Heat-stable Antigen Research Articles

Immunological detection of moulds in food: relation between antigen production and growth

In a study of Notermans and Heuvelman ( Int. J. Food Microbiol. (1985) 2, 247–258) an enzyme linked immunosorbent assay (ELISA) for detection of moulds was described. The ELISA used was based on detection of a heat stable, water extractable and genus specific antigen(s) produced by moulds. The results indicated the possibility of testing food products, whether or not heated, for mould contamination. In this study antigen(s) production by moulds was tested under different growth conditions. It was observed that production of antigen(s) was correlated with mycelium weight. Type of medium, including different fruit juices, did not affect the production of antigen(s). Also no differences between surface culturing and submerged culturing were observed. Both incubation temperature and water activity showed no significant effect on production of antigen(s). Within the genus Penicillium 43 out of 45 species tested produced identical antigen(s) in comparable quantities. The quantity of antigen(s), expressed as minimal detectable quantity of mould mycelium produced, varied from 6–108 ng/ml with an average of 32 ng/ml.

Isolation and identification of an exoantigen specific for Coccidioides immitis

Results of previous studies have established that mycelial-phase cells of Coccidioides immitis produce a heat-stable (HS) exoantigen that is specific for this fungus. In the present study, the HS exoantigen was isolated from a heterogeneous culture filtrate of C. immitis mycelia by using a combination of physicochemical procedures. Affinity chromatography of the culture filtrate on concanavalin A yielded two fractions: an effluent fraction that did not bind to the lectin and an eluate fraction that eluted in alpha-2-methylmannoside. Antigenic analyses by two-dimensional immunoelectrophoresis established that of the seven antigens detected in the unfractionated culture filtrate preparation, only two were present in the column effluent, and of these, only one was stable to heating at 56 degrees C for 30 min. Reactivity in the immunodiffusion assay for the HS exoantigen was demonstrable with the column effluent fraction and not the column eluate. The detection of only one precipitinogen in two-dimensional immunoelectrophoresis of the heat-treated concanavalin A effluent fraction, coupled with the reactivity of this fraction in the immunodiffusion assay for the HS antigen, provides strong, if not definitive, evidence that this antigen is the HS exoantigen. Purification of the HS antigen and the production of monospecific antiserum will provide the necessary reagents for the development of a sensitive and specific immunoassay for detecting the HS antigen in C. immitis cultures.

Use of a two-sited monoclonal antibody assay to detect a heat-stable malarial antigen in the sera of mice infected with Plasmodium yoelii.

Antigens, circulating in the blood during malarial infections, have been implicated in immune protection, immunosuppression, and immune-complex formation. We used a monoclonal antibody (MAb 7H8) to identify an antigen (Ag-7H8) in the sera of mice infected with Plasmodium yoelii. The major form of the antigen has a molecular weight of approximately 120,000 in P. yoelii, with minor components of 220,000; 65,000 to 75,000; and 45,000. Ag-7H8 remains antigenic after boiling for 5 min. A two-sited assay was developed with MAb 7H8 that demonstrated that the Ag-7H8 has at least two similar epitopes per molecule. The two-sited assay was used to follow Ag-7H8 in the blood of mice during lethal (strain 17XL) and nonlethal (strain 17XNL) P. yoelii infections. Ag-7H8 appeared on days 6 and 7 after infection with 10(6) and 10(4) 17XL P. yoelii parasites, respectively, and remained until the animals died. It was in plasma samples between days 6 and 14 after 17XNL P. yoelii injections in several inbred strains of mice, regardless of the course of parasitemia. Thus, the kinetics of antigenemia correspond with early stages of infection and not with the number of circulating parasites. Indirect immunofluorescence assays demonstrated that MAb 7H8 detects a cross-reactive antigen in other malarial parasites, including Plasmodium berghei and Plasmodium falciparum. Thus, this two-sited assay may have general application for the serodiagnosis of malaria and may be beneficial in determining the relationship of circulating antigens to malarial immunity.

Identification of Naegleria fowleri in fresh isolates of environmental amoebae using a staphylococcal coagglutination technique

A coagglutination test using Staphylococcus aureus Cowan strain 1 sensitized with specific antiserum successfully identified Naegleria fowleri in axenic and monoxenic culture by detection of a heat stable antigen released into the culture medium during growth. Strong coagglutination, visible to the naked eye, occurred within 30 sec when the sensitized reagent was mixed on a slide with medium from N. fowleri cultures. N. lovaniensis gave a weak cross-reaction which developed over one min clearly distinguishable from that of N. fowleri. N. gruberi and N. australiensis gave no reaction. The technique is proposed as part of a rapid and economical scheme for detecting N. fowleri in environmental samples very soon after primary isolation, with distinct advantages over conventional methods. Further development of the technique to detect other Naegleria spp. and the early diagnosis of primary amoebic meningoencephalitis are suggested as future applications.

Phenotypic characterization of ‘non‐T, non‐B’ acute lymphoblastic leukemia by a new panel (BL) of monoclonal antibodies

Peripheral blood and/or bone marrow leukemic cell suspensions from 49 patients with 'non-T, non-B' acute lymphoblastic leukemia (ALL) were analysed by flow cytometry using a new panel of four monoclonal antibodies. Anti-BL1 and anti-BL2 originating from NALM-6 and B35M lymphoblastoid cell lines, respectively. These antibodies recognize B-cell differentiation antigens: a heat stable non-immunoprecipitable antigenic determinant, and a 68 000 daltons glycoprotein molecule, respectively. BL5 and BL6 were derived by immunization with the promyelocytic cell line HL-60, recognizing antigens present on early hematopoietic cells: an 85 000 daltons MW glycoprotein (Pro-Im 1) and a heat stable antigen (Pro-Im2), respectively. All ALL patients studied had L1 or L2 morphology by the FAB classification and a blast count exceeding 50 per cent. There were 25 males and 24 females. Median age was 8 years (range 1-67 years). Thirty-nine cases were studied at initial presentation and 10 at relapse. Cells from 46/49 cases expressed BL2 and/or BL1, but were not reactive with BL5 or BL6. Three of 49 cases did not express BL1 or BL2. However, a small percentage of blasts from one case was positive for BL5 (13 per cent) and the other 2 cases were reactive with BL6 (20 per cent and 36 per cent, respectively). These were one adult and 2 pediatric patients that had other ALL markers and achieved a complete remission with appropriate ALL therapy. One of the BL6+ cases relapsed after 19 months with a change in phenotype to BL1+ BL2+ BL5- BL6-. This analysis shows that the majority of 'non-T, non-B' ALL's do express B-cell associated antigens (BL1/BL2) argumentative of their B-cell origin. A small subgroup does not express such antigens and may arise from a more immature cell, since they expressed antigens on early hematopoietic stem cells.

Identification of cell surface antigens present on murine hematopoietic stem cells.

Nine antigens found on murine bone marrow cells were examined to define their pattern of expression in murine hematopoietic differentiation. Lymphocyte function antigen (LFA-1), heat stable antigen (recognized by M1/69), common leukocyte antigen (CLA, T200, Ly-5) and Lgp100a (recognized by 30-C7) were present on early hematopoietic progenitors, BFU-E, CFU-E, CFU-GM, and CFU-M. All antigens found on progenitors were found on some immature precursor cells, myeloblasts, erythroblasts, or monoblasts, but their pattern of expression on identifiable hematopoietic cells varied. Three of these antigens, LFA-1, heat stable antigen recognized by M1/69, and CLA, were expressed on leukocytes of all stages of maturity but were lost from the erythroid lineage during differentiation. MAC-1, Forssman antigen, heat stable antigen (recognized by M1/75), anti-P-95 (recognized by M5/113), and Ia (recognized by M5/114) were found only on differentiated hematopoietic precursors or mature cells. The expression of these antigens was more lineage-specific. MAC-1 and heat stable antigen (recognized by M1/75) were restricted to either mature myeloid or erythroid cells, respectively. The marked differences in distribution of these antigens suggest that they may be useful in negative or positive selection experiments to enrich progenitors, and that some of them may have a functional role in differentiation.

Specificity analysis of mouse monoclonal antibodies defining cell surface antigens of human renal cancer.

Six mouse monoclonal antibodies (mAbs) defining separate systems of cell surface antigens of cultured human renal cancer were tested for reactivity with normal fetal and adult tissues and with neoplastic tissues. Five of the mAbs identified glycoproteins of Mr 160,000 (designated S4), Mr Mr 140,000 (F23), Mr 120,000 (S23 and S27), and Mr 115,000 (S22). The glycoprotein component of Mr 120,000 has been shown recently to be the adenosine deaminase binding protein (ADA-BP) and mAbS23 and mAbS27 define two distinct epitopes on ADA-BP. S22 was not detected on any normal fetal or adult tissues but was found on a subset of renal cancers. S4, F23, S23, and S27 defined distinct domains of the nephron: glomerulus (S4), proximal tubules (S4, F23, S23, and S27), and portions of Henle's loop (S23 and S27). mAbS4 also reacted with the interstitial matrix in the renal medulla and of other tissues, and mAbF23 reacted with fetal and adult fibroblasts. The S23 epitope of ADA-BP was expressed by placental trophoblasts and epithelial cells of breast, prostate, lung, and colon, whereas the S27 epitope was detected on a more limited range of cell types (trophoblasts and prostate epithelium). A panel of 20 renal cell carcinomas was typed for expression of these antigens; 7 phenotypes could be distinguished, with the S4+/F23+/S23+/S27+/S22+ or - phenotype (15 cases) being most common. The other antigenic system, V1, identified a heat-stable antigen that was widely expressed on cultured cell types but showed a restricted pattern of reactivity in tissues. V1 expression was limited to the adrenal cortex, Leydig cells, and the theca of ovarian follicles, and to adrenal cortical carcinomas.

Adaption of Elisa for the Detection oCampylobacter Antibodies and its Application in Seroepidemiological Studies in Sheep and Cattle Herds

ELISA was adapted for the study of antigenic relations among important Campylobacters and for the presence of anti-campylo-bacter antibodies in 394 sheep and 265 cattle. Rabbit anti-C. jejuni, C. coli, G. fetus subsp. fetus and C. laridis heat-stable antigen sera were evaluated against 29 Campylobacter strains and 6 other bacteria. Anti-C. jejuni and G. coli reacted strongly with homologous antigens and weakly with C. fetus subsp. fetus, C. laridis and C. fecalis antigens. C. fetus subsp. fetus serum reacted mainly with its homologous antigen. C. laridis serum showed closer reactivity to C. jejuni than to C. fetus subsp. fetus, C. coli and C. fecalis. Insignificant cross-reactions were observed with Y. enterocolitica, S. dublin and E. aerogenes heat-stable antigens, Ewes vaccinated with C. fetus subsp. fetus bacterin showed higher ELISA titers against C. fetus subsp. fetus antigens than non-vaccinated ewes or rams. Twenty-five percent of the vaccinated animals showed titers as low as 95 % of the non-vaccinated animals. In cattle the lowest antibody titers against C. fetus subsp. fetus, C. jejuni, C. coli and C. laridis antigens were exhibited by the precolostrum sera followed by the postcolostrum and adult sera. These studies demonstrated the applicability of the ELISA test in seroepidemiological investigations concerning the distribution and significance of Campylobacter antibodies in food animal sera.

Effect of heating on the sensitivity and specificity of an enzyme immunoassay (EIA) forErwinia carotovorasubsp.atrosepticaserogroup I

A double sandwich enzyme immunoassay (EIA) standardized for detection of Erwinia carotovora subsp. atroseptica serogroup I was serogroup specific only at the lower limit of sensitivity (105 to 106 cells/mL). At concentrations > 107 cells/mL strains representing subsp. atroseptica serogroups XVIII, XX, and XXII and subsp. carotovora serogroups II, III, IV, and V also gave positive but less strong reactions. Strains from 16 other E. carotovora serogroups and strains of Corynebacterium sepedonicum and Pseudomonas marginalis were all similar to the buffer controls. Heating suspensions of serogroup I strains prior to assay for at least 3 min at temperatures > 60°C enhanced A405 values. However the lower limit for detection was not reliably reduced below 105 cells/mL for all serogroup I strains. A405 values for strains known to share a common flagellar antigen (serogroups III and V) were reduced but not eliminated by heating. Strains having a related cell wall antigen (serogroups II and XVIII) were either unaffected by heating or had increased A405 values similar to the homologous strains. Heat-increased A405 values for serogroup XX and XXII strains suggest that they also have a heat-stable cell wall antigen in common with serogroup 1 strains.

Preparation and characterization of monoclonal antibodies recognizing three distinct differentiation antigens (BL1, BL2, BL3) on human B lymphocytes.

We report in this paper the generation and characterization of three monoclonal antibodies, designated alpha BL1, alpha BL2, and alpha BL3, that recognize distinctive antigens unrelated to complement, Fc, and mouse erythrocyte rosette receptors, which are preferentially expressed on B lymphocytes. alpha BL1 recognizes a heat stable nonimmunoprecipitable antigen, possibly glycolipid in nature. Alpha BL2 recognizes a nonreducible single polypeptide with a m.w. of 68,000 that occasionally co-precipitates with a p29,34 complex of HLA-DR antigens. Alpha BL3 recognizes a nonreducible single polypeptide with a m.w. of 105,000 with an acidic pI point. We demonstrated that BL1 is expressed on fetal liver hematopoietic cells, a small subset (5 to 15%) of Ficoll-Hypaque-separated normal bone marrow cells, and on a subpopulation of nonadherent, non-E rosette-forming cells and granulocytes. BL2 is expressed on fetal liver hematopoietic cells, on 3 to 7% of normal bone marrow cells, and on a majority (40 to 70%) of nonadherent, non-E rosette-forming cells with a distinctive pattern similar to that of HLA-DR. BL3 is expressed on a subpopulation of nonadherent, non-E rosette-forming cells, and on occasional cells in the monocyte-enriched adherent cell population. The peak fluorescence for BL2 is substantially higher than that of BL1 and BL3, indicating higher BL2 antigen density. All three antigens are absent from thymocytes and E rosette-positive T cell fractions obtained from various lymphoid tissues. Cellular distribution of the BL antigens on various well-characterized established hematopoietic cell lines, leukemias, and malignant lymphomas, in conjunction with the results of the in vitro activation and TPA-induction experiments, suggest that BL1 is expressed during early developmental stages of B cell differentiation, whereas BL3 is expressed at the later stages. BL2 expression spans immature and mature stages of B cell differentiation, with the exception of mature plasma cells. The alpha BL antibodies described here should prove to be useful in the investigation of B cell differentiation and in the clinical diagnosis of lymphoid neoplasms.

Analysis of Leptospira antigens by crossed immunoelectrophoresis.

Serovars icterohaemorrhagiae, canicola, and arboreae of pathogenic Leptospira interrogans and serovar patoc of saprophytic L. biflexa were examined by crossed immunoelectrophoresis. A close antigenic relationship was found between the interrogans serovars, particularly between icterohaemorrhagiae and canicola. To a much lesser extent cross-reactions were found between interrogans serovars and patoc. Comparison of three different antigenic preparations of icterohaemorrhagiae by tandem crossed immunoelectrophoresis and by absorption experiments with the patoc reference system showed the presence of several common broadly reactive antigens. Antigen 1 of the reference system, a genus-specific heat-stable protein antigen, was found in all antigenic preparations. It is a diagnostically important antigen.

Identification and preliminary characterization of a major heat-stable surface antigen of Actinomyces israelii by two-dimensional (crossed) immunoelectrophoresis

We have identified, employing two-dimensional (crossed) immunoelectrophoresis, a major heat-stable surface antigen in Actinomyces israelii with an electrophoretic mobility of 0.86 compared to bovine albumin. The surface component was protease sensitive and non-dialyzable and was found in relatively large amounts in two serotype 2 strains of A. israelii (WVU 307 and ATCC 23860). Serotype 1 strains either lacked antigen 0.86 (ATCC 12103 and CDC X522) or contained small quantities of a cross-reacting antigen which was not identical to antigen 0.86 (CDC W855). Thus, antigen 0.86 may be useful in the serotaxonomy of A. israelii. Our data also suggest the possibility that a greater degree of antigenic heterogeneity exists within the species than can be accommodated within the two currently recognized serotypes.

Relative Insensitivity of a Kenyan Strain of Leishmania donovani to Pentavalent Antimony Therapy in Hamsters

racidium and the eggshell retained stainability with immunofluorescence (Fig. 2). When nonheated sections were used for titration of sera by the IIF technique, the titer differed at different sites on the sections (Table II). The intraoval space showed stainability at higher dilutions than miracidia or host tissues. The positive titers against the antigen in this space ranged from 1: 80 to 1: 2,560. These titers were much higher than those measured by the COP test. When autoclaved sections were used, the titers of these sera against the antigen in the intraoval space were reduced at least fourfold. Furthermore, there seemed to be a correlation between the COP titer and the IIF titer detected on the autoclaved sections. Using histochemical techniques, Smith and von Lichtenberg(1967, Am. J. Pathol. 50:933-1007) have demonstrated a variety of substances associated with the contents of S. mansoni eggs, and they reported the existence of periodic acidSchiff (PAS) positive materials in the space beracidium and the eggshell retained stainability tween the miracidium and the eggshell. Booter et al. (1979, J. Immunol. 122: 39-43) isolated a polysaccharide antigen from S. mansoni eggs. Similarly, Long et al. (1981, Infect. Immun. 34: 389-396) obtained glycoprotein fractions from crude egg antigens of S. japonicum that contained COP inhibitory activity. We have demonstrated that there was amylase-resistant PAS positive material in the intraoval space of S. japonicum where the stainability by IIF technique was retained after autoclaving. We also demonstrated that the stainability with PAS was not altered by autoclaving (Fig. 3). These results show that the heat-stable antigen taking part in the COP reaction is most probably a polysaccharide and is located in the area between the miracidium and the eggshell. Infections of rabbits with S. japonicum and collections of the sera were done at the Schistosomiasis Control and Research Project (SCRP), Palo, Leyte, Philippines, for which we thank their staff.

Localization of Antigen within Eggs of Schistosoma japonicum That Participate in Circumoval Precipitin (COP) Reaction

The circumoval precipitin (COP) reaction was first reported by Oliver-Gonzales (1954, J. Infect. Dis. 95: 86-91) who observed precipitates around living schistosome eggs when they had been placed in homologous sera from infected humans or experimental animals. Since then, this reaction has been applied as an immunodiagnostic test for human schistosomiasis japonica (Yokogawa et al., 1967, Japan. J. Parasitol. 16: 77-84; Yogore et al., 1968, Am. J. Trop. Med. Hyg. 17: 65-71). Because the COP test requires only minimal equipment and has high specificity, this test is useful both for laboratory diagnosis and field survey in endemic areas (Tanaka, 1976, Southeast Asian J. Trop. Med. Publ. Hlth. 7: 176-179; Yogore et al., 1978, Southeast Asian J. Trop. Med. Publ. Hlth. 9: 344-355). Immunochemically, the COP was shown to be an antigen-antibody complex by demonstrating immunoglobulin in the precipitate (Sala et al., 1962, Proc. Soc. Exp. Biol. Med. 117: 921-923; Hillyer and Marrero, 1980, Am. J. Trop. Med. Hyg. 29: 1249-1253; Demaree and Hillyer, 1981, Am. J. Trop. Med. Hyg. 30: 402-405). Hillyer and Pelly (1980, Am. J. Trop. Med. Hyg. 29: 582-585) and Cruise et al. (1981, AJEBAK 59: 503-514) reported that some specific murine hybridoma-derived antibodies produced the COP reaction with S. mansoni eggs or S. japonicum eggs. However, the chemical and physical nature of the antigen(s) involved in the precipitate formation has not been clearly characterized. It is known the eggs retain reactivity in the COP test even after lyophilization (Sala et al., 1962, Am. J. Trop. Med. Hyg. 11: 199) or heating at relatively high temperature (Kamiya, 1980, Japan. J. Vet. Res. 28: 149-154). Interestingly, von Lichtenberg (1964, Am. J. Pathol. 95:75-93) has demonstrated that the autoclaved eggs possessed the antigenicity and induced egg-granulomas in the lung of injected mice. This may suggest a possible role of heat-stable antigen(s) within the eggs. Kamiya and Kamiya (1980, Japan. J. Vet. Res. 28: 155-1-60) indicated the location of immunofluorescent-stainable antigen in the eggs in paraffin sections of liver tissue. However they did not examine tissue heated at a temperature higher than 58 C. In the present study, we examined the heat-stability of the egg antigen in the COP reaction and attempted to define its localization within the eggs by the indirect immunofluorescent technique. The COP tests were carried out essentially as described by Oliver-Gonzales (1954, loc. cit.). Eggs of Schistosomajaponicum (Philippine strain) were isolated from the intestines of the dd strain of mice infected for 8 to 9 wk and were lyophilized according to the method of Matsuda et al. (1977, Japan. J. Exp. Med. 47: 369-375). Eggs of the same lot were used through the experiment and some of the eggs were autoclaved at 121 C for 15 min. The criteria of positivity and classification of precipitates in COP test proposed by Yokogawa et al. (1967, loc. cit.) were adopted. The titration of serum was done by determining the highest dilution at which positive reaction was still detectable. Cryostat sections of infected mouse liver containing S. japonicum eggs were examined by indirect immunofluorescence (IIF). Livers from the mice infected for 8 to 9 wk with S. japonicum (Philippine strain) were cut into small pieces (5 mm) and were embedded in 1.5% agarose (Sigma, St. Louis, Missouri, low gelling temperature type) in phosphate-buffered saline (PBS). They were then rapidly frozen in n-hexane with an acetone-dry-ice mixture at -70 C and were sectioned on a cryostat microtome at 5 um and placed on microscope slides. The specimens were fixed in 95% ethanol for 5 min and some of the fixed sections were autoclaved at 121 C for 15 min. The liver sections were layered with serial dilutions of test sera from rabbits for 30 min at 37 C in a moist chamber. After three separate,

Crossed immunoelectrophoretic analysis of Legionella pneumophila serogroup 1 antigens

By crossed immunoelectrophoresis, 85 different antigens were demonstrated in sonicated preparations of Legionella pneumophila serogroup 1 (Lp1). The precipitin patterns of 82 anodic-migrating antigens were numbered and were designated the Lp1 reference system. Eleven antigens were stable to boiling, and seven of these were shown to be surface antigens. One heat-stable surface antigen (antigen no. 61) was highly reactive with limulus amoebocyte lysates and formed a precipitin resembling lipopolysaccharide. Serum from an isolation confirmed case of Lp1 infection and serogroup-specific rabbit antiserum reacted specifically with antigen no. 61, which was designated the serogroup-specific antigen. Normal human and rabbit sera commonly had antibodies to antigen no. 66 of the Lp1 reference system. This antigen is antigenically related to the "common antigen" of Pseudomonas aeruginosa.

Separation of hemopoietic cells from adult mouse marrow by use of monoclonal antibodies

Primitive hemopoietic progenitor cells from adult mouse marrow have been substantially enriched by virtue of a negative selection procedure with monoclonal antibodies. It has been possible to segregate erythroid progenitor cells at distinct stages of differentiation on the basis of their cell surface antigens. This has been achieved with two monoclonal antibodies reactive with the mature elements of bone marrow. YBM 34.3 binds to a heat-stable antigen expressed on B lymphocytes, neutrophils, and cells of the erythroid lineage. YBM 6.1 reacts with cells of the neutrophil, eosinophil, and monocyte series but does not bind to colony- forming cells. Separation is achieved by indirect immunoadsorption (panning) with YBM 34.3 on Protein-A-coated plastic plates followed by FACS II cell sorting with YBM 6.1. The combined procedures yield a marrow population containing 58% immature cells (blasts, promyelocytes, and myelocytes) and 9.5% clonogenic cells. In addition, differential binding of YBM 34.3 can be used to segregate erythroid progenitor cells at distinct stages of differentiation (day 7 BFU-E, day 5 BFU-E and CFU- E) either by cell sorting or panning. It is shown that both techniques give a comparable degree of resolution of the different cell types with, however, an appreciable advantage of panning over cell sorting in allowing the rapid handling of large numbers of cells.

Separation of Hemopoietic Cells From Adult Mouse Marrow by Use of Monoclonal Antibodies

Primitive hemopoietic progenitor cells from adult mouse marrow have been substantially enriched by virtue of a negative selection procedure with monoclonal antibodies. It has been possible to segregate erythroid progenitor cells at distinct stages of differentiation on the basis of their cell surface antigens. This has been achieved with two monoclonal antibodies reactive with the mature elements of bone marrow. YBM 34.3 binds to a heat-stable antigen expressed on B lymphocytes, neutrophils, and cells of the erythroid lineage. YBM 6.1 reacts with cells of the neutrophil, eosinophil, and monocyte series but does not bind to colony-forming cells. Separation is achieved by indirect immunoadsorption (panning) with YBM 34.3 on Protein-A-coated plastic plates followed by FACS II cell sorting with YBM 6.1. The combined procedures yield a marrow population containing 58% immature cells (blasts, promyelocytes, and myelocytes) and 9.5% clonogenic cells. In addition, differential binding of YBM 34.3 can be used to segregate erythroid progenitor cells at distinct stages of differentiation (day 7 BFU-E, day 5 BFU-E and CFU-E) either by cell sorting or panning. It is shown that both techniques give a comparable degree of resolution of the different cell types with, however, an appreciable advantage of panning over cell sorting in allowing the rapid handling of large numbers of cells.

Role of heat labile antigens of Shigella flexneri in HeLa cell invasion.

In studies of the role of surface antigens of Shigella flexneri in HeLa cell invasion, three antisera were employed to block the invasion. Antisera against live (ALS) and boiled (ABS) S. flexneri blocked invasion very effectively. Reduction in the numbers of intracellular shigellae was always accompanied by reduction in the number adherent to the cells, indicating the importance of adhesion in the invasive process. Anti-live absorbed antiserum (ALAS) prepared by exhaustive absorption of ALS with boiled S. flexneri blocked adhesion and invasion at dilutions of 20 or 50; the efficiency of the absorption was indicated by absence of agglutinating and anti-lipopolysaccharide (LPS) antibodies. S. flexneri LPS did not block adhesion and invasion even at a concentration of 1.0 mg/ml. Hence it was concluded that heat-labile surface antigens are important in adhesion and invasion of HeLa cells by S. flexneri. Antiserum against heat stable antigen (ABS) probably blocks adhesion by steric hindrance.

アユのビブリオ病に対するＶｉｂｒｉｏ ａｎｇｕｉｌｌａｒｕｍの抽出リポ多糖経口ワクチンの効果

Previous papers on oral bacterin against vibriosis in cultured ayu plecoglossus altivelis showed that fish were effectively immunized with heated bacterin and that the agglutinin secreted in the body surface mucus of the immunized fish prevented pathogenic bacterium Vibrio anguillarum form adhering to the skin of ayu. These findings suggested that a heat stable antigen on the surface of the bacterium would play an important role in the protection against vibriosis. In this study it was investigated whether that lipopolysaccharide, a heat stable antigen in the cell wall, of V. anguillarium would be an effective immunogen or not. The lipopolysaccharide fraction extracted by WESTPHAL's method from the formalin killed bacterin was used as the extracted vaccine. Efficacy of the extracted vaccine was estimated by comparing mortality in the fish immunized with the extracted vaccine to that in the fish immunized with the formalin killed bacterin after challenge. Challenge test was executed by exposing fish to the bacteria discharged from the fish injected with the virulent strain. Administration of the extracted vaccine at a level of 25mg/fish kg/day for 9 days resulted in sufficient protection similar to that of the formalin killed bacterin at a level of 0.1g wet cells/fish kg/day for 9 days. Chemical analysis showed that the extracted vaccine contained a large percentage of lipopolysaccharide and nucleic acid but only a small percentage of protein and liped. From these results it is presumed that lipopolysaccharide is the protective antigen of the bacterin.

Topics on Bordetella pertussis vaccine

1. As reported previously, histopathological changes of adrenal glands in Wistar Rats (male, 100-200g) were observed with the injection of purified histamine-sensitizing-factor (HSF): Zona glomerulosa, the outer layer of zona fasciculata, and their lipid content were constant. Marked cytolysis in zona glomerulosa. Extraordinary depletion of lipids in the inner layer of zona fasciculata. Zig-zag formetion from the beneath the capsule reaching the deeper region of the inner layer of zona fasciculata (Fig. 1 and 2).It is well-known that diphtherial exotoxin induces marked hemorrhages of adrenal gland. In refference to this, histamine-sensitizing-factor (an exotoxin) of Bordetella pertussis might cause any change in the adrenal gland. Under this hypothesis, this experiment was carried out. Consequently, the changes mentioned above (Fig. 1 and 2) were recognized. These changes imply disturbance of secretion of hormones (Cortisol, Aldosteron, Adrenal Androgens). I drew inferences from these facts that these were reported as the results of “stress”. I got a lot of letters from everywhere in the world. Unexpectely, among them, the comment of Prof. M.S. Zakharova at Moscow, USSR, was valuable. She critisized that my findings are not only results of stress, but also they are functional changes of the whole host body. It is of the very importance for establishment of prevention against whooping cough infection. This suggestive comment gave me a hint.No doubt, the changes (Fig. 1 and 2) express only static stage, followed by rapid repairement, accompanying hyperfunction and production of antitoxin. So that, the forced definition “preventive antigen” is completely useless.Recently, Arai & Munoz could crystallize HA (Fimbrial Hemagglutinin), whick lacks completely preventivity. Also, Arai & Munoz could succeed in crystallization of “pertussigen” (Package of HSF, LPF & thermo-labile lethal antigen). The future of pertussigen deserves the keen interest.2. Is IC (INTRACEREBRAL CHALLENGE) of mice the one and only as a test?IC method is spread all over the world like the Bible. Oblivious experiments by Standfast whose work I have learned from Dr. Margeret Pittman of NIH, Md, NSA. Standfast's experiments (1) Usual pertussis vaccine gave good effect on IC mouse protection as well as field trial. (2) Contrary to this, pertussis vaccine heated at 100C for one hour do not any effect on IC mouse protection at all. However, this heated vaccine gave good effect on IN (INTRANASAL CHALLENGE) mouse protection as well as field trial (human being). This undeniable facts are of the very importance, because packkage-pertussigen (a group of exotoxins-thermolabile) was vanished away by heating. Hence, one can obtain the most safe pertussis vaccine without any side effect. It reminded of my heat-stable neurotoxin.I do not know whether it is an exotoxin or not. In spite of unknown character, it shows charactristic whooping cough syndromes. My working hypothesis suggests that “heat-stable antigen” by Standfast might coincident with my “heats-table neurotoxin”.