Clostridium perfringens Beta toxin. Crude toxin prepared by ammonium sulphate precipitation of culture supernatants was purified by chromatography on Sephadex G50, Sephadex G100 and DEAE cellulose. This material, although highly purified was not homogeneous on polyacrylamide gel electrophoresis. It had a toxicity of 800 000 mouse MLDs/mg N, a typical protein absorption spectrum in the UV region, an iso-electric point of 5, 6 and the main component had a molecular mass of 42 000 +/- 2 000 (estimated by electrophoresis in sodium dodecyl sulphate containing polyacrylamide gels).     

柞蚕蛹溶菌酶的诱导与提取

本文报道热空气、大肠杆菌K_(12)D_(31)诱导柞蚕蛹(Antheraea pernyi)血淋巴溶菌酶活力提高的最适条件以及从柞蚕蛹免疫血淋巴中提取溶菌酶.以Sephadex G-50和CM-Sepharose柱层析法从柞蚕蛹免疫血淋巴中提取溶菌酶,并首次获得柞蚕蛹溶菌酶结晶,溶菌酶制剂比活力达到57353单位/mg蛋白,提高了257倍,总活力回收79.2%.首次应用选择性热变性,等电点沉淀、DEAE-纤维素离子交换层析、包被甲壳素-纤维素亲和层析等分离技术从柞蚕蛹免疫血淋巴中提取溶菌酶.溶菌酸制剂比活力达25 375单位/mg蛋白,提高了140倍,活力回收51%.用15%、pH4.0聚丙烯酰胺凝胶电泳(PAGE)和3.0-100%.pH6.7-8.9SDS-PAGE鉴定了酶制剂的纯度,并测定了柞蚕蛹溶菌酶的部分氨基酸组成.     

Studies on Aspergillus Fumigatus; Hydrolysis of Polyamino Acids by Mycelial Filtrate and Chromatographic Fractionation of the Filtrate

Mycelial filtrates from Aspergillus fumigatus (AF), shown to possess haemolytic, toxic, casein precipitating, and protein hydrolyzing activity, hydrolyzed poly-L-lysine and poly-L-glutamine in the pH range 4.6—5.3. Incipient activity against poly-L-lysin was observed at pH 9. Owing to spontaneous hydrolysis of the polyamino acid at pH > 10, no activity optimum could be traced.Gel filtration of mycelial filtrate on Sephadex G-75 or G-100 columns offered no definite information whether the protein hydrolyzing activity, using haemoglobin as substrate, at the optimum pH values, 2.9, 4.6 and 10, shows the activity of a single enzyme with more than 1 pH optimum or of more than 1 enzyme active at different pH values. Certain results of the investigations seem to indicate that the protein hydrolyzing activity at pH 2.9 was not caused by enzymes identical with the enzyme (s) causing the protein hydrolyzing activity at pH 4.6 or pH 10.Casein precipitating and protein hydrolyzing activity occurred, following gel filtration on a Sephadex G-100 column, in identical fractions whereas neither haemolysin nor toxin could be found in samples of 0.5 ml fraction solution from any of the fractions after filtration on Sephadex G-75 or G-100 columns.By using antiserum to a crude filtrate from a homologous AF strain it could be shown, applying immuno-electrophoresis, that dialyzed mycelial filtrate contained 8 precipitating antigens whereas proteinase purified by gel filtration and displaying protein hydrolyzing activity at pH 2.9, pH 4.6 and pH 10 contained 4 such antigens.     

鸡免疫球蛋白G Fc(IgG Fc)片段的分离纯化及其抗血清的制备 Ⅱ.豚鼠抗鸡IgG Fc片段血清的制备

经硫酸铵盐析、DEAE 32-纤维素和Sephadex G 200柱色谱法分离得到纯化的鸡血清IgG。然后用木瓜蛋白酶水解IgG,再经DEAE 32-纤维素柱色谱纯化制得IgG Fc片段,并以IgG Fc片段免疫豚鼠制备豚鼠抗鸡IgG Fc血清。     

家蚕γ-谷氨酰环化转移酶的纯化及其性质的研究

首次从家蚕(Bombyx mori L.)蛹体脂肪织织及真皮细胞分离得到催化丫-L-谷氨酰-2-L-氨基丁酸反应生成吡咯烷酮羧酸和2-L-氨基丁酸的Y-谷氨酰环化转移酶。并采用硫酸铵沉淀、葡聚糖G-75柱层析、DEAE-纤维素DE_(32)柱层析和羟基磷灰石柱层析等步骤,将该酶提纯了1,285倍。高度纯化后的酶,对Y-L-谷氨酰-2-L-氨基丁酸具有最适pH7.2-7.4,最适酶反应温度为47℃,米氏常数(Km)为0.04M。在分离纯化过程中未发现蚕体内有该酶的同功酶存在,纯化后的酶溶解于pH8.0、0.01M的Tris—HCl缓冲掖中,-30℃经过一个月没有发现明显的失活现象,但在4℃中一个月后丧失活性76％。     

猪SIgA分泌片的分离纯化及其多克隆抗体的制备

为制备抗分泌片多克隆抗体,采用分子筛凝胶层析、离子交换层析和硫酸铵盐析法从猪初乳中分离纯化分泌片和SIgA。SDS—PAGE鉴定表明,纯化的SIgA呈现分泌片、重链和轻链三条带,大小约为70ku;凝胶薄层扫描分析SIgA和分泌片纯度分别为90％和89％;琼脂扩散鉴定表明,SIgA和分泌片与抗分泌片单克隆抗体发生了特异反应。以纯化分泌片为抗原,制备抗分泌片多克隆抗体,琼脂扩散检测多抗的效价为1：32。高纯度分泌片的提取及高效价抗分泌片多抗的制备为粘膜负．疫研究奠定了物质基础。     

Purification of infectious bronchitis coronavirus by Sephacryl S-1000 gel chromatography

A procedure was developed to purify infectious bronchitis virus (IBV) by gel chromatography (GC) with a Sephacryl S-1000 column. Virus samples concentrated by centrifugation were applied to a Sephacryl S-1000 column and eluted by 0.02 M phosphate buffer (pH 7.2) containing 0.15 M NaCl. Virus particles were recovered mainly in the first peak. Purity of the samples was evaluated by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electron microscopy. Using electron microscopy, it was found that there were more spike-rich particles in the virus samples purified by GC than in those purified by sucrose density gradient centrifugation (SDGC). In addition, the hemagglutination unit [log10 (infectivity titer/hemagglutination titer)] of GC-purified virus samples was approximately 10 times lower than that of SDGC-purified virus samples. These results indicate that Sephacryl S-1000 gel chromatography is useful for purification of IBV.     

Isolation of C-reactive protein from cat serum

C-reactive protein (CRP) was isolated from sera from healthy cats by calcium-dependent affinity chromatography on phosphorylcholine derivatives of bovine albumin-coupled Toyopearl, followed by an anion-exchange chromatography using DEAE cellulose. It was identified as CRP by its immunochemical cross reactivity with human CRP. The molecular weight of cat CRP was approximately 100 kilodaltons (kDa) and composed of two glycosylated subunits (23 kDa) and three non-glycosylated subunits (20 kDa) with non-covalent association. Under electron microscopic examination, cat CRP had a pentameric disc-like configuration which is characteristic of CRP. Immunoelectrophoresis and isoelectric focusing showed that cat CRP is an acidic α₁-globulin (pi 4-1 to 4-3). Serum concentrations of CRP in cats and kittens were measured by single radial immunodiffusion. In 12 healthy cats from various sources, values ranged from 38 to 168 μg/ml. In kittens, serum CRP levels also showed a wide distribution, 81 per cent of them were less than 40 μg/ml.     

鸡肠道抗菌肽的提取及活性研究

取新鲜鸡肠黏膜超声波破碎,乙酸浸提,经Sephadex G-50层析后,收集具有抑菌活性的组分进行超滤离心,抑菌实验表明滤过物具有抑菌活性.滤过物经SDS-PAGE分析为一个条带,分子量约为6.078KD,得到电泳纯的鸡肠道抗微生物肽.该肽具有一定的耐热性,但经90℃以上热处理后活性下降较大;pH值对其活性影响较大,在pH6～8时活性最强.     

Hypophosphatemia Induced by Dietary Aluminium Hydroxide Supplementation in Growing Pigs: Effects on Erythrocytes,Myocardium, Skeletal Muscle and Liver

Håglin, L., B. Essén-Gustavsson and A. Lindholm: Hypophosphatemia induced by dietary aluminium hydroxide supplementation in growing pigs: Effects on erythrocytes, myocardium, skeletal muscle and liver. Acta vet. scand. 1994, 35, 263-271.– Three groups of pigs were studied during and after 10 weeks of treatment with either Al(OH)₃ (Al[OH]₃-group, n=8) to induce hypophosphatemia, A1P0₄ (AlP0₄-group, n=8, aluminium control without hypophosphatemia) or no addition to the feed (control group, n=8). Blood samples were taken at the start of the experiment and after 3, 6 and 10 weeks and were analyzed for phosphate, calcium and 2,3-diphosphoglycerate (2,3-DPG). Samples from myocardium, skeletal muscle and liver were obtained in connection with exsanguination and analyzed for glycogen, adenosine-tri-phosphate (ATP), creatine phosphate (CP), glucose-6-phosphate (G-6-P) and lactate. The Al(OH)₃-group became hypophosphatemic and hypercalcémie with low levels of 2,3-DPG in erythrocytes within 3 weeks and showed a retarded growth rate. After 10 weeks the Al(OH)₃-group had low levels of ATP in myocardium as compared with the control-group and low levels of G-6-P as compared with the AlP0₄-group. No disturbances on electro-cardiograms registered at rest could be documented. G-6-P concentration was low in the biceps muscle in the Al(OH)₃-group as compared with the AlP0₄-group and in the liver low G-6-P concentration was seen in addition to high lactate concentration. The fibre type composition in M. Longissimus did not differ between groups, but the Al(OH)₃-group had, due to retardation in growth, smaller mean fibre-areas than pigs in the AlP0₄-group. Hypophosphatemia gave rise to high serum calcium levels, low concentration of 2,3-DPG in erythrocytes and influenced G-6-P concentration in skeletal muscle, G-6-P and ATP in myocardium, G-6-P and lactate in liver. Retarded growth was one serious consequence of hypophosphatemia and the disturbed energy metabolism.