Bordetella pertussis agglutinogens in cultivation dynamics

Bordetella pertussis growth phases during homogenous batch dynamic cultivation in the liquid medium as well as during the static cultivation on the solid medium were established. The maximal activity of agglutination reaction with antisera to B. pertussis agglutinogens 1, 2, and 3 was detected in bacterial culture at the end of exponential phase of growth. The activity of agglutination reaction decreased when cultures in stationary and death phases were used. During transition from exponential to death phase level of antibodies to agglutinogen 2 decreased by4 - 32 times. 2 - 4-fold decrease of antibodies level was observed when antiserum to agglutinogen 3 was used. Activity of agglutination reaction with antiserum to agglutinogen 1 was high and did not depend from phase of growth. When polyvalent antiserum to B. pertussis was used 4-fold decrease of antibody titers was observed in parallel with change of growth phases. Sera from rabbits immunized with B. pertussis cultures from the middle of exponential growth phase, the end of this phase, and begin of the death phase had high (maximal) level of agglutinating antibodies (6400), which was detected on 101 day after immunization with the former culture and on 31 day after immunization with either of the two latter cultures. To the end of experiment (292 day) titers decreased to 800, 3200, and 1600 respectively. These findings confirm an advisability of use of exponential growth culture for immunization of rabbits in order to obtain highly active diagnostic antisera to B. pertussis.     

Development of a microslide agglutination assay with the aid of an inexpensive projection microscope

A microslide agglutination assay was developed involving the mixing of 2.5 microl each of antiserum and a cell suspension of Listeria monocytogenes. Cell agglutination in the final volume of 5.0 microl was visually observed at a direct magnification of 22 x on the projection screen of an inexpensive 20 US dollar projection microscope. The procedure has the advantage of increasing by a factor of 20 the number of agglutination assays that can be performed with a given volume of antiserum with the use of an inexpensive optical projection system.     

Reactions of antibodies against ferredoxin, ferredoxin-NADP+ reductase and plastocyanin with spinach chloroplasts

Purified antisera against ferredoxin, ferredoxin-NADP+ reductase and plastocyanin agglutinated osmotically shocked and washed spinach chloroplasts, prepared according to standard procedures. The monomeric antibody (immunoglobulin G fraction) of the reductase antiserum agglutinated chloroplasts specifically and directly, indicating that protruding structures (for example, the coupling factor) do not act as steric hindrances as has been suggested. With ferredoxin antiserum, the presence of a pentameric antibody (immunoglobulin M fraction) was obligatory to observe a positive agglutination reaction. Immunoglobulin G only inhibited ferredoxin-dependent reactions, like NADP+-photoreduction, but did not cause agglutination. Ferredoxin seems to be located in depressions of the membrane, possibly caused by a partial release of this protein in shocked chloroplasts. Similar results were obtained with purified immunoglobulins from a plastocyanin antiserum. Again the immunoglobulin G fraction inhibited electron transport reactions catalyzed by plastocyanin, whereas immunoglobulin M showed a positive agglutination, but had no influence on electron transport. It is concluded that ferredoxin, ferredoxin-NADP+ reductase and plastocyanin are peripheral electron transport components, located at the outer thylakoid membrane.     

Trypanosoma brucei: in vitro propagation of metacyclic forms derived from the salivary glands of Glossina morsitans

1 Metacyclic forms of Trypanosoma brucei obtained from the salivary glands of the tsetse fly, Glossina morsitans have been cultured for the first time in their infective forms for more than 200 days in continuous culture. The parasites were grown at 25 C and 30 C on a bovine embryonic spleen (BESP) feeder layer in buffered RPMI 1640 medium supplemented with 20% heat-inactivated bovine fetal serum (BFS) and 5% lactalbumin hydrolysate. Initial growth rate was enhanced when normal, noninfected, salivary glands were added to the cultures. The parasites thus cultured appeared like slender or intermediate blood stream forms which were infective to rats and mice. Addition of rat anti-T. brucei specific antiserum to the cultures caused agglutination of the parasites and rendered them noninfective. This study opens up new areas of investigating sleeping sickness. The cultured metacyclic parasites have the potential of being applied as antigens for controlling African trypanosomiasis.     

Immunological Methods for the Detection of a Coryneform Isolated from Ratoon Stunted Sugarcane

A coryneform bacterium, isolated from ratoon stunted sugarcane, has been obtained in pure culture. Its identification by immunological means was investigated. Optimum conditions for the light microscopic observation of bacterial agglutination by specific antiserum, immunofluorescence and the microcapillary haemagglutination of IgG-sensitized erythrocytes by the bacterium were established. All three assay procedures are simple and rapid. By comparison, more samples can be handled at any one time bythe microcapillary haemagglutination method. Based on bacterial agglutination, the antiserum is specific for the coryneform isolated from ratoon-stunted sugarcane.     

Characterization of a cytotoxic pilin subunit of Xenorhabdus nematophila

Xenorhabdus nematophila is an insect pathogenic bacterium, known to produce protein toxins that kill the larval host. We have described a cytotoxic pilin subunit of X. nematophila, which is expressed on the cell surface and also secreted in the extracellular medium associated with outer membrane vesicles. A 17kDa pilin subunit was isolated and purified from X. nematophila cell surface. The protein showed cytotoxicity to larval hemocytes of Helicoverpa armigera in an in vitro assay, causing agglutination of the cells, and releasing cytoplasmic enzyme lactate dehydrogenase in the medium. The pilin protein was able to bind to the surface of larval hemocytes. The binding and cytotoxicity of the purified 17kDa protein to hemocytes was inhibited by antiserum raised against the pilin protein. The study demonstrates for the first time a cytotoxic structural subunit of pilin from an entomopathogenic bacterium X. nematophila that is excreted in the extracellular medium with outer membrane vesicles.     

Serological method for identification of Vibrio parahaemolyticus from marine samples

Use of agglutination with antiserum against lateral flagella (H-agglutination) for the identification of Vibrio parahaemolyticus was studied. Sucrose-negative bacteria were isolated from seawater, and their characterization was carried out by traditional biological tests and slide agglutination with antiserum specific to lateral flagella of V. parahaemolyticus. Of 135 strains isolated, 78 were identified as V. parahaemolyticus by biological tests and were agglutinated with the above serum. Fifty-five strains did not agglutinate with the serum, and their biological characteristics were different from those of V. parahaemolyticus. Two strains also differed from V. parahaemolyticus in some biological characteristics but agglutinated with the antiserum. All clinically isolated V. parahaemolyticus strains also agglutinated with the above serum. These results suggest that our serological method is useful for the identification of V. parahaemolyticus, especially for samples in which there are many organisms related to V. parahaemolyticus, because many biological tests can be omitted.     

Specific activity of colored variants of N. meningitidis colonies

The character of fluorescence of the colonies formed by serogroup A meningococci (229 strains) in oblique light and their activity in the agglutination test with group specific S and RD antisera to the meningococcal dissociant of the same serogroup were studied. Orange and orange-green colonies were found to have pronounced group-specific activity, and faint gray colonies changed the character of their agglutination with the group-specific antiserum up to the loss of agglutinability; and simultaneously their capacity for agglutination with the antiserum to the dissociant was revealed. The study of the character of fluorescence and the group-specific activity of meningococcal colonies belonging to other serogroups provided similar results.     

A comparative study of 14 strains of Naegleria australiensis demonstrates the existence of a highly virulent subspecies: N. australiensis italica n. spp

Fourteen strains of Naegleria australiensis, including the type strain, were compared for virulence for mice, maximum growth temperature, lectin agglutination, isoenzyme pattern, and total protein banding pattern. Their relation to other species of Naegleria also was compared by immunoelectrophoretic analysis. Strains with high virulence, comparable to that of N. fowleri, were found to be different in concanavalin A agglutination as well as with regard to zymograms and total protein patterns. Although serologically different from N. fowleri and reacting with N. australiensis antiserum in the fluorescent antibody test, these high-virulence strains differed in number of immunoelectrophoretic precipitin bands. Because of these results, the high-virulence strains are considered to be a subspecies of N. australiensis. The low-virulence strains showed minor differences from the type strain. Thus, N. australiensis does not appear to be as homogenous a species as N. fowleri. Pathogenic N. australiensis also seems to be more widespread than previously thought.     

Serological method for identification of Vibrio parahaemolyticus from marine samples.

Use of agglutination with antiserum against lateral flagella (H-agglutination) for the identification of Vibrio parahaemolyticus was studied. Sucrose-negative bacteria were isolated from seawater, and their characterization was carried out by traditional biological tests and slide agglutination with antiserum specific to lateral flagella of V. parahaemolyticus. Of 135 strains isolated, 78 were identified as V. parahaemolyticus by biological tests and were agglutinated with the above serum. Fifty-five strains did not agglutinate with the serum, and their biological characteristics were different from those of V. parahaemolyticus. Two strains also differed from V. parahaemolyticus in some biological characteristics but agglutinated with the antiserum. All clinically isolated V. parahaemolyticus strains also agglutinated with the above serum. These results suggest that our serological method is useful for the identification of V. parahaemolyticus, especially for samples in which there are many organisms related to V. parahaemolyticus, because many biological tests can be omitted.     

The effects of nutrient limitation and growth rate in the chemostat on the immunogenicity of Vibrio cholerae

The possibility was examined of using the chemostat to produce cholera vaccine of immunogenicity similar or superior to that of conventional vaccine produced from organisms grown on nutrient agar. The immunogenicity in mice of vaccines prepared from organisms grown under carbon, nitrogen, magnesium and phosphate limitation each at low and high growth rates was inferior to that of conventional vaccine. It was concluded that under the conditions used in this study the chemostat could not be used to produce cholera vaccine of acceptable immunogenicity. The sensitivity of organisms grown under the same conditions in the chemostat to agglutination by specific agglutinating antiserum showed phenotypic variation and under carbon limitation was growth rate related, although the increased sensitivity to agglutination of carbon-limited organisms at high growth rates was not paralleled by increased ability of these organisms to induce agglutinating antibodies.     

The agglutination test of coracidia of pseudophyllidean cestodes and its use in cestodological research

Ability of coracidia to agglutination in homologous antiserum was established. The method of reaction of a direct agglutination of coracidia (RDAC) in the culture of alive larvae was described. When comparing the RDAC results in homologous and heterologous variants a dependence of the agglutination pattern on the degree of antiserum homology was established. On the example of RDAC of the cestodes Diphyllobothrium dendriticum and D. ditremum in polyvalent and species-specific antisera to D. dendriticum possibility was shown of RDAC usage in the studies on specific taxonomy of pseudophyllid cestodes.     

Use of microsphere-antibody conjugates in microparticle-enhanced nephelometric immunoassay.

A new microparticle-enhanced nephelometric immunoassay has been recently described as a sensitive, accurate, and easy-to-perform competitive immunoassay for various analytes. As initially described, this test is based on the nephelometric quantification of the inhibition, by the antigen to be assayed, of immunoagglutination of microparticle-antigen conjugates. Its applicability as a competitive immunoassay is thus limited by the necessary availability of pure antigens to prepare microparticle-antigen conjugates. In this paper, we report an adaptation of this initial test, where microparticles are coated by monoclonal antibodies, eliminating the need for purified antigens. The new configurations of particle agglutination-based immunoassays described include use of these microparticle-antibody conjugates with microparticle-antigen conjugates, free antigen, and anti-mouse immunoglobulins antiserum. The feasibility of such configurations is studied with human chorionic gonadotropin hormone, human thyroid stimulating hormone, and human myoglobin as antigens. Capture of the analyte by microparticle-antibody conjugates is evidenced by inhibition of their agglutination with microparticle-antigen conjugates and by agglutination in a sandwich assay with a complementary monoclonal antibody or polyclonal antiserum. The use of a second xenogenic antibody enhances the agglutination process and increases the assay sensitivity. Microparticle-antibody conjugates may extend the applications of microparticle-enhanced nephelometric immunoassays to unavailable analytes.     

Secretion of a TGF-beta-like growth inhibitor by normal rat mammary epithelial cells in vitro

We have examined conditioned medium (CM) from cultures of normal rat mammary epithelial (RME) cells for growth factor activity on fresh RME cell cultures. RME cell-derived CM contained potent growth inhibitory activity toward fresh RME cell cultures when the medium was acidified by dialysis against 1% acetic acid prior to concentration. Dialysis of the CM at neutral pH resulted in CM that had growth stimulatory activity and no inhibitory activity. The acid-activated growth inhibitor was heat and acid stable, protease sensitive, and eluted from a Bio-Gel p60 column with a peak of activity in the 28 kDa range. Incubation of the acidified-concentrated CM with neutralizing antiserum (affinity purified IgG) against transforming growth factor (TGF)-beta completely abolished the inhibitory activity of the CM. Furthermore, RME cell growth in the presence of the growth inhibitor plus TGF-beta antiserum was greater than that observed in growth medium alone. Subsequent experiments demonstrated that addition of TGF-beta antiserum alone to serum-free medium enhanced RME cell growth, whereas addition of nonimmune IgG was without effect even at 25-fold higher concentrations. Zymographic analysis of RME-CM revealed the presence of plasminogen activator proteases that may mediate the partial activation of the latent growth factor. These results indicate that normal RME cells secrete a latent TGF-beta-like growth factor into conditioned medium. Furthermore, the results indicate that some of the latent growth factor is activated in situ and contributes to the growth potential of the cells in primary culture in an autocrine manner.     

A bacterial-induced lectin which triggers hemocyte coagulation in Manduca sexta

An inducible hemagglutinin termed M13, was purified from M. sexta hemolymph. M13 is a glucose-specific lectin which in addition to erythrocyte agglutination, can activate dedifferentiation of various hemocytes into a filamentous coagulation network. When lectin activity was inhibited with glucose or antiserum, neither erythrocyte agglutination or hemocyte coagulation occurred. When M13 was boiled or trypsin treated, hemocyte activation was lost, but erythrocyte agglutination remained. Hence M13 activity appears to be bimodal, possessing both a lectin activity and a hemocyte-coagulating activity.     

Detection of Hepatitis Associated Antigen by the Latex Agglutination Test

Hepatitis associated antigen may be detected quickly and reliably by the latex agglutination test, using antiserum from guinea pigs immunized with the antigen. The latex test has a sensitivity comparable to the counter current immunoelectrophoresis technique.     

A microagglutination test for blood typing rhesus monkeys, Macaca mulatta

We have developed a microagglutination test for typing rhesus monkey erythrocytes that is sensitive, accurate and easy to perform. The technique requires only microliter quantities of antiserum and cells, and agglutination is easily detected using an inverted microscope. An advantage of this technique is that the typing plates can be stored at -70 degrees C without loss of activity. The results of typing over 400 rhesus blood samples with this technique were 95% concordant with results using the standard microtitre agglutination technique. Preliminary results indicate that this test is also adaptable to typing human blood.     

A microagglutination test for blood typing rhesus monkeys, Macaca mulatta

Summary. We have developed a microagglutination test for typing rhesus monkey erythrocytes that is sensitive, accurate and easy to perform. The technique requires only μ1 quantities of antiserum and cells, and agglutination is easily detected using an inverted microscope. An advantage of this technique is that the typing plates can be stored at -70°C without loss of activity. The results of typing over 400 rhesus blood samples with this technique were 95% concordant with results using the standard microtitre agglutination technique. Preliminary results indicate that this test is also adaptable to typing human blood.     

Modifications of the Growth Inhibition Test and its Application to Human T-Mycoplasmas

Several factors were found to influence the growth inhibition test. Inoculum size and amount of antiserum are well known variables, but the method of applying the antiserum, the incubation temperature, and the pH of the agar medium also play significant roles. The growth inhibition test modified according to these findings was found to be specific and well suited for the classification and identification of human T-mycoplasmas.     

Isolation of N-Acety-β-Hexosaminidase from Acanthamoeba castellanii

ABSTRACT. The lysosomal enzyme N-acetyl-β-hexosaminidase (βhex) has been purified from Acanthamoeba castellanii growth medium by a three step procedure. The enzyme was precipitated with ammonium sulfate, partially purified on a DE52 column and purified to homogeneity on an affinity column. The purified βhex appeared to be a monomer with a molecular mass of 58 kDa and a pI of approximately 5.8. The enzyme activity in growth medium at RT was stable for several months. The purified βhex was enzymatically deglycosylated and injected, into two rabbits to make polyclonal antibodies. One antiserum was specific for βhex, but the other stained many bands on immunoblots of whole cell preparations. Using fluorescently labelled secondary antibodies we have determined that both antisera stain digestive vacuoles in the Acanthamoeba cytoplasm, and do not stain the contractile vacuole. The multi-specific antiserum had high avidity for βhex, but also stained the carbohydrate portion of other molecules. These other molecules may be lysosomal enzymes as well, since the activity of several other lysosomal enzymes was partially immunoprecipitable with the antiserum. We plan to use these antibodies to study traffic patterns among the variety of vacuolar structures in Acanthamoeba cytoplasm.