Isolation and characterisation of toxin A-negative, toxin B-positive Clostridium difficile in Dublin, Ireland

Clostridium difficile is a major cause of infectious diarrhoea in hospitalised patients. Most pathogenic C. difficile strains produce two toxins, A and B; however, clinically relevant toxin A-negative, toxin B-positive (A- B+) strains of C. difficile that cause diarrhoea and colitis in humans have been isolated worldwide. The aims of this study were to isolate and characterise A- B+ strains from two university hospitals in Dublin, Ireland. Samples positive for C. difficile were identified daily by review of ELISA results and were cultured on selective media. Following culture, toxin-specific immunoassays, IMR-90 cytotoxicity assays and PCR were used to analyse consecutive C. difficile isolates from 93 patients. Using a toxin A-specific ELISA, 52 samples produced detectable toxin. All isolates were positive using a toxin A/B ELISA. Similarly, all isolates were positive with the cytoxicity assay, although variant cytopathic effects were observed in 41 cases. PCR amplification of the toxin A and toxin B genes revealed that 41 of the previous A- B+ strains had a c. 1.7-kb deletion in the 3'-end of the tcdA gene. Restriction enzyme analysis of these amplicons revealed the loss of polymorphic restriction sites. These 41 A- B+ isolates were designated toxinotype VIII by comparison with C. difficile strain 1470. PCR ribotyping revealed that all A- B+ isolates belonged to PCR-ribotype 017. A- B+ C. difficile isolates accounted for 44% of the isolates examined in this study, and appeared to be isolated more frequently in Dublin, Ireland, than reported rates for other countries.     

An enzyme-linked immunosorbent assay (ELISA) for the major crustacean allergen, tropomyosin, in food

Shellfish are a common cause of food reactions in hypersensitive individuals and are among the eight foods that account for over 90% of food allergies. At present, the only way to prevent these serious consequences of food allergies is to avoid the foods that trigger the reactions. A sandwich-ELISA kit has been developed for the detection of crustacean meat in food, based on the major heat-stable shellfish allergen, tropomyosin. Tropomyosin was purified from whole prawn (Penaeus latisulcatus) and used to immunize rabbits after confirming its identity by MALDI-TOF MS. A sandwich-ELISA based on the rabbit antibodies takes less than 2 h to perform, including the food extraction, and has a detection limit of 1 ppm crustacean (prawn, lobster), without detectable cross-reactivity with fish or mammalian meat.     

Penicillin-specific antibodies: Monoclonals versus polyclonals in ELISA and in an optical biosensor

Two penicillin-specific monoclonal antibodies mAb 19C9 and mAb 9H3 and the penicillin-specific polyclonal antibodies pAb K2 were evaluated for their use in a competitive ELISA and in the BIAcore™ optical biosensor. In the ELISA, an ampicillin-protein conjugate was used as a coating molecule. For the biosensor assay, ampicillin was immobilized on a CM5 chip. With both monoclonal antibodies and in both test systems, ampicillin, amoxicillin and benzylpenicillin were better recognized than oxacillin, cloxacillin and dicloxacillin. Because the reproducibility was better in the biosensor (CV = 1.6%) than in the ELISA (CV = 8.9%), the limit of detection for ampicillin in buffer solution using mAb 19C9 was lower in the biosensor (46 ng ml^-1) as compared to the ELISA (356 ng ml^-1). Ampicillin can thus be detected below the MRL (50 ng ml^-1) in the biosensor assay but not in the ELISA. Both the ELISA and biosensor assay using the polyclonal antibodies pAb K2 were more sensitive as compared to the assays with the monoclonals. The ELISA using pAb K2 allowed the detection of all tested penicillins below the MRL. In the biosensor assay, ampicillin was also detected below the MRL (IC50 = 10 ng ml^-1). In contrast to the binding of the monoclonals, no spontaneous dissociation was observed after injection of the polyclonal antibodies in the biosensor. Whereas the monoclonals were completely removed from the sensor surface using ampicillin in the buffer solution as regeneration solution, stronger conditions were necessary for the pAb binding.     

Analysis of sensitization to carboxymethylcellulose: Identification of high risk group using ELISA and histamine release experiment

Objective and Design: Carboxymethylcellulose (CMC) has been considered to be inert and is commonly used as an additive in medicines, foods and cosmetics. However, we experienced a patient who developed an anaphylactic reaction to CMC after an upper gastrointestinal examination using a barium meal containing CMC. Therefore, we examined the incidence of sensitization by CMC in healthy subjects, and categorized the high risk group prone to developing anaphylactic response to CMC.Methods: An ELISA for detecting CMC-specific IgE antibody was developed using serum from the patient as a positive control. In the ten subjects exhibiting high anti-CMC IgE among 387 normal populations, histamine release from isolated leukocytes was performed.Results: Five of ten subjects with a high IgE titer showed a significant CMC-induced histamine release from leukocyte preparations in vitro as observed in the patient, and were classified as high risk group. There was a correlation between sensitization by CMC and that by Japanese cedar pollen. The incidence of sensitization in females was 2.4 fold higher than that in males.Conclusions: The combination of ELISA and histamine release experiment made it possible to identify the high risk group for developing anaphylactic response. The administration of high dose CMC as a suspending agent in barium sulfate or injectable corticosteroids to this group should be avoided to prevent anaphylactic reactions in the clinic.Received 18 August 2003; returned for revision 29 September 2003; accepted by M. J. Parnham 10 December 2003     

An enzyme-linked immunosorbent assay for thyroxine in dried blood spotted on filter paper

We have developed a rapid and sensitive enzyme-linked immunosorbent assay (ELISA) for thyroxine (T4) in dried blood samples spotted on filter paper. The assay is carried out on microtiter plates without extraction or centrifugation steps. The detection limit of the assay is 5 pg/disc/well, equivalent to 1.25 micrograms/1 of whole blood or 2.5 micrograms/1 of serum. Intra- and inter-assay coefficients of variation for various T4 concentrations are 2.4-9.0% and 5.9-17.5% respectively. Correlation between the proposed ELISA method and the RIA is good (r = 0.900, n = 62, y(RIA) = 0.99x(ELISA) + 9.90). The ELISA method is useful for mass-screening of neonatal congenital hypothyroidism using dried blood samples on filter paper, is very simple and one person can assay more than 300 samples per day.     

Detection by ELISA of IgA and IgM antibodies in secretion and IgM antibodies in serum in primary lower respiratory syncytial virus infection

Twenty-six infants and children with primary lower RS virus infection, diagnosed by the detection of RS virus in nasopharyngeal secretion (NPS) by use of immunofluorescent antibody (FA) technique, were studied with respect to the presence of IgA and IgM antibodies. Samples of NPS and serum obtained during the first 3-4 months following the beginning of illness, were investigated. Employing a reverse ELISA technique, we found IgM antibodies in the acute, but not during the convalescent, phase of illness in NPS from 20 of the patients and in serum from 21 of the patients. The majority of the IgM antibody conversions observed occurred in NPS as well as in serum on days 5-8 following the illness. RS virus IgA antibodies, also detected by a reverse ELISA technique, were demonstrated in NPS in 22 of the patients, with antibody conversions being found in 19 of the patients on days 5-8 following the beginning of the illness. Two patients still had IgA antibodies in NPS approximately 3 months FSOI. By comparison, RS virus was detected in acute-phase NPS by double-antibody sandwich ELISA in 25 of the 26 patients investigated.     

GRO-alpha in Human Serum: Differences Related to Age and Sex

PROBLEM: Human GRO-alpha (GRO-α) is a new member of the chemokine family that is supposed to play an important role in inflammatory and immune reactions. We established a sandwich enzyme-linked immunoassay (ELISA) system with polyclonal antibodies against human GRO-α and investigated the serum level of healthy donors to establish normal ranges for this chemokine in adults. METHODS: GRO-α concentrations were measured cross-sectionally in the sera of 240 healthy adults. The variability of serum GRO-α levels was also measured in normal volunteers, samples from whom were obtained by sequential venipunctures or by a small plastic cannula with a heparin-saline lock, to determine short-term variability. RESULTS: Whereas there was no difference between the concentration of human GRO-α from men (logarithmic mean, 77.6 pg/ml, n = 120) and that from women with normal menstrual cycles (log mean, 71.6 pg/ml, n = 73), the concentration from postmenopausal women (log mean 45.0 pg/ml, n = 31) was lower than that from women with normal menstrual cycles (log mean 71.6 pg/ml, n = 73). However, we could not detect any significant difference between healthy donors' serum levels and those of donors with acute inflammation. Fewer variations were recognized in the case of the sequential venipunctures method than in that of the heparin-saline lock method. CONCLUSION: We found that the GRO-α concentration of postmenopausal women was significantly lower than that of women with normal menstrual cycles. These results suggest the GRO-α serum levels of normal healthy women may have some correlation with sex hormones.     

Humoral Immunity to Dietary Antigens in Atopic Dermatitis

Enzyme-linked immunosorbent assays (ELISA) employing a biotin-avidin amplification step are described for the quantification of human serum IgG antibodies to the dietary antigens ovalbumin (OA) and beta-lactoglobulin (BLG). The analytical quality of these assays was acceptable. Antibodies were measured in 16 patients with mild or moderate atopic dermatitis (AD), in 31 patients with a history of AD, and in closely matched controls. Levels of serum anti-OA antibodies did not differ in patients and controls, whereas anti-BLG antibodies tended to be higher in patients with mild or moderate AD than in controls (P less than 0.05).     

Factors influencing the sensitivity of herpes simplex virus detection in clinical specimens in a simultaneous enzyme-linked immunosorbent assay using monoclonal antibodies

A rapid simultaneous enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies was investigated for herpes simplex virus (HSV) detection. All HSV isolated (n = 127) were detected, whereas no response was obtained with HSV negative preparations. Equivalent results were obtained from 275 of 277 clinical specimens in the monoclonal ELISA and in an ELISA using polyclonal antibodies, confirming that appropriately selected monoclonal antibodies may be as efficacious as polyclonal antibodies in antibody-based assays. In clinical specimens, the rate of HSV detection (sensitivity) relative to tissue culture isolation was low for both assays, and the major factor responsible for this was the low concentration of virus present in some specimens. The sensitivity of ELISA obtained in routine use varied with different panels of unselected specimens and was related to the speed of development of the cytopathic effect. These results emphasise the need for caution in assigning a definitive sensitivity level to ELISA tests evaluated on different panels of specimens.     

建立一种用重组蛋白检测抗口蹄疫病毒抗体的间接ELISA方法

目的研究以重组蛋白作为包被抗原检测口蹄疫病毒（FMDV）感染后的动物血清中特异性抗体的可能性,为建立一种非病毒颗粒的ELISA检测试剂盒提供实验依据。方法利用自行构建表达的O型口蹄疫病毒VP1表位肽重组蛋白（VP1epi）作为包被抗原,采用间接ELISA方法确定抗原的最佳工作浓度和包被方法,优化各项实验条件,并以FMDV感染后的豚鼠血清作为标准阳性血清确定ELISA方法的特异性和灵敏度。结果FMDV感染后的阳性豚鼠血清可以很好地识别VP1epi重组蛋白,用此蛋白包被检测抗FMDV抗体的灵敏度可达1∶3200,并证明所检测的抗体是FMDV特异性的。结论VP1epi重组蛋白可以替代FMDV颗粒用于建立检测抗FMDV抗体的ELISA试剂盒。