Erythrocytic diagnostic agents for determining antibodies to Proteus O antigens

Highly sensitive and specific erythrocyte diagnostic agents (ED) for the determination of antibodies to Proteus O-antigens have been obtained by the sensitization of formolated sheep red blood cells (SPBC) with activated lipopolysaccharides (LPS) without the use of mediators. The tannin treatment of formolated SRBC and/or the increase of temperature from 45 degrees C to 100 degrees C in the process of the preparation of ED have been found to produce no increase in effectiveness. Antibody ED permitting the detection of Proteus O- and H-antigens has been obtained by the sensitization of formolated chick red blood cells with immunoglobulin preparations to Proteus hydroxylamine antigens, carried out with the use of amidol. The experiments have shown the possibility of using this antibody ED for the determination of O-antibodies in the antigen neutralization test with nonactivated LPS used as an agglutinating agent. The passive hemagglutination test with antibody ED has proved to be a more sensitive method for the detection of O-antibodies than the antigen neutralization test with antigenic ED. The determination of Proteus etiology in the passive hemagglutination test with the use of antigenic ED has been shown to be highly effective in the examination of patients with chronic osteomyelitis at the stage of exacerbation.     

Comparative characteristics of the adhesive properties of microorganisms isolated from the urine of children with chronic obstructive pyelonephritis

The adhesive properties of 215 cultures, including 215 Escherichia coli strains, 43 Klebsiella pneumoniae strains and 60 Pseudomonas aeruginosa strains isolated from the urine of 124 children with chronic obstructive pyelonephritis were studied in the direct hemagglutination test simultaneously with those of 30 E. coli strains and 20 K. pneumoniae strains isolated from the feces of 50 healthy children, as well as 60 P. aeruginosa strains isolated from children with parenteral infections of other localization. E. coli and K. pneumoniae strains isolated from the urine of children with chronic obstructive pyelonephritis were found to have D-mannose-resistant hemagglutinins (68% and 37.2%) and a combination of mannose-sensitive and mannose-resistant adhesins (44.6% and 13.3% respectively). P. aeruginosa strains isolated from the urine of urological patients in the postoperative period showed the presence of mannose-resistant hemagglutinins to a greater extent (76.6%) than those isolated from children with parenteral infections of other localization (45%).     

Host-Specific Hemagglutination of Influenza A (H1N1) Virus

H1N1 strains of influenza A virus isolated during the influenza season of 1991–92 were divided into two groups according to the property of host-specific hemagglutination. Group 1 viruses agglutinated human and chicken red blood cells. Group 2 viruses agglutinated human but not chicken red blood cells. The viruses of both groups, however, showed the same antigenic structure determined with ferret antisera. The virus clones which were plaque-purified twice from a group 2 virus retained the characteristic of host-specific hemagglutination after five successive passages in MDCK cells, indicating that this phenomenon is genetically determined. However, the amino acid, sequences of the hemagglutinin (HA) polypeptides deduced from the nucleotide sequences of the HA gene of the two groups did not show any differences between them. This suggests a difference in amino acids in some other polypeptide(s), which affects the host-specific hemagglutination.     

DNA uptake in competent Streptococcus pneumoniae requires ATP and is regulated by cytoplasmic pH

We analyzed the ability of 120 encapsulated strains of B. fragilis to agglutinate guinea pig and human red blood cells. Sixteen strains showed a strong hemagglutination (HA) ability, 21 strains a moderate HA ability, 7 strains a weak HA ability and 74 strains did not agglutinate the tested red blood cells. Six strains tested from each HA group were able to adhere to cheek epithelial cells and to a cultured human intestinal cell line. Hemagglutinating strains were the most adhesive. By electron microscopy, pilus-like structures were found in three of the encapsulated adhesive strains. Treatment of the bacterial cells with pronase E reduced both HA ability and adherence of piliated encapsulated, and of piliated non-encapsulated strains. Glucosidase treatment of cells reduced HA activity and adherence of piliated encapsulated and of non-piliated encapsulated strains. Finally, it was found that hemagglutinating strains are more frequently isolated from clinical specimens (55%) than from feces of healthy donors (20%).     

Serological studies on lithotrophic,ammonia oxidizing bacteria

Rabbit antisera were prepared against living cells of six different ammonia oxidizing nitrifying bacteria. They were examined as to cross-reactivity in the agglutination test (Microtiter-system) with 24 nitrifier strains, including members of all known genera.Usually distinct cross-reactions were obtained only within the genera, but some exceptions were noticed. There was stated a clear cross-reaction between the two anti-Nitrosospira-antisera and the four tested Nitrosolobus strains. In some cases cross-reactions between cells of the Nitrosovibrio strains and the anti-Nitrosospira- as well as the anti-Nitrosococcus-antisera could be observed. The interpretation of the results obtained with the Nitrosomonas group was complicated by the fact that all strains showed positive zero titers with the control sera. In seven cases lipopolysaccharides were isolated and tested in the passive hemagglutination test to their cross-reactivity with the above mentioned antisera. hemagglutination could only be observed in the homologous system, cross-reactivity was never expressed.Abbreviations LPS Lipopolysaccharide(s) - G+C Guanine + cytosine - PBS Phosphate-buffered saline - SRBC Sheep red blood cells     

The determination of Escherichia virulence on nutrient media]

The virulence of 250 pathogenic Escherichia strains secreted from humans and from various environmental objects has been studied on nutrient media with stains, ram erythrocytes and in hemagglutination reactions. The highest percentage of strain virulence is revealed when using crystal violet and in the blood agar (44.0 and 27.2%, respectively), evidently, due to nonspecific reactions. The medium with congo red showed a positive result in 20.3 + 3.1%, and the d-mannose-resistant hemagglutination reaction--in 16.5 + 2.3% cases. In practice it is recommended to use several methods which enable estimating correctly the risk to infect humans and to conduct timely preventive measures.     

Hemagglutinating and adhesive capacities of Klebsiella and Enterobacter strains

The authors analyze the data of studies on the hemagglutinating and adhesive capacity of 290 cultures, including 118 K. pneumoniae strains and 64 E. cloacae strains isolated from sick children, as well as 59 K. pneumoniae strains and 49 E. cloacae strains isolated from healthy children. The hemagglutinating properties of the strains were determined in the hemagglutination test with fresh, formalin- and tannin-treated red blood cells, the adhesive properties were studied by light microscopy. Among K. pneumoniae and E. cloacae strains isolated in acute intestinal infections, mannose-sensitive hemagglutination and pronounced adhesive activity were prevalent in most cases. Poorly adhesive and nonadhesive strains were characteristic of K. pneumoniae and E. cloacae cultures isolated from healthy children. The strains isolated from sick and healthy children differed only by the prevalence of adhesive cultures.     

Utilization of a differential hemoglobin elution procedure as a rapid assay for identification of embryonic and adult chicken red blood cells.

1. A hemoglobin elution-staining procedure has been developed for distinguishing embryonic chick red blood cells from adult chicken red blood cells. 2. Adult hemoglobin is eluted from red blood cells with 1.9 M potassium phosphate buffer, pH 7.2; whereas, embryonic hemoglobin is retained within the cells and gives positive staining with erythrosin B. 3. The hemoglobin elution-staining pattern during development can be correlated with two embryonic hemoglobins as detected by polyacrylamide gel electrophoresis. 4. The series of red blood cells staining with erythrosin B correspond to the primary erythrocyte series suggesting that hemoglobin expression during development is correlated with different cell populations.     

Correlation between cell-associated mannose-sensitive hemagglutination by Vibrio parahaemolyticus and adherence to a human colonic cell line Caco-2

Abstract Cell-associated hemagglutination (cHA) activity with human erythrocytes was examined for 468 clinical and 71 environmental strains of Vibrio parahaemolyticus . Approximately 95% of the strains tested were cHA positive irrespective of source or Kanagawa phenomenon. 75% of clinical strains showed relatively strong mannose-sensitive hemagglutination (MSHA), whereas 88% of the environmental strains showed relatively weak mannose-resistant hemagglutination (MRHA). Adherence of V. parahaemolyticus to Caco-2 cells was also determined. A clear positive correlation between cell-associated MSHA and adherence to Caco-2 cells was observed.     

Specificity of antierythrocyte autoantibodies secreted by a NZB-derived hybridoma and NZB peritoneal cells

The specificity of monoclonal IgM antierythrocyte autoantibody produced by a NZB-derived hybridoma and the specificity of autoantibodies produced by uninduced NZB peritoneal cells in culture were determined. Supernatant fluids from cultures of hybridoma and peritoneal cells reacted in direct hemagglutination assays with bromelin-treated mouse erythrocytes, and, to a lesser extent, with sheep red blood cells; no agglutination was observed with intact mouse red blood cells or human O⁺ erythrocytes. These results suggest the presence of previously characterized anti-HB, but not anti-X or cold reactive autoantibodies, with a cross-reaction between antigenic constituents on sheep and bromelin-treated mouse erythrocytes. Specificity was affirmed by neutralization of agglutination or of direct hemolysis of bromelin-treated mouse erythrocytes with partially purified SEA-HB, the soluble plasma analog of the erythrocyte-bound HB autoantigen. Plaque formation in direct plaque-forming cell assays by both hybridoma and peritoneal cells was specifically inhibited by SEA-HB. These results demonstrate that NZB-derived hybridoma as well as NZB peritoneal cells secrete anti-HB autoantibody, an autoantibody that spontaneously appears in the serum of NZB as well as other strains of mice.     

舍蝇(Musca domestica vicina)凝集素的分离、纯化和部分性质研究

舍蝇蛹体液经抽提后上Sepharose 4B亲和层析柱纯化制得舍蝇凝集素。制剂经聚丙烯酰胺凝胶电泳和在SDS-PAGE上均呈单一蛋白带,表观分子量为33400。它能凝集人B型红细胞,亦能凝集小白鼠及兔血红细胞。其专一结合的糖为半乳糖与D-及L-岩藻糖。     

Serological diagnosis and immunological aspects of Proteus infection. V. Design and trial of polyvalent antigenic preparations

The main principles of the development of Proteus polyvalent antigen and erythrocyte diagnosticum, including the selection of the initial strains according to the clinical importance of their H-antigens and to the variety of their partial factors, the combination of monoantigens to form a polyvalent antigenic preparation with due regard to the potency of each component and the use of the preparation in serological tests in accordance with its summary potency, as well as the simultaneous loading of formaldehyde-fixed and tannin- or bisdiazo benzidine-treated sheep red blood cells with sensitins, have been worked out. The diagnostic value of these preparations has been confirmed by the study of serum samples from 579 patients with intestinal and urological infections (among them 153 patients releasing Proteus) and 245 healthy persons.     

The hemagglutinating activity of bacteria in the genus Klebsiella and the morphofunctional characteristics of their fimbriae]

The hemagglutinating activity of 77 Klebsiella strains from the international collection, grown in a culture medium prepared on the basis of soy-bean flour enzymatic hydrolysate, was studied. These strains could be divided into four groups according to their capacity for synthesizing different types of hemagglutinins on their surface: 2 strains carried mannose-sensitive hemagglutinins, 18 strains had mannose-resistant K-type hemagglutinins, 48 strains exhibited the signs indicating the presence of both mannose-sensitive and mannose-resistant hemagglutinins, and 9 strains showed no hemagglutinating activity. The hemagglutinating activity of strains K-74, K-79, K-80, K-81 and K-82 was characterized. Of the reference strains under study, 22 strains were found to have mannose-resistant hemagglutinating activity with respect to fresh chick red blood cells. The occurrence of hemagglutinins in Klebsiella was shown to depend on the temperature of cultivation and the consistency of the culture medium. The formation of large-sized capsules in Klebsiella grown in the Werfel-Fergusson medium with a considerable content of saccharose was shown to cause the absorption of their fimbrial structures by the capsular substance and, as a consequence, the suppression of their hemagglutinating activity.     

Sugar binding properties of the vitellogenic proteins in the Colorado beetle,demonstrated by hemagglutination and precipitation experiments

Summary The sugar binding properties of 2 important vitellogenic proteins in Colorado beetle hemolymph were demonstrated by hemagglutination and precipitation experiments. The agglutination of human red blood cells by the hemolymph of reproducing females was observed up to a hemolymph dilution of 1/256, irrespective of the blood-group. It increased significantly after trypsinization of the crythrocytes. Vitellogenin 1 was identified as the hemagglutinin. Hemagglutination and hemagglutination inhibition tests showed that this protein has a low affinity for hexosamines and a higher affinity for sulfated polysaccharides. Precipitation tests demonstrated that besides vitellogenin, another major yolk protein, chromoprotein 2, reacts with sulfated polysaccharides. The possibility that there is a specific reaction of the vitellogenic proteins with well defined saccharides on the oocyte surface is discussed. This lectintype reaction may explain the selectivity of yolk precursor endocytosis.     

鲤鱼不同品种（系）红血球抗原特异性的初步研究

用血球凝集和血清吸收试验的方法,对若干个鲤鱼品种及鲤鱼人工雌核发育系的红血球中的某些同种异型抗原纯合性进行了初步研究。①以抗雌核发育红鲤抗血清(abs／R)为试剂,三个雌核发育系(R8305,R8413,RMGF1)的RBC反应值分别为1521—2360,1521-2028,853—1521,普通红鲤、镜鲤、白鲢的反应值在80—782。②abs／8305,与abs／8413经靶细胞相互吸收后的残留活性显著降低(均为320),而以白鲢RBC吸收后抗血清活性不变。作者认为这部分抗原在鲤鱼种以下的品种和品系中具有特异性,可考虑将其作为鱼类品系鉴定的一种手段。     

Detection by Hemagglutination of Antibodies to Group A and Group E Streptococci by the Use of O-Stearoyl Derivatives of Their Cell Wall Carbohydrate-grouping Antigens

The streptococcal group A and group E cell wall polysaccharide antigens were extracted with trichloroacetic acid from the cell or cell wall and esterified with stearic acid. The stearoyl derivatives contained 5 to 8% (by weight) of the ester. Sheep or human red blood cells were sensitized with the esterified antigens and were shown to agglutinate in the presence of specific rabbit antisera. Sera from (i) children hospitalized with group A streptococcal respiratory disease and (ii) swine possessing group E streptococcal lymphadenitis were shown to possess antibody titers significantly higher than the controls. The use of the two esterified antigens as controls for each other established the specificity of the reaction in each case. The general shape of the antigen-antibody precipitin curves was not changed when the stearoyl antigens were used; however, the quantitative aspects differed markedly. Oligosaccharides which inhibit the normal antigen-antibody precipitin reaction did not inhibit the hemagglutination reaction. The adsorption of antisera with whole streptococcal cells reduced the hemagglutination titer in relation to the quantity of cells employed. Data are given on the (i) optimal concentration of stearoyl antigen for sensitization, (ii) time of adsorption of antigen to red cells, (iii) use of albumin as diluting fluid, and (iv) condition of red cells. Properties of the esterified antigens and the mechanism of the agglutination reaction are discussed. The results indicate that polysaccharide antigens of other bacteria may be esterified and employed in a similar manner.     

Indirect hemagglutination test of Rickettsia orientalis: its specificity for reference strains, Karp, Gilliam and Kato.

Amberlite XE-64 and bovine serum albumin-treated rickettsia suspension, which was prepared from the chicken yolk sacs infected with each of 3 reference strains of Rickettsia orientalis, Karp, Gilliam and Kato, was sonicated at 10 KC for 15 min at 4 degrees C and centrifuged at 10,000 rpm for 40 min at 0 degrees C. The supernatant was used to sensitize the formalinized and tanned sheep red blood cells (SFTSRC). This antigen (2.5%) could be preserved for at least 1 wk at 4-8 degrees C and at least a month, if merthiolate (1:10,000) was added. Each SFTSRCP of 3 reference strains was found to be specific in indirect hemagglutination (IHA) reaction with each homologous immune serum and there was little cross reaction with the heterologous one. This minor cross reaction might be due to the presence of a small amount of soluble antigen in SFTSRC. No cross IHA reaction with the typhus fever immune serum was observed. The IHA test was considered to be useful for the diagnosis of scrub typhus.     

Studies of the antigenic properties of fowl erythrocytes

Summary While in all the hemagglutination reactions with viruses the properties showed by complete fowl erythrocytes are exclusively situated in the cell walls, the authors have studied the antigenic properties of both elements of fowl red cells obtained after hemolysis. Cell walls provoke in the blood serum of rabbits formation of hemolysin and agglutinin just as entire normal red cells do. Probably hemolysate contains a protein fraction identic with one in the blood serum. All the antigenic properties of fresh cell walls are preserved after lyophilization.     

Adhesion pili in Yersinia pestis

Y. pestis cells cultivated at 37 degrees C are capable of agglutinating red blood cells of some animals, which is due to the appearance of pili. The adhesion pili consist of protein subunits with a molecular weight of the order of 12000 daltons, their isoionic point being at pH 4.7. The reaction of hemagglutination was inhibited by the mixture of ganglyosides, while the preliminary treatment of red blood cells with neuraminidases increased its effectiveness. The pili are supposed to take part in the expression of virulence.     

A third example of haemolytic auto-anti-Vel

A non-transfused, 43 year old Caucasian female presented with acute haemolytic anaemia and splenomegaly. Sections of bone marrow showed erythroid hyperplasia. The patient's red blood cells gave a negative reaction with polyspecific antiglobulin serum, but a positive reaction with specific anti-IgM. A heat eluate prepared from her red cells showed anti-Vel specificity. Her serum agglutinated only Vel-positive cells including her own. All papain pre-treated red cells including her own and Vel-negative cells were completely haemolysed at 37 degrees C. The percentage of haemolysis of Vel-positive cells was greater than that of Vel-negative cells.