Specificity of the serological diagnosis of dysentery using erythrocytic reagents

The serological specificity of the diagnosis of dysentery, made by different methods with the use of antigenic and antibody erythrocyte diagnostic agents under the conditions of the circulation of different Shigella species and subspecies in a given locality, has been compared. The method for the determination of the diagnostic titer of the total serum antibody activity has proved to be the least specific. The methods for the detection of Shigella antigens, especially over time, in patients' excretions and for the determination of serum antibody activity show complete specificity. The combination of specificity and sensitivity makes the detection of Shigella antigens in feces by means of the passive hemagglutination test and the antibody neutralization test the method of choice for the diagnosis of dysentery.     

自体红细胞凝集试验研究进展

自体红细胞凝集试验是一种快速、简便,成本低廉的免疫学检测方法。用于自体红细胞凝集试验的主要成分是一种双功能性抗体。介绍了自体红细胞凝集试验的基本原理和主要特点,以及如何建立自体红细胞凝集试验检测体系;简要综述了双功能性抗体的活性、稳定性、特异性、敏感性等方面的研究进展,以及自体红细胞凝集试验检测方法的应用前景。     

Results of a trial of a plague monoclonal antibody erythrocyte diagnostic agent

Plague antibody monoclonal erythrocyte diagnosticum was studied in serological tests simultaneously with commercial plague antibody erythrocyte diagnosticum prepared on the basis of hyperimmune horse serum and with commercial plague antigenic erythrocyte diagnosticum. In this investigation the suspensions of numerous strains of Yersinia pestis, other closely related and heterologous organisms, experimentally infected wild and laboratory animals, as well as samples of materials obtained from small rodents caught in several natural foci of plague, were studied. The monoclonal diagnosticum was, practically, not inferior to the similar commercial preparation with respect to the frequency of positive results and the activity of the materials under study in serological tests, but showed greater specificity, as it reacted strictly with Y. pestis capsular antigen.     

结核感染T细胞斑点试验对肺结核病的临床诊断价值研究

目的：评价结核感染T细胞斑点试验（T-SPOT．TB）对肺结核病的临床诊断价值。方法：选择2012年2月～8月湘雅医院呼吸科住院病人中92例可疑肺结核患者进行T—SPOT．TB检测、结核菌素试验（PPD试验）、结核抗体、血沉及影像学检查及病史收集。分析和比较T．SPOT．TB与传统结核诊断方法的阳性率、特异度、灵敏度并对其检测结果进行相关性分析。结果：92例患者中,48例被确诊为肺结核,其中41例T．SPOT．TB结果阳性,44例非肺结核患者中5例T．SPOT．TB结果阳性。T—SPOT．TB检测的敏感度为85．4％,特异度为88．6％。T-SPOT．TB检测在结核组的阳性检出率（85．4％）显著高于传统检查方法PPD（37．5％,P〈0．01）、结核抗体（16．7％,P〈0．01）、血沉（66．7％,P〈0．05）,在非结核病组中的特异性（88．6％）显著高于血沉（36．6％,P〈0．0J）。PPD与T-SPOT．TB联合可提高诊断结核的阳性率（89．6％）。T．SPOT．TB检测仅与PPD试验的结果存在显著性差异（P〈0．05）。结论：T-SPOT．TB诊断肺结核的敏感性及特异性较传统的PPD实验、结核抗体更高,具有重要的I临床应用价值。     

Preparation and properties of antisera to glycolipid of guinea pig erythrocyte membrane

Antibodies against a glycolipid of guinea pig erythrocyte membranes were prepared in rabbits by immunization with guinea pig erythrocyte stroma or the purified glycolipid, gangliotriaosylceramide. The antibodies agglutinated guinea pig erythrocytes. The specificity of antibodies could be revealed by several immunochemical methods, including inhibition of hemagglutination, immunodiffusion, agglutination of liposomes, and complement fixation. The antibodies were specific for gangliotriaosylceramide.     

Dissociation of fibronectin from gelatin-agarose by amino compounds.

Affinity chromatography of radioiodinated solubilized erythrocyte stroma and of radioiodinated purified Band 3 on an anti-(blood-group I)-adsorbent column showed blood-group-I activity associated with a subpopulation of Band 3. The specificity of binding was confirmed by inhibition with known blood-group substances in radioimmunoassays.     

Equine peptidases: Correspondence with human peptidases and polymorphism for erythrocyte peptidase a

Equine erythrocyte peptidases were compared to the six human erythrocyte peptidases, A, B, C, D, E, and F, regarding substrate specificity, relative activity, and electrophoretic mobility. Five equine erythrocyte peptidases appeared homologous to human peptidases A, B, D, E, and F. In contrast to human, equine peptidase C was absent in red cells, although it was weakly active in white cells. On the other hand, an equine peptidase, probably homologous to human peptidase S, was weakly active in red cells as well as present in white cells. Polymorphism for equine erythrocyte peptidase A is reported.     

Association of human erythrocyte membrane glycoproteins with blood-group Cad specificity.

Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of erythrocyte membranes from a blood-group-B individual with the rare Cad phenotype indicates a lower-than-normal mobility of the main sialoglycoproteins, suggesting an increase in apparent molecular mass of 3kDa and 2kDa respectively for glycoprotein alpha (synonym glycophorin A) and glycoprotein delta (synonym glycophorin B). Since the chief structural determinant of Cad specificity is N-acetylgalactosamine, the membrane receptors have been isolated by affinity binding on immobilized Dolichos biflorus (horse gram) lectin. The predominant species eluted from the gel was the abnormal glycoprotein alpha, whereas in control experiments no material could be recovered from the adsorbent incubated with group-B Cad-negative erythrocyte membranes. After partition of the membranes with organic solvents, the blood-group-Cad activity was found in aqueous phases containing the sialoglycoproteins, but not in the organic phases containing simple or complex glycolipids, which, however, retained the blood-group-B activity. The carbohydrate composition of highly purified lipid-free glycoprotein alpha molecules prepared from Cad and control erythrocytes was determined. Interestingly the molar ratio of N-acetylneuraminic acid to N-acetylgalactosamine was equal to 2:1 in the case of controls and equal to 1:1 in the case of Cad erythrocytes. Taken together these results suggest that Cad specificity is defined by N-acetylgalactosamine residues carried by the alkali-labile oligosaccharide chains attached to the erythrocyte membrane sialo-glycoproteins.     

The action of phosphatidylinositol-specific phospholipases C on membranes.

A phospholipase C prepared from lymphocytes readily hydrolysed pure phosphatidyl-inositol but was relatively ineffective against phosphatidylinositol in erythrocyte "ghosts" and rat liver microsomal fraction and also against sonicated lipid extracts from these membranes. In contrast, a phospholipase C prepared from Staphylcoccus aureus readily hydrolysed phosphatidylinositol in sonicated lipid extracts but had only low activity against purified phosphatidylinositol. Unlike the enzyme from lymphocytes, the S. aureus phospholipase C did not require Ca2+ for its activity and was inhibited by cations. The previously reported specificity of this enzyme was confirmed by our observation of hydrolysis of approx. 75% of the phosphatidylinositol in ox, sheep and cat erythrocyte "ghosts" together with no detectable effect on the major erythrocyte membrane phospholipids. The phosphatidylinositol of rat liver microsomal fraction was hydrolysed only to a maximum of 15%. Some preliminary experiments showed that approx. 60% of the phosphatidylinositol of ox or sheep erythrocytes could be hydrolysed without causing substantial haemolysis.     

Characterization of the hydroperoxide-reducing activity of human plasma

A peroxidase was identified in human plasma using a novel peroxidase assay. In this assay both the substrate 5-phenyl-4-pentenyl hydroperoxide (PPHP) and its reduction product, 5-phenyl-4-pentenyl alcohol (PPA) are quantitated by HPLC. Substrate specificity studies indicated that the peroxidase requires glutathione as reducing substrate. No reduction was detected using the classical heme peroxidase reducing substrates, phenol and hydroquinone. Peroxidase activity was not due to glutathione transferases. Failure to saturate the peroxidase activity with reduced glutathione and inhibition by Cd+2 indicated that it is probably selenium dependent. The enzyme appears to be different from erythrocyte glutathione peroxidase based on kinetic and immunological experiments. The apparent Km values for PPHP are 25 microM for erythrocyte peroxidase and 54 microM for plasma peroxidase at 0.5 mM reduced glutathione. Anti-peroxidase prepared against bovine erythrocyte glutathione peroxidase partially inhibited human erythrocyte peroxidase but did not inhibit human plasma peroxidase.     

Serologic diagnosis of intestinal yersiniosis. The design of a stable erythrocyte preparation for the indirect hemagglutination reaction

Nine types of erythrocyte diagnostica of serovars O3 and O9, differing in the methods of obtaining sensitins and the physical state of erythrocytes, were put on trial. The preparations were used for the titration of hyperimmune sera and blood sera obtained from about 500 healthy persons, 300 patients with Yersinia enteric infection and 300 patients with other diseases. Freeze-dried diagnostica, when compared with liquid ones, were found to be less sensitive, but more stable and specific. Sensitins isolated by the methods of Westphal ad Boivin showed the highest degree of specificity. The authors believe freeze-dried sheep red blood with activated Boivin's antigen adsorbed onto them to be the optimal preparation for use in the passive hemagglutination (PHA) test. The preparation was found to retain its serological activity for as long as 2-3 years. The titer 1:160 (1:200) in the PHA test is recommended as the minimal diagnostic indicator. Erythrocyte diagnosticum is more sensitive, specific and stable than bacterial one. Since 1984 dried Yersinia erythrocyte diagnostica (serovars O3 and O9) have gone into quantity production at the Leningrad Research Institute for Vaccines and Sera.     

Erythrocyte reagents for detecting antibodies to species-specific staphylococcal antigens

Antigenic species-specifics (S. aureus and S. epidermidis) erythrocyte diagnosticums have been obtained with the use of different loading methods. The cross reaction of passive hemagglutination with homologous and heterologous sera have demonstrated that conjugation with amidole ensures the maximum effectiveness and species specificity of diagnosticums in comparison with other conjugation methods.     

Phenothiazine antagonism of calmodulin: a structurally-nonspecific interaction

Phenothiazine antagonism of calmodulin-stimulated (Mg²⁺ + Ca²⁺)-ATPase activity in erythrocyte ghosts was examined to determine the structural specificity of the interaction. Four analogs of chlorpromazine, differing in the position of the chlorine substitution of the aromatic ring, were about equally potent in antagonizing calmodulin activation, while only the 2-chloro analog (chlorpromazine) has tranquilizer activity and antagonizes dopamine-sensitive adenylate cyclase. As all four analogs have similar hydrophobicity and surface activity, the results indicate that the antagonism of calmodulin by phenothiazines is unrelated to their pharmacological specificity and occurs at a structurally-nonspecific hydrophobic site on the protein.     

Interaction of human red cell membrane acetylcholinesterase with phospholipids

Liposomes of phospholipids fully sustain the enzyme activity of the amphiphile-dependent dimers of human erythrocyte membrane acetylcholinesterase; no head group specificity exists. Diacylglycerides, glycerophosphorylcholine, or free fatty acids do not sustain the catalytic activity. It could be shown that the dimeric acetylcholinesterase with an exposed hydrophobic region can penetrate the lipid bilayer of liposomes and thus becomes stabilized by the surrounding phospholipid molecules.     

Characterization of 12-L-hydroperoxyeicosa-5,8,10,14-tetraenoic acid peroxidase in platelets by monoclonal antibody against glutathione peroxidase

In the present investigation, 12-L-hydroxyeicosa-5,8,14-tetraenoic acid (12-HPETE) peroxidase in the platelet 12-lipoxygenase pathway was characterized by using a monoclonal antibody to erythrocyte glutathione peroxidase. Pure glutathione peroxidase was used for the immunization of mice. Monoclonal antibody directed against the erythrocyte glutathione peroxidase was obtained from hybridomas, following fusion of mouse NS-1 myeloma cells with spleen cells from a mouse immunized with the enzyme. The subclass of monoclonal antibody was immunoglobulin M with kappa-light chain. Enzyme activity assays using cumene hydroperoxide and [1-14C]12-HPETE as substrates were employed. The monoclonal antibody reacted with glutathione peroxidase in the cumene hydroperoxide assay. In order to see whether platelet 12-HPETE peroxidase reacts with the monoclonal antibody, platelet cytosol and glutathione peroxidase were incubated with the monoclonal antibody and the antibody was precipitated by goat anti-mouse immunoglobulin M. The activities of platelet 12-HPETE peroxidase and glutathione peroxidase remaining were then assayed by using [1-14C]12-HPETE as substrate. The ability of glutathione peroxidase to transform 12-HPETE to 12-HETE was removed by the monoclonal antibody; however, the activity of platelet cytosol was not removed by the antibody. The results indicated that the antigenic specificity of 12-HPETE peroxidase in the platelet 12-lipoxygenase pathway is different from that of erythrocyte glutathione peroxidase.     

Comparative study of cholinergic activity of some tropolone and isoquinoline alkaloids

Comparative study was performed of action of a group of 15 isoquinoline homoproaporphine and homoaporphine alkaloids, 10 tropolone colchicine alkaloids and their lumoderivatives that were isolated from corms of a representative of the lily family, the showy autumn crocus Colchicum speciosum Stev. on activity of mammalian erythrocyte acetylcholinesterase and serum butyrylcholinesterase. The studied compounds have turned out to be moderate-potency reversible inhibitors of the studied cholinesterases and to show to a certain degree some specificity of action towards different enzymes both quantitatively, by the ratio of total inhibitor constant values, and qualitatively, by the type of mechanism of the enzymatic activity inhibition. The overwhelming majority of the studied alkaloids revealed certain specificity towards butyrylcholinesterase. An exception was colchamine.     

The stereospecificity of protein kinases

To test whether cellular protein kinases exist that phosphorylate D-amino acid residues, a method was developed for separating O-phospho-D-serine from O-phospho-L-serine and O-phospho-L-tyrosine from O-phospho-D-tyrosine. This was accomplished by converting these amino acids to the L-leucyl dipeptide derivatives followed by separation of the diastereomers by anion-exchange high-performance liquid chromatography. The enantiomeric content of these D- and L-residues were measured in hydrolysates of 32P-labeled proteins produced by the protein kinases of human erythrocytes and the tyrosyl protein kinase of the Abelson leukemia virus. We found no measurable D-phosphoserine in erythrocyte membrane proteins under conditions where a 1% content of this residue relative to L-phosphoserine would have been detected. These values can be used to place an upper hypothetical limit on the fraction of erythrocyte protein kinase activity that is specific for serine residues in the D-configuration. In separate experiments, we examined the specificity of the tyrosyl protein kinases. We found that all of the phosphotyrosine that we isolated from the erythrocyte band 3 NH2-terminal fragment and from the autophosphorylation of the Abelson virus tyrosyl kinase was in the L-configuration.     

Cultural conditions forAeromonas hydrophila affect the production of haemolysins with differing host specificities

Five strains ofAeromonas hydrophila were studied for production of haemolysin specific for erythrocytes of various animal species using three cultural methods. All the strains produced haemolysin for all the erythrocyte species when the organisms were cultured on blood agar.Using cellophane overlay method, all the strains produced haemolysin for fish erythrocytes and variable activity to mammalian erythrocytes. Only one strain produced haemolytic activity for various though not all of the erythrocyte species when grown in brain heart infusion broth.Data suggest thatA. hydrophila produces multiple haemolysins with specificities for erythrocytes of different animals. This was confirmed for trout and horse erythrocyte targeted haemolysins, by using iso-electric focussing separation and by measuring the effect of addition of ammonium sulphate to the growth medium.     

Partial purification and characterization of an endo-alpha-N-acetylgalactosaminidase from the culture of medium of Diplococcus pneumoniae.

The culture medium of Diplococcus pneumoniae contains enzymic activity that cleaves Galbeta1 leads to 3GalNAc from desialized human erythrocyte membrane glycoprotein. The enzyme was purified 180-fold by ammonium sulfate fractionation, gel filtration through a Sephadex G-200 column, and DEAE A-25 Sephadex chromatography. The purified enzyme liberates Galbeta1 leads to 3GalNAc from glycopeptides and glycoproteins with Galbeta1 leads to 3GalNAcalpha1 leads to Ser and Thr moieties. The optimum pH of this enzyme is 6.0. Using glycopeptides obtained by trypsin digestion of human erythrocyte membrane glycoprotein as a substrate, a Km of 0.20 mM (on the basis of the amount of Galbeta1 leads to 3GalNAc residues) was obtained. So far, the enzyme appears to have a strict specificity for Galbeta1 leads to 3GalNAcalpha1 leads to Ser and Thr structures, because no oligosaccharides larger than trisaccharides were liberated from porcine submaxillary mucin.