A recombinant antigen with potential for serodiagnosis of Echinococcus granulosus infection in dogs

Antibodies specific for Echinococcus granulosus were affinity purified from dog serum on transfer blots containing putative serodiagnostic antigens. These antibodies and serum pools derived from dogs with E. granulosus infections were used to screen a lambda gt11 cDNA library constructed using E. granulosus protoscolex mRNA. Nine definitive antigenic clones were isolated and characterized, of which one (c10P1) gave strong specific reactions in plaque immunoassay with sera from E. granulosus infected dogs. These clones were all subcloned into the plasmid vector pGEX-1. Antigenicity of clones was confirmed in colony immunoassay and/or immunoblot. Glutathione S-transferase (GST) fusion proteins of individual subclones were produced in Escherichia coli, purified by affinity chromatography and evaluated in ELISA using sera from dogs with infections of E. granulosus, Taenia spp. or nematodes, and helminth-free dog sera. The GST fusion protein 10P1 showed a specificity of 100% in ELISA for diagnosis of E. granulosus infection in dogs despite its relatively low sensitivity. Further investigations aim to identify additional recombinant antigens and test 10P1 expressed in alternative expression systems to increase diagnostic sensitivity of the ELISA.     

Prévalence de l'infection cervicale à Chlamydia trachomatis dans une population féminine consultant pour contraception.

OBJECTIVES: To determine the prevalence and risk indicators of cervical infection due to Chlamydia trachomatis among female patients consulting for contraception and to evaluate an enzyme immunoassay for the detection of C. trachomatis in this setting. DESIGN: Prevalence study. Endocervical specimens were analysed by means of culture and enzyme immunoassay. C. trachomatis infection was diagnosed through culture. SETTING: A hospital family planning clinic in Trois-Rivières, Que. SUBJECTS: All 533 female patients who consulted for contraception between November 1986 and March 1988. Results of culture were available for 495 patients. MAIN OUTCOME MEASURE: Demographic, epidemiologic and clinical information was collected by means of a standard questionnaire and a gynecologic examination. MAIN RESULTS: The prevalence rate of chlamydial infection was 9% (45/495). Enzyme immunoassay detected 37 (82%) of the infections. The mean age of the patients was 19.8 years, and 98% of the infections were diagnosed in those aged 25 years or less. The variables significantly associated with C. trachomatis infection were having more than one sexual partner in the preceding year (odds ratio [OR] 2.9; 95% confidence limits [CL] 1.7 and 5.0) and having more than one partner in the preceding 3 months (OR 2.3; 95% CL 1.2 and 4.3). These two indicators would have detected 58% and 22% of the infections respectively. CONCLUSIONS: Screening for C. trachomatis infection by means of enzyme immunoassay should be proposed to all female patients aged 25 years or less consulting for contraception in our clinic. Such screening may prove to be an effective preventive measure in other similar clinical settings.     

Biological activity of polar lipids from bifidobacteria

Fractions of polar lipids have been isolated from bifidobacteria, and the immunoreactivity and serological specificity of glycolipids and phospholipids have been studied. Enzyme immunoassay (dot-EIA) of polar lipids demonstrates that the fractions of glycolipids and phospholipids of bifidobacteria are highly immunoreactive. Pronounced reactions of major glycolipids and phospholipids with a homologous polyvalent antiserum against B. adolescentis 94-BIM have been observed at antigen concentrations of approximately 100 ng. The antiserum contained high titers of specific antibodies against glycolipids and phospholipids of bifidobacteria, as demonstrated by heterogeneous solid-phase enzyme immunoassay (ELISA). Experimental data confirming the presence of subpopulations of specific antibodies (antiglycolipid and antiphospholipid) in the blood sera of immunized animals lead to the conclusion that the major glycolipids and phospholipids of bifidobacteria are specific markers appropriate for serological diagnostics.     

三种药物联合治疗假丝酵母菌性阴道炎的临床疗效评价

目的:研究克霉唑阴道片、硝夫太尔制霉素阴道软胶囊联合定君生栓治疗复发性假丝酵母菌性阴道炎的临床疗效,为临床治疗提供依据。方法:选取2014年9月到2015年6月我院就诊的复发性假丝酵母菌性阴道炎患者60例,按照随机数字表法将患者分为实验组和对照组,每组30例,实验组给予克霉唑阴道片、硝夫太尔制霉素阴道软胶囊联合定君生栓治疗,对照组给予克霉唑阴道片和定君生栓治疗,治疗前检测所有患者病原菌分布情况,治疗后随访3个月,分析两组临床疗效和不良反应。结果:细菌培养结果共分离出158株假丝酵母菌,其中白色假丝酵母菌最多,占66.46%;实验组总有效率为93.33%,显著高于对照组的76.67%,两组比较差异具有统计学意义(P0.05);两组不良反应发生率比较无统计学意义(P0.05)。结论:克霉唑阴道片、硝夫太尔制霉素阴道软胶囊联合定君生栓治疗复发性假丝酵母菌性阴道炎具有较好的临床疗效,且无明显不良反应。     

Biological Activity of Polar Lipids from Bifidobacteria

Fractions of polar lipids have been isolated from bifidobacteria, and the immunoreactivity and serological specificity of glycolipids and phospholipids have been studied. Enzyme immunoassay (dot-EIA) of polar lipids demonstrates that the fractions of glycolipids and phospholipids of bifidobacteria are highly immunoreactive. Pronounced reactions of major glycolipids and phospholipids with a homologous polyvalent antiserum against B. adolescentis 94-BIM have been observed at antigen concentrations of approximately 100 ng. The antiserum contained high titers of specific antibodies against glycolipids and phospholipids of bifidobacteria, as demonstrated by heterogeneous solid-phase enzyme immunoassay (ELISA). Experimental data confirming the presence of subpopulations of specific antibodies (antiglycolipid and antiphospholipid) in the blood sera of immunized animals lead to the conclusion that the major glycolipids and phospholipids of bifidobacteria are specific markers appropriate for serological diagnostics.     

Development and application of an immunoassay diagnostic technique for studying Hematodinium infections in Nephrops norvegicus populations.

Patent Hematodinium infections of the Norway lobster Nephrops norvegicus can be detected with a morphological method (pleopod diagnosis), but this fails to identify low-level haemolymph (sub-patent) and any tissue-based (latent) infections. The current study describes the development and application of an immunoassay for the detection of antigens of the parasite Hematodinium in the Norway lobster N. norvegicus. Infected tissue and haemolymph samples were detected as multiple-band reactions to a polyclonal antibody (anti-Hematodinium). The sensitivity limit of the method was 204 parasites mm(-3), approximately 10 times more sensitive than the pleopod diagnosis method. Use of the immunoassay on tissue samples taken from catches in the Clyde Sea area, Scotland, UK, showed that the pleopod method considerably under-diagnosed infection prevalence in the early part of the season, though this under-diagnosis decreased as infected lobsters in the field progressed from latent and sub-patent to patent infections. However, the immunoassay failed to detect any infected lobsters during the summer months, suggesting that infection may not be carried over from one season to the next. The data presented suggest that this immunoassay allows for the accurate estimation of Hematodinium infection prevalence in the field and should be employed, where possible, for the routine monitoring of infection prevalence in N. norvegicus.     

免疫标记技术的进展及纳米技术在其中的应用

酶免疫标记、胶体金免疫标记及荧光免疫标记技术是目前应用最广泛的免疫反应检测技术。随着科学技术,尤其是纳米技术的发展和生物医药等方面的需求,人们在提高检测灵敏度、简化操作步骤、有效缩短检测时间等方面,取得了很多可喜的进展。本文将分别针对酶免疫标记、胶体金免疫标记及荧光免疫标记技术三方面的进展情况,尤其是纳米标记技术在免疫反应检测中的应用进展进行综述。     

Impaired Cellular Immunity in Kwashiorkor with Improvement after Therapy

Children with kwashiorkor showed a high incidence of deranged cellular immunity as evidenced by impairment of delayed cutaneous hypersensitivity reactions to candida and diphtheria toxoid antigens and of lymphocyte transformation after phytohaemagglutinin stimulation. This may contribute to their susceptibility to infection. A correlation was shown between the degree of impairment of tests of cellular immunity and the severity of the kwashiorkor. Once recovery was initiated the skin tests gave the expected positive results and the lymphocyte transformation index improved. Protein deprivation may result in impaired deoxyribonucleic acid (DNA) synthesis and in atrophy of both the thymus and the lymphoid tissue.     

Immune abnormalities associated with chronic mucocutaneous candidiasis

Twenty six patients with chronic mucocutaneous candidiasis (CMCC) have been studied. Four immunological patterns emerged.Five patients failed to produce migration inhibitory factor (MIF) in vitro although their lymphocytes were normally activated to DNA synthesis after challenge with candida antigen. Four of these patients were unable to mount delayed hypersensitivity (DH) reactions to candida antigen (CAg), purified protein derivative (PPD), or dinitrochlorobenzene (DNCB). The lack of DH in these patients is thought to reflect their inability to produce MIF.Another group of nine patients with absent DH to candida also failed to produce MIF after challenge with candida antigen. Their lymphocytes were, however, not activated in vitro by this antigen probably due to a factor present in their serum, which specifically inhibited candida induced transformation of lymphocytes from healthy individuals.Two patients were able to produce MIF in vitro but they were unable, nevertheless, to mount DH reactions. Furthermore, they did not show delayed inflammatory response to intradermal injections of a MIF preparation. It is postulated that these patients have defective macrophage function.In 10 patients no significant abnormalities in cellular or humoral immunity were revealed.It is concluded that chronic mucocutaneous candidiasis is a syndrome associated with several distinct immunological abnormalities. The pathogenesis of the syndrome is discussed and it is emphasized that chronic mucocutaneous candidiasis is a model which can be used for advancing our knowledge of the immune system.     

Detection of antibodies to preparations of Staphylococcus aureus peptidoglycan, teichoic acids and polysaccharide in patients' sera

Enzyme immunoassay systems on the basis of S. aureus teichoic acids, peptidoglycan and polysaccharide-containing preparation are incapable of reliably diagnosing the staphylococcal nature of postoperative complications in oncological patients.     

Itraconazole (Orungal) in the treatment of mycotic infections in oncologic patients]

Clinical and microbiological activity of itraconazole (Orungal, solution for oral use, "Janssen-Cilag") for treatment of oncological patients with inhospital infections associated with candida was evaluated. The trial included 50 patients with oncological pathology of respiratory tract, as representatives of risk group for candida contamination. Two groups of patients were formed: when candida contamination was proved patients of the test group were treated by antifungal agent itraconazole (Orungal, "Janssen-Cilag"). Patients in control group were treated by fluconazole (Diflazon, "KRKA") according to standard therapy regime. When no clinical efficacy was achieved patients in both groups were treated by amphotericin B (Fungizone, "Bristol-Myers Squibb"). Clinical efficacy was demonstrated at 13 patients and microbiological efficacy (pathogen elimination) at 12 patient of 14 of itraconazole group. Clinical efficacy was demonstrated at 9 patients and microbiological efficacy at 7 of 11 patients of fluconazole group. Treatment with amphotericin B was effective at all 4 patients treated with this antibiotic, microbiological effect was demonstrated for 3 patients of the group. Frequency of side effects was significantly less in itraconazole group. The course price was also significantly lower at itraconazole group. All this data are important for elaboration of optimal pharmaco economic policy regarding inhospital infections at critical care units.     

Comparison of quantity of thyroxine in thyroglobulin and the peptide hormones derived from chromatography and immunoenzyme assay

An evaluation of the thyroxine content in thyroglobulin and different derived thyroxine-containing peptides by enzyme immunoassay is presented. Results correlate very well with those obtained by ion-exchange chromatography which served as a reference method: r = 0.997, p less than 10(-6). Enzyme immunoassay is more rapid and sensitive than ion-exchange chromatography and then suitable for routine use. Furthermore, this paper shows, for the first time, that enzyme immunoassay can be used to perform direct thyroxine estimation in small peptide (Mr less than 2,500) without previous total enzymatic hydrolysis.     

Non-secretion of blood group antigens and susceptibility to infection by Candida species

Abstract One of the innate defences against superficial infections by Candida species appears to be the ability of an individual to secrete the water-soluble form of his ABO blood group antigens into body fluids. There was a significantly higher number of non-secretors (48.9%) among 174 patients with either oral or vaginal candida infections compared with the proportion of non-secretors in the local population (26.6%).
The protective effect afforded by the secretor gene might be due to the ability of glycocompounds in the body fluids of secretors to inhibit adhesins on the surface of the yeast. In attachment studies, preincubation of blastospores with boiled secretor saliva significantly reduced their ability to bind to epithelial cells. Non-secretor saliva did not reduce the binding and often enhanced the numbers of attached yeasts.
Possible host-parasite interactions underlying the susceptibility of non-secretors to candida and other infections are discussed.     

A new periparturient disease in Eastern Europe, Clostridium difficile causes postparturient sow losses

Postparturient sow losses caused by Clostridium difficile have not been reported in the veterinary literature. Recently in Croatia, in a large outdoor production unit with suboptimal environmental conditions, a sudden increase in postparturient sow mortality was diagnosed. After postpartal application of enrofloxacine to postparturient mastitis metritis agalactia (MMA) suffering sows, diarrhea, respiratory distress, and mortality of these sows were recorded. While 13% of MMA suffering and treated sows died, only 0.4% of the non-treated (no MMA suffering) sows died postpartum. Gross pathology revealed mesocolonic edema, hydrothorax, and ascites. Microscopic examination showed scattered foci of suppuration in the colonic lamina propria and accumulation of neutrophils and fibrin on colonic mucosa. Anaerobic cultures of the colon yielded heavy growth of C. difficile. Enzyme immunoassay revealed C. difficile toxins A and B. C. difficile infections of postparturient MMA suffering sows may be associated with environmental stress, the application of antibiotics, or both. C. difficile infections are an impending danger in Eastern Europe and does not only raise animal welfare issues, but seriously inflict the economical well being of outdoor production units.     

Non-secretion of blood group antigens and susceptibility to infection by Candida species

One of the innate defences against superficial infections by Candida species appears to be the ability of an individual to secrete the water-soluble form of his ABO blood group antigens into body fluids. There was a significantly higher number of non-secretors (48.9%) among 174 patients with either oral or vaginal candida infections compared with the proportion of non-secretors in the local population (26.6%). The protective effect afforded by the secretor gene might be due to the ability of glycocompounds in the body fluids of secretors to inhibit adhesins on the surface of the yeast. In attachment studies, preincubation of blastospores with boiled secretor saliva significantly reduced their ability to bind to epithelial cells. Non-secretor saliva did not reduce the binding and often enhanced the numbers of attached yeasts. Possible host-parasite interactions underlying the susceptibility of non-secretors to candida and other infections are discussed.     

The production of specific immunoenzyme conjugates to human immunoglobulins by the glutaraldehyde method]

The results of studies aimed at obtaining class-specific conjugates to human immunoglobulins to be used in the enzyme immunoassay (EIA) are presented. At the first stage of the studies purified IgA, IgM and IgG preparations were obtained. These preparations were used for obtaining immunologically active immunosorbents on the basis of bromocyanic Sepharose. Specific antibodies to human IgA, IgM and IgG were isolated from animal sera by the method of affinity chromatography. These antibodies were conjugated with peroxidase by the glutaraldehyde method. The specific activity of the conjugates were determined in EIA. The results thus obtained revealed that all preparations exhibited high specific activity and gave no cross reactions with immunoglobulins of other classes.     

Purification, immunoassay, and tissue distribution of rat C1-tetrahydrofolate synthase

C1-tetrahydrofolate synthase (C1-THF synthase), a eukaryotic trifunctional enzyme, catalyzes three sequential folate-mediated one-carbon interconversions. These three reactions supply the activated one-carbon units required in the metabolism of purines, thymidylate, and several amino acids. In order to study the regulation of C1-THF synthase expression in mammals, we have purified the enzyme to homogeneity from rat liver, raised polyclonal antisera to it in rabbits, and developed a sensitive solid-phase immunoassay for the enzyme. The enzyme was purified approximately 600-fold to a specific activity of 24.6 U/mg protein based on 10-formyl-THF synthetase activity. Western blot analysis indicated that the antisera is specific for one protein in crude liver extracts which comigrates with purified C1-THF synthase. Using the solid-phase immunoassay, as little as 200 pg of immunoreacting protein can be detected in tissue homogenates. Several rat tissues were examined for the three C1-THF synthase enzymatic activities and immunoreactive protein. The results indicated that the level of C1-THF synthase is regulated in a tissue-specific manner. Enzyme assays revealed that certain tissues differ by more than 100-fold in enzyme activity, with liver and kidney containing the highest levels, and lung and muscle the lowest. However, immunoassay of these same tissues indicated only a 10-fold difference in C1-THF synthase concentration. This apparent masking of enzyme activity was observed in all tissues, but to varying degrees. These results emphasize the advantages of an immunoassay in studying the regulation of C1-THF synthase.     

Simplified Extraction Procedure for Serological Grouping of Beta-Hemolytic Streptococci

An adaptation of the nitrous acid extraction of streptococci proved to be a reliable and practical method for the preparation of extracts for routine serological group identification. The extracts of all groups tested gave strong capillary precipitin reactions as well as reactions of double diffusion in gel. For routine grouping, extracts were prepared from the first one-half-plate subculture of the initial throat culture. The technique is simple and reliable, and it requires a minimum of technical skill, reagents, and equipment. Its use would facilitate epidemiological surveillance of group A streptococci and rapid diagnosis of streptococcal infections at a low cost.     

Characterization of two 3-ketothiolases possessing differing substrate specificities in the polyhydroxyalkanoate synthesizing organism Alcaligenes eutrophus

Abstract Two constitutive acetyl-CoA acetyltransferases (3-ketothiolases A and B) were purified from Alcaligenes eutrophus . Enzyme A was active with only acetoacetyl-CoA and 3-ketopentanoyl-CoA, whereas enzyme B was active with all the 3-ketoacyl-CoAs (C₄−C₁₀) tested. Enzyme A appeared to be a tetramer ( M _r 70 000) with identical subunits ( M _r 44 000) and enzyme B had a similar M _r of 168 000 (containing M _r 46 000 subunits). Enzymes A and B had isoelectric points of 5.0 and 6.4, respectively. The stoichiometry of the reactions catalysed by each enzyme was confirmed. K _m values of 44 μM and 394 μM for acetoacetyl-CoA, and 16 μM and 93 μM for CoA, were determined with enzymes A and B, respectively. Enzymes A and B gave K _m values of 1.1 mM and 230 μM, respectively, for acetyl-CoA. The condensation reaction was potently inhibited by CoA in both cases.     

Serum antibodies to the hepatitis A virus in persons following a history of the infection

Enzyme immunoassay was used for titration of serum antibodies in subjects with a history of clinically pronounced or asymptomatic hepatitis A infection. Titers of hepatitis A virus antibodies (anti-HAV) essentially decreased during 3-4 years after the disease, then the rate of this decrease slowed down and antibody titers stabilized at low levels. After clinically pronounced hepatitis A anti-HAV levels were considerably higher than after the asymptomatic form of this infection.