Development of a competitive format sorbent assay for the determination of parathion in water using molecular imprinted polymer as specific sorbent carrier

A rapid,simple,and reliable competitive molecular imprinted polymer sorbent assay（MIPSA） was developed and validated for measurement of parathion in water samples.This assay employed a molecular imprinted polymer（MIP） that was synthesized with non-covalent imprinting method as capture reagent and p-aminoparathion conjugate of horseradish peroxidase（para-HRP） as an enzyme label.The assay depended on a competitive binding reaction between the enzyme conjugate and analyte for the binding sites of the MIP.The optimized analysis conditions of 10 ng mL-1 para-HRP and 10 mg mL-1 polymer were found.The assay was acceptable to detect parathion in water samples under the optimized conditions,with a limit of detection of 50 ng mL-1.Mean analytical recoveries of added parathion in water samples ranged from 101.2%to 105%.The precision of the assay was satisfactory; relative standard deviation ranged from 4.3%to 6%.

仓储小麦中脱氧雪腐镰刀菌烯醇检测技术的优化

脱氧雪腐镰刀菌烯醇(Deoxynivalenol,DON)是小麦仓储过程中的重要危害因子,其污染水平是仓储公司定期检测的重要指标。为提高DON检测的平行性和准确度,文章对仓储小麦DON检测过程中样品制备和酶联免疫吸附法(ELISA)检测关键技术进行优化。结果表明:样品制备过程中,将扦样后的样品先粉碎再分样的处理方法(方法 2)明显优于先分样再粉碎的方法(方法 1),测定样品的相对标准偏差由25.28%降至7.42%。另外,将ELISA检测过程中两种不同的移液枪使用方法(前进移液法和反向移液法)进行比较,发现前进移液法在10次测定中结果相对标准偏差较小,平行性较好;且加样过程中,采用不贴壁加样方式会使检测值更加准确,平行性也较好。优化后的仓储小麦中DON毒素ELISA快速检测技术具有较好的平行性和准确度,可用于实际仓储小麦中DON毒素的准确、快速检测。

Analysis of immune responses against H pylori in rabbits

AIM： To investigate the immunogenicity of H pylori proteins, to evaluate the production rate of anti H pylori IgG antibodies in relation to time and to demonstrate the fidelity of newly optimized in-house enzymelinked immunosorbent assay （ELISA） technique as an alternative for H pylori infection assay. METHODS： In the present study, 100 μg of formalinfixed H pylori whole cell antigens was injected into an experimental animal （New Zealand white female rabbit） intramuscularly on d 0, 16, 27 and 35. The first two doses were injected with adjuvants. On d 0, a serum sample was collected from the rabbit before immunization and this pre-immunized serum was used as a negative control for the whole study. To evaluate the immunogenic responses of the injected antigen, serum samples were collected from the rabbit at regular intervals up to d 42. The sera were analyzed using inhouse ELISA and Western blot techniques.RESULTS： The production of anti Hpylor/IgG antibodies in the rabbit in response to the injected antigen increased almost exponentially up to d 14 and after that it was maintained at the same level until the last day （d 42）. By analyzing the immune profiles of immunized sera, 11 proteins were identified to be immunogenic, among them 2 （approximately 100 kDa and 85 kDa） were most prominent. CONCLUSION： Analysis of the immune responses against pathogenic microorganisms like H py/ori is necessary for the development of various diagnostic and preventive approaches. The results of this experiment reveal that the formalin-fixed H pylori whole cell antigens injected into the rabbit are highly immunogenic. These prominent proteins （approximately 100 kDa and 85 kDa） might have higher immunogenic effects among humans infected with H pylori and some of these immunogenic proteins can be included in diagnostic approaches based on serology and also for vaccine formulation. The in- house ELISA is a promising alternative compared to invasive techniques.

Field Evaluation of Alternative Testing Strategies for the Detection of HIV Infection in Beijing

To identify a cost-efficient altemative antibody testing strategy for screening and confirmation of HIV infection by rapid simple tests （RSTs） and enzyme-linked immunosorbent assays （ELISAs）. Methods Four RSTs （RST1, RST2, RST3, and RST4 ） and five ELISAs （ELISA1, ELISA2, ELISA3, ELISA4, and ELISA5） were evaluated in two phases by using banked and serum specimens prospectively collected at regional hospitals and voluntary counseling and testing （VCT） centers in Beijing. A total of 200 banked serum specimens were included in the first phase, including 62 HIV-positive, 127 HIV-negative and 11 indeterminate specimens. All specimens were tested by four RSTs and five ELISAs respectively. The second phase involved prospective testing of 389 routine specimens, including 92 HIV-positive, 287 HIV-negative, and 10 indeterminate specimens. All the specimens were tested by two RSTs （RST2 and RST4） and three ELISAs （ELISA1, ELISA3, and ELISA4）, which were selected for their respective excellent sensitivity and/or specificity. Westem blot （WB） was used as a gold standard for confirming the reactivity of all the specimens. Results Sensitivity, specificity, and efficacy were calculated for each assay in two phases. In the first phase, four assays （ELISA4, RST2, RST3, and RST4） had a specificity of 100%. For the determination of efficacy, ELISA4, RST2, and RST4 were selected in the second phase. ELISA1 and ELISA3 which have a sensitivity of 95.9% and 93.2% respectively also entered this phase. In the second phase, all the five assays （ELISA1, ELISA3, ELISA4, RST2, and RST4） had a sensitivity and specifity of over 90%. ELISA1 had a sensitivity of 99% and ELISA4 a specificity of 99%. Conclusion The sensitivity ELISA1 and the specificit of ELISA4 are comparable to ELISA/WB standard strategy. Application of this alternative testing strategy provides a cost-effective method for determining HIV prevalence in Beijing.

Iron Status of Multiple Transfused Sickle Cell Anaemia Patients Attending a Sickle Cell Clinic in Benin City, Nigeria

This study was aimed at evaluating the iron status of multiple blood transfused sickle cell anaemia (SCA) patients in Benin City, Nigeria. A total of 86 subjects participated in the study, comprising of 30 multiple transfused SCA subjects, 30 of rarely transfused SCA subjects and 26 of age and sex matched healthy control subjects. Serum ferritin was determined by enzyme linked immuno sorbent assay technique. Serum iron and Total iron binding capacity was determined by spectrophometric method. The mean serum ferritin concentration was elevated in the sickle cell anaemia patients whose multiple transfusions (MT) are more than those who were rarely transfused (RT) as compared with the control groups (p < 0.001). There was a positive correlation between the serum ferritin and the number of units of blood transfused (r = 0.719, p = 0.000). This study revealed that a high level of serum ferritin, percentage transferrin saturation and a reduction in total iron binding capacity were observed in sickle cell anaemia patients who received ≥3 units of packed cells in one year.

Evaluation of purified recombinant spike fragments forassessment of the presence of serum neutralizing antibodiesagainst a variant strain of porcine epidemic diarrhea virus

Since 2010, variant strains of porcine epidemic diarrhea virus(PEDV) have caused disasters in the pork industry. The spike(S) protein, as the major immunity-eliciting antigen, has previously been used for serological testing and has been found to correlate significantly with the results of the serum neutralization(SN) test. However, further evaluation of this method is needed as new epidemic strains of PEDV emerge. Hence, the main objective of this study was to assess sow sera and determine the correlation between enzyme-linked immunosorbent assay(ELISA) results(involving a newly isolated GDS01 virus-based ELISA and ELISAs based on seven recombinant fragments comprising overlapping S1 and partial S2 sequences) and SN titers. Furthermore, we determined the reliability of the ELISAs based on receiver operating characteristics(ROC) curve analyses. For the most promising ELISA, i.e., the SP4 ELISA, the correlation coefficient(r) and the area under curve(AUC) were determined to be 0.6113 and 0.8538, respectively. In addition, we analyzed the homology of the SP4 sequences obtained from different strains(including vaccine strains) and found that various strains showed a high degree of homology in this region. Thus, we conclude that SP4 is a promising serological testing protein for use in the field.