Microplate Enzyme Immunoassay Research Articles

Dehydroepiandrosterone sulfate (DHEAS) in excreta is a good indicator of serum DHEAS in Japanese macaques (Macaca fuscata)

We developed a microplate enzyme immunoassay (EIA) to measure dehydroepiandrosterone sulfate (DHEAS) in the blood, urine, and feces of Japanese macaques and evaluated the relationship between serum DHEAS and excreta DHEAS. Our DHEAS EIA using heterological DHEA derivatives conjugated with enzyme was highly sensitive, and linearities and recoveries for all matrices of Japanese macaques were reliable. For the biological evaluation of the metabolism of DHEAS in Japanese macaques, dissolved DHEAS was injected into the subjects, and consecutively collected serum, urine, and fecal samples were analyzed. The peaks of serum DHEAS were observed 6 h after the administration, while those of urine and feces were observed after 24 h. The fluctuation of those in urine and feces were significantly correlated with serum DHEAS levels. In addition, we measured pregnanediol-glucuronide (PdG), and estrone-conjugate (E1C) in urine and fecal samples to investigate the effects of administrated DHEAS on these progesterone and estrogen metabolites. The peak of PdG was observed 24 h after administration, then declined sharply. The concentration of E1C increased 1 week after administration in two out of three subjects. Our results suggest that measuring urinary and fecal DHEAS with our EIA provides a non-invasive alternative to assessing DHEAS levels in the serum of Japanese macaques.

Study of thyroid disorders in vitiligo.

Vitiligo, an acquired sometimes familial depigmentary disorder of the skin and hair that results from selective destruction of melanocytes or pigment cells. It is the single most important non-neo plastic disease that involves both the immune system and melanocytes which are subsequently destroyed and the affected area turns pale and becomes white. The prevalence of the disease is between 1% and 2% in general population. It is a prospective, randomized, and controlled study. Over 90 vitiligo patients attending Dermatology OPD and vitiligo clinic are enrolled in the study. About 35 apparently healthy, age and sex matched individuals are selected to serve as control. A prescribed proforma containing all the demographic data, relevant questionnaire were recorded for each and every case with brief clinical history suggestive of any thyroid disease as well as those referred by the clinicians. P value <0.05 is considered significant. The Quantitative Determination of Thyroglobulin (Tg) Autoantibodies in Human Serum or Plasma by a Microplate Enzyme Immunoassay. Vitiligo group, thirty four (37.78%) patients have clinical hypothyroidism while 9 (10%) patients have clinical hyperthyroidism. This difference in the distribution is statistically significant (P < 0.05) with a Chi-square value of 10.08. The data are entered, analyzed and computed with SPSS version 15 software and well-known statistical test like Chi-square, students' 't' test have been advocated wherever found applicable. P value <0.05 is considered significant. There is increased incidence of autoimmune thyroid diseases among vitiligo patients. The reason being vitiligo usually precedes the onset of thyroid dysfunction.

SCREENING OF THYROID AUTOANTIBODIES ANTI-TG, ANTI-TPO AND ITS CORRELATION WITH CLINICAL THYROID DISEASES IN VITILIGO IN THE NORTH-EASTERN REGION OF INDIA

Background and aims: Vitiligo is an acquired sometimes familial depigmentary disorder of the skin and hair that results from selective destruction of melanocytes or pigment cells. It is the single most important non-neo plastic disease that involves both the immune system and melanocytes which are subsequently destroyed and the affected area turns pale and becomes white. The precise cause of vitiligo is complex but some evidences are always suggesting that it is caused by a combination of autoimmune, genetic and environmental factors. Over half of the people with vitiligo have acquired some loss of pigment cells before the age of 20 years. The prevalence of the disease is between 1-2% in general population. Therefore, the present study is taken up to evaluate the prevalence of thyroid diseases in vitiligo patients. Methods:It is a prospective, randomized and controlled study. 90 vitiligo patients attending the Dermatology OPD and vitiligo clinic are enrolled in the study. Thirty five apparently healthy, age and sex matched individuals are selected to serve as control. A prescribed proforma containing all the demographic data, relevant questionnaire were recorded for each and every case with brief clinical history suggestive of any thyroid disease as well as those referred by the clinicians.interpretationsare made. P value &lt; 0.05 is considered significant. The Quantitative Determination of Thyroglobulin (Tg) Autoantibodies in Human Serum or Plasma by a Microplate Enzyme Immunoassay. Results:Vitiligo group, thirty four (37.78%) patients have clinical hypothyroidism while 9(10%) patients have clinical hyperthyroidism. This difference in the distribution is statistically significant (P&lt;0.05) with a chi-square value of 10.08. The datas are entered, analysed and computed with SPSS version 15 software and well known statistical test like Chi-square, students t test have been advocated wherever found applicable. The necessary interpretations are made. P value &lt; 0.05 is considered significant. Conclusion:There is increased incidence of autoimmune thyroid diseases among vitiligopatients. Various thyroid antibodies were detected in thyroid disorders, suggesting that these thyroid antibodies could act as sensitive markers for detection of early and subclinical autoimmune disorders of thyroid gland including Graves disease and Hashimotos thyroiditis. The reason beingvitiligo usually precedes the onset of thyroid dysfunction. Therefore, these thyroid autoantibodies specially anti-TPO, anti-TG should be included along with thyroid parameters TSH, T3,T4 in routine investigation of vitiligo, as association of vitiligo with autoimmune thyroid disease is very much common in the North - Eastern region of India.

Prevalence and risk factors for hyperhomocysteinemia: a population-based cross-sectional study from Hunan, China

ObjectivesHyperhomocysteinemia is an independent risk factor for cardiovascular diseases. We aimed to investigate the prevalence and risk factors for hyperhomocysteinemia, especially modifiable lifestyle factors, such as smoking behaviour and dietary...

Impact of Coenzyme Q10 on Hormonal Profile in Male Sprague-Dawley Rat Exposed to Sub-Chronic Concentrations of Cypermethrin

Aim: This study was aimed at evaluating the impact of Coenzyme Q10 on hormonal profile in male Sprague-Dawley rat exposed to sub-chronic concentrations of Cypermethrin.&#x0D; Experimental Design: A completely randomized experimental design using standard methods for analysis. Hormonal assay was carried out by Microplate Enzyme Immunoassay using their respective test kits. Including AccuBindTM Microplate EIA Test system from Monobind Inc. Lake Forest CA 92630 USA while statistical analysis was carried out using one-way Analysis of Variance (ANOVA); where significant differences were found, Pair-wise comparisons conducted with Tukey test using SPSS 20 software.&#x0D; Location and Duration of Study: This study was carried out in the Department of Biology, Ignatius Ajuru University of Education Rumuolumeni, Port Harcourt, Rivers State, Nigeria. GPS 4o48ʹ14ʹʹ N 6o59ʹ12ʹʹ E. The study lasted for 28days.&#x0D; Methodology: Thirty male Sprague-Dawley rats were randomly assigned to five groups, A-E(n=6/group).Group A was given cool clean water and standard rat pellet ad libitum. Group B,C and D were administered Cypermethrin @ 10mg/kg/bw,20mg/kg/bw,30mg/kg/bw respectively along with 10mg/kg/bw of coenzyme Q10 (CoQ10). Group E received 30mg/kg/bw of cypermethrin only without Coenzyme Q10. All animals were allowed access to cool clean water and standard rat pellet ad libitum. Bodyweight of the animals were taken twice a week and recorded in grams. Twenty-four hours before the animals were euthanized with ethyl ether inhalation, feed was withdrawn from the animals. Blood samples were collected by cardiac puncture between the hours of 7:00 and 9:00am into plain sample tubes according to the approved protocol of blood collection techniques. Analysis for the quantitative determination of all androgens was by Microplate Enzyme Immunoassay using the AccuBindTM Microplate EIA Test system from Monobind Inc. Lake Forest CA 92630 USA and expressed as their respective units. The animals were latter dissected and the vital organs harvested and weighed. The values were subjected to statistical analysis using SPSS software 20.&#x0D; Results: Results showed that there was no significant difference between the bodyweight and organ weight of animals in the treatment group compared with the control. Also, treatment with Cypermethrin only in group E, reduced the level of all the androgens considered in exposed animals. However, with concurrent administration of coenzyme Q10, the production of all androgens especially the production of Follicle Stimulating Hormone, Luteinizing Hormone and Progesterone in groups B,C,D and Estrogen in group B were significantly (P=.05) increased to values higher than the control.&#x0D; Conclusion: This elevation indicates the role of Coenzyme Q10 as an effective antioxidant to boost hormonal level production especially the level of androgens.

Detection of Cortisol Level via Enzyme Immunoassay Influenced by Exercise

Cortisol is among the human body’s most necessary hormones. On any given day, cortisol’s primary function is to control how our body creates and utilizes energy. This study examined the influence of exercise on the cortisol levels.Twenty female subjects had their cortisol levels determined before their morning exercise (Pre) and after their morning exercise (Post) using microplate enzyme immunoassay method. The result of study showed that there was significant difference (P&lt;0.05) in the cortisol levels before exercise 13.49 ± 0.90µg/dl Compared with after exercise 15.10±1.08µg/dl. It was also observed that there was no significant difference in the cortisol level (P&gt;0.05) obtained from the female subjects before and after exercise between the ages of 21-23, 24- 26 and 27-29 years. Collectively, these results showed that exercise provoked increased in cortisol level. Keyword: Cortisol, Immunoassay, Exercise, Hormone

Orthopaedic Section Poster Presentations (Abstracts OPO1-OPO300).

These abstracts are presented here as prepared by the authors. The accuracy and content of each abstract remain the responsibility of the authors. In the identification number above each abstract, OPO designates an Orthopaedic Section poster presentation. J Orthop Sports Phys Ther 2018;48(1):A67-A202. doi:10.2519/jospt.2018.48.1.A67.

Variation in the limit-of-detection of the ProSpecT Campylobacter microplate enzyme immunoassay in stools spiked with emerging Campylobacter species

Campylobacter enteritis in humans is primarily associated with C. jejuni/coli infection. The impact of other Campylobacter spp. is likely to be underestimated due to the bias of culture methods towards Campylobacter jejuni/coli diagnosis. Stool antigen tests are becoming increasingly popular and appear generally less species-specific. A review of independent studies of the ProSpecT® Campylobacter Microplate enzyme immunoassay (EIA) developed for C. jejuni/coli showed comparable diagnostic results to culture methods but the examination of non-jejuni/coli Campylobacter spp. was limited and the limit-of-detection (LOD), where reported, varied between studies. This study investigated LOD of EIA for Campylobacter upsaliensis, Campylobacter hyointestinalis and Campylobacter helveticus spiked in human stools. Multiple stools and Campylobacter isolates were used in three different concentrations (104–109CFU/ml) to reflect sample heterogeneity.All Campylobacter species evaluated were detectable by EIA. Multivariate analysis showed LOD varied between Campylobacter spp. and faecal consistency as fixed effects and individual faecal samples as random effects. EIA showed excellent performance in replicate testing for both within and between batches of reagents, in agreement between visual and spectrophotometric reading of results, and returned no discordance between the bacterial concentrations within independent dilution test runs (positive results with lower but not higher concentrations). This study shows how limitations in experimental procedures lead to an overestimation of consistency and uniformity of LOD for EIA that may not hold under routine use in diagnostic laboratories. Benefits and limitations for clinical practice and the influence on estimates of performance characteristics from detection of multiple Campylobacter spp. by EIA are discussed.

Evaluation of a novel chemiluminescent microplate enzyme immunoassay for hepatitis B surface antigen detection

Hepatitis B virus surface antigen (HBsAg) is an important biomarker used in the diagnosis of hepatitis B virus (HBV) infection, but false-negative results are still reported in the detection of HBsAg using commercial assays. In this study, we evaluated the qualitative properties of a novel HBsAg chemiluminescence enzyme immunoassay (CLEIA) assay – WTultra. WHO standard sample dilution series and samples from low-level HBsAg carriers (<1ng/mL) were used to evaluate the sensitivity of the WTultra assay. Boston Biomedica, Inc. (BBI) hepatitis B seroconversion panels were used to assess the ability of the WTultra assay to detect the window period. In addition, dilution series of 22 serum samples with different genotypes, serotypes and HBsAg mutations were used to assess the WTultra assay, and these were compared with other commercial assays. The lower detection limit of the WTultra assay was 0.012IU/mL, and it showed a high sensitivity (97.52%, 95% CI, 94.95–99.00) in the detection of 282 low-level HBsAg carriers (<1ng/mL). In samples with various HBV genotypes, serotypes and HBsAg mutations, the WTultra assay yielded 117 positive results in 132 samples, which was significantly higher than the results with the other four commercial assays (89, 83, 65 and 45, respectively, p<0.01). In the assays of mutant strains, the WTultra assay detected 82 positive results in 90 samples, which was significantly better than the results for the Hepanostika HBsAg Ultra (58 positive) and Architect (55 positive) (p<0.01) assays, which in turn were significantly better than the Murex V.3 (41 positive, p=0.026) and AxSYM V2 (29 positive, p<0.01) assays. However, in the detection of 42 samples of wild-type strains with various genotypes and serotypes, no significant differences were observed among the WTultra (35 positive), Architect (28 positive) and Hepanostika HBsAg Ultra (31 positive) assays. However, the WTultra assay detected significantly more samples than the Murex V.3 (24 positive, p<0.01) and AxSYM V2 (16 positive, p<0.01) assays. In conclusion, the WTultra HBsAg assay has a high detection sensitivity and presents excellent results for the detection of mutants.

Competitive photometric enzyme immunoassay for fullerene C60 and its derivatives using a fullerene conjugated to horseradish peroxidase

The article describes a highly sensitive single-step microplate enzyme immunoassay of the ELISA type for fullerene C60 and its derivatives. Monoclonal anti-fullerene antibodies and a conjugate between fullerene and horseradish peroxidase were used as specific reagents. A direct competitive ELISA was carried out that was based on antibodies immobilized in the well of a microtiter plate, a peroxidase-labeled antigen, and detection via the dye formed from 3,3′,5,5′-tetramethylbenzidine and hydrogen peroxide. Both pristine fullerene C60 and its water-soluble forms can be determined. The detection limits are 1.5 ng∙mL−1 for fullerene C60, and between 0.1 and 1.3 ng∙mL−1 for its derivatives. This ELISA format allows for almost two-fold reduction of the time needed for the assay in comparison to indirect scheme with labeled antibodies.

Performance of a new rapid test for detecting anti-hepatitis E virus immunoglobulin M in immunocompetent and immunocompromised patients

BackgroundHepatitis E virus (HEV) infections are widespread in both developing and developed countries. It is, therefore, important to assess the performance of systems for rapidly detecting anti-HEV immunoglobulin M antibodies in human sera. ObjectivesTo evaluate the diagnostic value of a new immunochromatographic assay, the HEV IgM rapid test (Wantai). Study designBlood samples were taken from 30 acutely infected immunocompetent and 30 acutely infected immunocompromised patients, all with HEV RNA in their blood. Specificity and cross reactivity was assessed using samples from 30HEV RNA negative immunocompetent patients who had acute Epstein–Barr virus (EBV) or cytomegalovirus (CMV) infections and 30HEV RNA negative immunocompromised patients. The performance of the HEV IgM Rapid Test was compared to that of a conventional microplate enzyme immunoassay. ResultsThe sensitivity of the rapid test in immunocompetent patients was 90% (95% CI: 72.1–100%), similar to that of the Wantai microplate assay (sensitivity: 96.6%, 95% CI: 78.77–100%; p=0.61). The sensitivity of the rapid test in immunocompromised patients was 73.3% (95% CI: 55.4–91.2%) and that of the microplate assay was 83.3% (95% CI: 65.44–100%; p=0.53). The rapid test produced no false positive reactions with samples from HEV RNA negative patients; while the microplate assay gave two false positive results (3.3%). ConclusionThe new Wantai HEV IgM rapid test is easy to use and suitable for rapidly detecting acute hepatitis E infections in both immunocompetent and immunocompromised patients. However, HEV RNA must be detected using a molecular assay for diagnosing an HEV infection in anti-HEV IgM negative patients.

Anti-spermatogenic and estrogenic effects of Escravos crude oil in Chinchilla rabbits

This study aimed at investigating the effects of Escravos crude oil on spermatogenesis in relation to serum concentrations of estradiol and total cholesterol. A total of 30 male Chinchilla rabbits within the age range of 12 to 14 weeks and weighing 1.2 kg to 1.45 kg were used in this study. The doses of 00, 15, 20, 25 and 30 mg kg-1 body weight of Escravos crude oil were orally administered to groups A, B, C, D and E, respectively for 28 days. Serum concentrations of estradiol and total cholesterol were estimated using the microplate enzyme immunoassay and enzymatic end point methods, respectively. The SPSS software package (version 16) was used for the statistical analyses and results expressed in mean ± Standard Deviation. The results showed dose dependent significant increases in estradiol (18.32 +/- 6.46 to 69.41 +/- 12.89 pg/ml) and total cholesterol (1.32 +/- 0.24 to 2.92 +/- 0.34 mmol/l) concentrations (p 0.05) in the relative weight of the testis (3.80 +/- 0.40 to 5.50 +/- 0.90 g). The histology of the testes revealed decreased spermatogenic activity. The findings of this study suggest that Escravos crude oil could be a potential endocrine disruptor and anti-spermatogenic agent. Keywords : Escravos crude oil, Estradiol, Anti-Spermatogenesis, Total cholesterol, Chinchilla rabbits.

Systematic serological testing for hepatitis E virus in kidney transplant recipients.

Hepatitis E virus (HEV) genotype 3 is endemic in Europe and hyperendemic in southern France. Recent reports of a high prevalence of HEV RNA in blood donations and in culinary specialties from this geographical area confirmed the endemicity of HEV and sources of viral transmission in this geographical area. HEV causes acute and chronic hepatitis in solid organ transplant recipients. Since March 2012, we have implemented systematic HEV serological testing in our cohort of kidney transplant recipients (KTRs) in Marseille in southeastern France. The aim of our study was to assess HEV exposure in this cohort between March 2012 and May 2014. During these 27 months, we found that 39% of the patients who underwent kidney transplantation had an anti-HEV IgG response using a sensitive microplate enzyme immunoassay. This seroprevalence was approximately 43% at both 1 and 8 years after, using the same assay. In addition, systematic HEV serological testing detected 6 cases of HEV infection among 578 KTRs (1%) during the 27 months of the study, with 5 at an acute stage and 1 at a chronic stage. In conclusion, continuous HEV monitoring in this population is useful for better understanding the epidemiology of HEV in France, because these patients are a well-monitored population. Moreover, HEV monitoring in KTRs is clinically relevant because HEV represents a clinical threat in these patients. Nevertheless, HEV serological testing may be more fruitful for identifying HEV infections when performed in cases of biological liver abnormalities than when performed systematically.

Dengue viremia in blood donors in Northern India: Challenges of emerging dengue outbreaks to blood transfusion safety.

Backdround:Emerging infectious diseases pose threats to the general human population; including recipients of blood transfusions. Dengue is spreading rapidly to new areas and with increasing frequency of major outbreaks. Screening blood for dengue antigens in dengue-endemic countries would be costly and should, therefore, be recommended only after careful assessment of risk for infection and cost.Aim:A prospective study was conducted to establish the magnitude of the threat that dengue poses to blood safety where it is sporadic with seasonal variations, to quantify risk and to assess that whether screening is feasible and cost-effective.Materials and Methods:Nonstructural protein 1 (NS1) antigen test was done on 1709 donations during dengue outbreak in the months August to November 2013 as an additional test using Bio-Rad Platelia Dengue NS1AG test kit which is one step sandwich format microplate enzyme immunoassay using murine monoclonal antibodies for capture and revelation. Chi-square test was used to find statistical significance.Results and Conclusions:Majority cases were whole blood, replacement, male donors with 76.10% donors in <35 years age group. About 17.85% were single donor platelet donations. NS1 antigen in all donors was negative. In the past, dengue affected mainly children who do not donate blood. With the changing trend, mean age of infection increased affecting the population that does donate blood, further reducing blood donation pool. Further studies need to be done in different geographic regions of the country during dengue transmission season to establish maximum incidence of viremic donations, rates of transfusion transmission and clinical consequences in recipients. If risk is found to be substantial, decision will be taken by the policymakers at what threshold screening should be instituted to ensure safe blood transfusion.

Histopathological, Hemochromatotic, Hypercholesterolemic, and Androgenic Effects of Escravos Crude oil on the Testis in Male Chinchilla Rabbits

Aim: This study aimed to investigate the effects of Escravos crude oil on serum concentrations of testosterone, total cholesterol, body weight, relative weight and microanatomical architecture of the testis using male Chinchilla Rabbits. Place and Duration of Study: This study was carried out at the Department of Medical Laboratory Science, Nnamdi Azikiwe University, Nnewi Campus between May and June 2013 (28 days). Methodology: A total of thirty male Chinchilla Rabbits aged 12 to 14 weeks and weighing 1.2kg to 1.45kg was used. Crude oil was administered orally at the doses of 15, 20, 25 and 30mg/kg body weight to groups designated B, C, D and E respectively for 28 days, while group A was given normal saline. Serum concentration of testosterone and total Original Research Article British Biotechnology Journal, 4(6): 649-658, 2014 650 cholesterol were estimated using the microplate enzyme immunoassay and enzymatic end point methods, respectively. The SPSS software (version 20) was used for the statistical analysis and the result expressed in mean ± SD. Result: The results showed dose dependent effects on the hormone and biochemical assays, especially at the concentration of 25mg/kg body weight of the administered crude oil. Significant increases in serum concentrations of testosterone (0.32+/-0.05 to 0.46+/0.14) and total cholesterol (1.35+/-0.17 to 1.76+/-0.15) concentrations (p≤.05), and insignificant increase (p≥.05) in the relative weight of the testis (4.80+/-0.40 to 6.50+/0.90) were observed. The histology of the testes revealed hypertrophy of the seminiferous tubules, atrophy of the basal lamina and interstitial cells, and hemochromatosis. The histological findings agree with the hormonal and biochemical findings. Conclusion: These findings suggest that Escravos crude oil might be a potential endocrine disruptor and toxic substance which can affect the micro-anatomical architecture of the testis.

Sensitive and reliable micro-plate chemiluminescence enzyme immunoassay for okadaic acid in shellfish

A highly sensitive and reliable analytical micro-plate chemiluminescence enzyme immunoassay based on a monoclonal antibody against okadaic acid was developed and validated for the detection of okadaic acid from a shellfish matrix.

Micro-Plate Chemiluminescence Enzyme Immunoassay for Determination of Zeranol in Bovine Milk and Urine

Zeranol (α-Zearalanol, α-ZAL), well-known as an anabolic promoter, was officially banned in Europe due to its potential carcinogenic and endocrine-disrupting biological activities. In this study, a method for the determination of zeranol residues in bovine milk and urine was developed based on micro-plate chemiluminescence enzyme immunoassay (CLEIA). The limit of detection (LOD) was 50 ng/L, and the linear range was between 100 ng/L and 4510 ng/L, with a correlation coefficient (R2)0.9982. The recoveries of zeranol in bovine milk and urine were between 84.7% and 123.6%. This study showed that CLEIA was a reliable, convenient, and sensitive method for screening zeranol residues in bovine milk and urine.

ImmunoCard STAT! cartridge antigen detection assay compared to microplate enzyme immunoassay and modified Kinyoun's acid-fast staining technique for detection of Cryptosporidium in fecal specimens

Cryptosporidium species infect humans and a wide range of animals worldwide; outbreaks of cryptosporidiosis have been reported in several countries. Routine diagnostic methods may be insufficient to demonstrate the presence of these organisms. The study assessed the diagnostic accuracy of the antigen detection immuno-cartridge test, ImmunoCard STAT! (Meridian Bioscience Inc., Cincinnati, OH, USA), compared to the combined gold standard: modified Kinyoun's acid-fast technique confirmed with the microplate enzyme immunoassay (EIA) for the detection of Cryptosporidium in fecal specimens. Three hundred fifteen formalin-fixed stool specimens were submitted for testing. The Kinyoun's acid-fast-stained smear revealed 24 positive samples for Cryptosporidium (of which 23 specimens were confirmed by the EIA) and 291 negative samples (of which 289 were negative by EIA). Agreement between the three used tests was shown in 22 positive and 288 negative samples for Cryptosporidium. Kappa score of agreement between the immuno-cartridge test and EIA was 0.957, p = 0.000. The sensitivity of the immuno-cartridge test was 96% (95% confidence interval (CI), 87% to 104%) and the total accuracy of the test was 97% (95% CI, 93-103). The ImmunoCard STAT! Cryptosporidium cartridge assay is easy to use and does not require specialized training or equipment and is useful in routine diagnosis and screening for Cryptosporidium especially where rapid, point of care testing is needed or where other reliable tests are unfeasible with a performance comparable to the EIA and acid-fast technique.

Micro-plate chemiluminescence enzyme immunoassay for aflatoxin B1 in agricultural products

In this work, a micro-plate chemiluminescence enzyme immunoassay by antibody-coated for the determination of aflatoxin B1 (AFB1) in agricultural products has been established. Aflatoxin B1 antibody (AFB1-Ab) was adsorbed physically on polystyrene micro-plate hole as solid phase antibody, which took place immunity-reaction between antigen and antibody with AFB1 standard solution or samples by direct competition. Luminol–hydrogen peroxide chemiluminescence system catalyzed by horseradish peroxidase (HRP) with p-iodophenol enhancement was used as signal detecting system. The effects of several factors, including composition and pH of coating solution, dilution ratio and amount of antibody and enzyme labeled antigen, time of antibody-coating, incubation and chemiluminescence reaction, and other relevant variables upon the immunoasaay were studied and optimized. The linear range of proposed method for AFB1 was 0.05–10.0 ng g −1 with a correlative coefficient of −0.9997. The sensitivity of the proposed method was 0.01 ng g −1. The RSDs of intra- and inter-assay were less than 12.2% and 10.0%, respectively. This method has been successfully applied to the evaluation of AFB1 in agricultural products with recoveries of 79.8%, 101.9% and 115.4% for low, middle and high concentration samples, respectively. It shows a good correlation with the commercial available ELISA kit for AFB1 with correlative coefficient of 0.9098 indicating that the established CLEIA method can be used to determine AFB1 in real samples.

Lower genital tract infections in HIV-seropositive women in India

The presence of STD facilitates shedding of HIV and increases HIV-1 disease progression, possibly by increasing plasma viremia. Our aim was to study the presence of various associated Sexually transmitted disease/Reproductory tract infections in HIV-seropositive women in India. The study included 40 HIV-seropositive women attending the antiretroviral therapy (ART) clinic at Lok Nayak Hospital. An informed consent was taken from all subjects. All cases were subjected to detailed gynecological examination and two types of swabs, i.e., a vaginal swab and a cervical swab were taken for STD/RTIs evaluation. The vaginal swabs were used for preparation of wet mount and KOH mount for diagnosis of trichomoniasis and candidiasis; to make a vaginal smear for Gram staining to diagnose bacterial vaginosis (BV) as per Nugent's criteria; for culture of aerobic bacteria and Candida spp. The cervical swab was used for isolation of Neisseria gonorrhoeae by culture and for detection of Chlamydia trachomatis antigen by Chlamydia microplate enzyme immunoassay kit (BIORAD). All data were analyzed using appropriate statistical tests. All 40 cases were evaluated for the presence of STD/RTIs associated with HIV infection. The women belonged to the reproductive age group (15-45 years) and majority (40%) of them were para 2. Most of the women (14, 35%) were in World Health Organization (WHO) stage I and maximum number (28, 70%) had their CD4 cell count more than 200 cells/ml. There was no significant correlation between WHO stage of HIV-seropositive women and their CD4 cell count (P=0.092). Out of 40 cases, 15 (37.5%) were on ART with maximum cases (53.3%) in WHO stage III. The duration of ART was more than 6 months in 9 (60%) cases. The most common presenting complaint was vaginal discharge in women with WHO stage II and III and 27.5% women showed vaginitis on per speculum examination. Laboratory tests showed high prevalence of BV (30%), mixed infection (30%), and candidiasis (10%) among HIV-seropositive women (P<0.001 in both). Women with BV were mostly in WHO stage I (38.4%) and stage II (36.3%), while those with mixed infection were mainly in WHO stage III (36.3%) and stage IV (40%).Women with candidiasis were mainly in WHO stage III. C. trachomatis antigen was found only in one subject (prevalence 2.5%). Both WHO stage and CD4 cell count had no significant correlation with presence of BV (P=0.056 and 0.063, respectively) and candidiasis (P=0.492 and 0.530, respectively). Maximum number of patients on ART had mixed infection (53.3%), while most of the patients (36%) not on ART had BV. There was no significant association between duration of ART and the presence of vaginal infections. The prevalence of gynecological symptoms and RTIs in HIV-seropositive women is high enough to warrant routine gynecologic evaluation and RTI screening in these patients. However, larger studies and trials are needed to evaluate the effects of ART on these abnormalities as well as to choose the best screening tool in HIV-seropositive women.