Competitive ELISA Method Research Articles

Production and evaluation of anti-BP26 monoclonal antibodies for the serological detection of animal brucellosis.

Brucella BP26 proves to be a highly immunogenic antigen with excellent specificity in brucellosis detection. In China, the authorized use of the Bp26-deleted vaccine M5ΔBP26 for preventing small ruminant brucellosis highlights the importance of developing accurate detection methods targeting BP26, particularly for the diagnosis of differentiation between infected and vaccinated animals (DIVA). Using the traditional mouse hybridoma technique, we successfully obtained 12 monoclonal antibodies (mAbs) targeting BP26. The efficacy of these mAbs in detecting various animal brucellosis cases using the competitive ELISA method was evaluated. Among them, only the E10 mAb exhibited significant efficiency, being inhibited by 100, 97.62, and 100% of brucellosis-positive sera from cattle, small ruminants, and canines, respectively. The E10-based competitive enzyme-linked immunosorbent assay (cELISA) outperformed the BP26-based indirect enzyme-linked immunosorbent assay (iELISA) in accuracy, particularly for cattle and small ruminant brucellosis, with cELISA sensitivity reaching 97.62% compared to 64.29% for iELISA for small ruminants. Although cELISA showed slightly lower specificity than iELISA, it still maintained high accuracy in canine brucellosis detection. The epitope of mAb E10 was identified in the amino acid sequence QPIYVYPDDKNNLKEPTITGY, suggesting its potential as a diagnostic antigen for brucellosis. In conclusion, the E10-based cELISA presents an effective means of detecting animal brucellosis, particularly significant for DIVA diagnosis in China, where the BP26-mutant vaccine is widely used.

Effectiveness of Self-Management Training, “EDUDARA”, on Psychological Wellbeing and Cortisol Levels in Breast Cancer Patients during the COVID-19 Pandemic

Background: Women diagnosed with breast cancer often experience emotional distress, leading to a decrease in psychological well-being. Online self-management training during the COVID-19 pandemic is crucial for maintaining psychological well-being, as it is closely related to stress hormone cortisol levels, which play a vital role in boosting the body’s immune system. However, research on self-management strategies for breast cancer patients, particularly regarding psychological aspect and its relationship to cortisol levels during the COVID-19 pandemic, remains limited.Purpose: The purpose of this study was to measure the effectiveness of self-management training on psychological well-being and salivary cortisol levels in breast cancer patients.Methods: This study used a pre-post-test quasi-experimental design with a control group. The participants included 70 breast cancer patients undergoing chemotherapy at a hospital in Malang City, East Java, Indonesia, recruited using simple random sampling. Self-management training using the EDUDARA (Edukasi Kanker Payudara) was administered to the participants in the intervention group for six weeks. Data on wellbeing were collected using the Ryff Psychological Wellbeing Questionnaire, while salivary cortisol levels were measured with the DBD cortisol kit using the competitive ELISA method. SPSS with t-tests was used to analyze the data.Results: The average post-test psychological well-being score in the intervention group was 90.3, while in the control group, it was 82.69. The results of statistical tests showed a significant difference in psychological well-being between the two groups, with the intervention group showing greater improvement after the training. Additionally, the mean salivary cortisol level post-training was 4.531 in the intervention group and 6.169 in the control group (p=0.001). This indicates a greater reduction in cortisol levels in the intervention group after training, showing that participants’ psychological condition following the “EDUDARA” training was better than that of the control group.Conclusion: Self-management training had a positive effect on increasing psychological well-being and decreasing salivary cortisol levels among breast cancer patients. Therefore, structured self-management training for these patients can be used for nursing intervention

Development of anti-PEG IgG/IgM/IgE ELISA assays for profiling anti-PEG immunoglobulin response in PEG-sensitized individuals and patients with alpha-gal allergy

Polyethylene glycol (PEG) is frequently used in various protein and nanomedicine therapeutics. However, various studies have shown that select PEGylated therapeutics can induce production of anti-PEG antibodies (APA), potentially culminating in rapid clearance from the systemic circulation, loss of efficacy and possibly increased risks of allergic reactions. Although IgE is a frequent cause of immediate hypersensitivity reactions (IHR), the role of IgE APA in PEG-related IHR is not well understood, due in part to a lack of standardized assays for measuring IgE APA. Here, we developed a rigorous competitive ELISA method to measure the concentrations of various APA isotypes, including IgE, with picomolar sensitivities. In a small number of serum samples from patients with known PEG allergy, the assay allowed us to detect a strong correlation between IgG and IgE APA in individuals with history of allergic reactions to PEG or PEGylated drugs, but not between IgM and IgE APA. We detected appreciable levels of IgG and IgM APA in individuals with history of alpha-gal allergy, however, they were not elevated relative to those detected in other healthy controls, and we found no pre-existing IgE APA. While preliminary and should be further investigated, these results suggest that differences in the route and mechanism of PEG exposure may drive variability in APA response.

Preparation of Anti-Zearalenone IgY and Development of an Indirect Competitive ELISA Method for the Measurement of Zearalenone in Post-Fermented Tea.

Post-fermented tea (PFT) is one of the most commonly consumed beverages worldwide. Rapid microbial growth and significant changes in the microbial composition of PFT during processing and storage pose a potential risk of contamination with mycotoxins such as zearalenone (ZEN). Screening for ZEN contamination in a simple, rapid, and inexpensive manner is required to ensure that PFT is safe for consumption. To monitor ZEN in PFT, ZEN was conjugated with bovine serum albumin to prepare egg yolk immunoglobulins (IgY). A specific indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) based on IgY was developed and validated. ZEN was extracted with acetonitrile and water (50:50, v/v) containing 5% acetic acid and purified using a mixture of primary and secondary amines and graphitized carbon black to remove matrix interference from the PFT samples. Under optimal conditions, the linear range of this assay was 13.8-508.9 ng mL-1, the limit of detection was 9.3 ng mL-1, and the half-maximal inhibitory concentration was 83.8 ng mL-1. Cross-reactivity was negligible, and the assay was specific for ZEN-related molecules. The recovery rate of ZEN in the control blanks of PFT samples spiked with a defined concentration of ZEN of 89.5% to 98.0%. The recovery and accuracy of the method were qualified for PFT matrices. No significant differences were evident between the results of the actual PFT samples analyzed by high-performance liquid chromatography and ic-ELISA. The collective data indicate that the developed ic-ELISA can be used for the rapid and simple detection of ZEN in PFT products.

Measurement of Urinary Gc-Globulin by a Fluorescence ELISA Technique: Method Validation and Clinical Evaluation in Septic Patients-A Pilot Study.

A major complication of sepsis is the development of acute kidney injury (AKI). In case of acute tubular damage, Gc-globulin, a known serum sepsis marker is increasingly filtrated into the urine therefore, urinary Gc-globulin (u-Gc) levels may predict septic AKI. We developed and validated a competitive fluorescence ELISA method for u-Gc measurement. Serum and urine samples from septic patients were collected in three consecutive days (T1, T2, T3) and data were compared to controls. Intra- and interassay imprecisions were CV < 14% and CV < 20%, respectively, with a recovery close to 100%. Controls and septic patients differed (p < 0.001) in their u-Gc/u-creatinine levels at admission (T1, median: 0.51 vs. 79.1 µg/mmol), T2 (median: 0.51 vs. 57.8 µg/mmol) and T3 (median: 0.51 vs. 55.6 µg/mmol). Septic patients with AKI expressed higher u-Gc/u-creatinine values than those without AKI at T1 (median: 23.6 vs. 136.5 µg/mmol, p < 0.01) and T3 (median: 34.4 vs. 75.8 µg/mmol, p < 0.05). AKI-2 stage patients exhibited more increased u-Gc/u-creatinine levels at T1 (median: 207.1 vs. 53.3 µg/mmol, p < 0.05) than AKI-1 stage individuals. Moderate correlations (p < 0.001) were observed between u-Gc/u-creatinine and se-urea, se-creatinine, se-hsCRP, WBC, u-total protein, u-albumin, u-orosomucoid/u-creatinine, and u-Cystatin C/u-creatinine levels. U-Gc testing may have a predictive value for AKI in septic patients.

Correlation analysis between camrelizumab trough concentration levels and efficacy or safety in East Asian patients with advanced lung cancer.

Camrelizumab combined with chemotherapy is approved across tumor types. However, only a fraction of patients benefits from immunotherapy, and biomarkers such as the expression of PD-L1, tumor mutational burden, and CXCL11 are expensive and suboptimal specificity for cancer patients. An exposure-response (E-R) relationship has been reported in many immune checkpoint inhibitors (ICIs), and the trough concentrations and other drug exposure metrics are broadly used to guide dosing decisions, assess exposure-outcomes relationships, and ultimately predict outcomes based on those relationships. However, the potential use of trough concentration levels for camrelizumab is still not clear. Blood samples were obtained at trough levels after doses 3 and 4 from 77 patients with advanced lung cancer who received camrelizumab (200mg Q3 W) monotherapy or combined with chemotherapy. We optimized a competitive ELISA method to measure the trough concentration. We found that the trough concentration was steady after 3 dose cycles, and the trough concentration level of camrelizumab was higher in patients who developed immune-related adverse effects (irAEs) than in those who did not (P < 0.05) but was not observed in disease progression and PFS (P > 0.05). Age (< 65 years old), no smoking history, and efficacy evaluation after 4-dose treatment were associated with PFS (P < 0.05), but no significance was observed in other clinical characteristics. Total bilirubin and albumin had an influence on trough concentration, and monocytes and albumin were independent risk factors for PFS (P < 0.05). Our results suggest that the trough concentration level of camrelizumab might be a risk factor for the occurrence of irAEs in advanced lung cancer, and using the immunotherapy as early as possible may bring better clinical outcomes.

Assessment of the Efficiency of Technological Processes to Modify Whey Protein Antigenicity.

Whey is a by-product that represents a cheap source of protein with a high nutritional value, often used to improve food quality. When used as a raw material to produce hypoallergenic infant formulas (HIF), a processing step able to decrease the allergenic potential is required to guarantee their safe use for this purpose. In the present paper, thermal treatments, high hydrostatic pressure (HHP), and enzymatic hydrolysis (EH) were assessed to decrease the antigenicity of whey protein solutions (WPC). For monitoring purposes, a competitive ELISA method, able to detect the major and most allergenic whey protein β-lactoglobulin (BLG), was developed as a first step to evaluate the efficiency of the processes. Results showed that EH together with HHP was the most effective combination to reduce WPC antigenicity. The evaluation method proved useful to monitor the processes and to be employed in the quality control of the final product, to guarantee the efficiency, and in protein antigenicity reduction.

Frequency of HEV Antibodies in Pregnant Women and Its Association with Existent Risk Factors, Sudan: A Cross-Sectional Study

Background: Hepatitis E virus (HEV) infection is an increasing global problem and high endemicity is observed in many developing countries. Pregnancy predisposes women to chronic infection and various complications.&#x0D; Objective: This study aimed to investigate sero-positivity of HEV and existent risk factors among pregnant women in Gezira State, Sudan.&#x0D; Methods: A cross-sectional design was followed, and a non–probability convenience sampling technique was used to collect 120 venous blood samples from pregnant women. Separated serum specimens was used for the quantitative detection of specific anti-HEV IgM and IgG by competitive ELISA method (MIKROGEN GmbH - Germany).&#x0D; Results: The frequency of positive results for HEV IgG antibodies in the study population amounted to 44.2 (53/120). Of the 53 HEV IgG positive pregnant participants 47.2% (25/53) were in the third trimester period, 39.6% (21/53) had preterm labor, 39.6% (21/53) had a history of abortion and 24.5% (13/53) with jaundice. Significant association was found between HEV IgG positive women and preterm labor (p-value 0.004), and history of jaundice (p-value 0.009).&#x0D; Conclusion: This preliminary study highlighted the high frequency of sero-positivity of hepatitis E virus in pregnant women in the studied area of Sudan.

Zearalenone in feed, urine and meat from three pig farms in Croatia

Zearalenone (ZEN) is a mycotoxin of the genus Fusarium which belongs to the group of macrocyclic lactones. ZEN contamination occurs during cereal harvest or in the early phase of storage if drying was insufficient. The aim of this study was to determine the level of ZEN in feed mixtures given to pigs during the fattening period at three different farms in the Republic of Croatia, as also to determine ZEN levels in urine and meat taken from the same animals. The study also examined correlation between ZEN concentrations in urine and meat with the estimation of ZEN intake in the human body through meat consumption, expressed as a percentage of the Tolerable Daily Intake (TDI). In total, 9 feed mixtures (3 samples per farm), 45 urine and 45 meat samples (from 15 animals per farm) were taken during 2021 from three pig farms located in eastern and central Croatia. ZEN concentrations were determined by the competitive enzyme ELISA method. All values in feed from all three farms were within maximum recommended limit (MRL) given in EU Recommendation, i.e., 250 μg/kg, though at one far, the levels recorded were just under the MRL. Monitoring of ZEN levels in urine can be used as an indicator for the detection of feed contamination with this mycotoxin. Although pigs were fed with feed with near the MRL level of contamination, a negligible percentage of TDI value was obtained for this mycotoxin for humans through meat consumption. However, since meat is just one component of the human diet, and in view of the fact that ZEN can be present in a number of foodstuffs, especially cereals, its total intake could be significantly higher than estimated herein.

Development and Validation of the ELISA Method for Neutralizing Anti-trastuzumab Antibodies Detection in Human Blood Serum

Introduction. Trastuzumab is the first known anti-HER2 agent, which revolutionized the treatment of one of the most common cancer types – breast cancer. Despite trastuzumab being approved long time ago, further improvement of related analytical methods remains relevant primarily due to the emergence of new biosimilars. For instance, immunogenicity – adverse reaction which is usually associated with biological drugs, can still be relevant for trastuzumab. Anti-drug antibodies, including neutralizing antibodies, caused by trastuzumab therapy, can affect drug effectiveness and safety profile.Aim. The aim of this study was to develop and validate the analytical method for neutralizing anti-trastuzumab antibodies determination in human blood serum.Materials and methods. The neutralizing anti-trastuzumab antibody determination was carried out by the competitive ELISA method, using spectrophotometric detection in the visible range of the spectrum.Results and discussion. The developed method was validated for cut-point, selectivity, sensitivity, specificity, precision and stability (short-term and long-term). To decrease the background noise from non-specific binding of sera components, the minimum required dilution value was determined at 0.5 % serum. The calculated value for cut-point was 14.62 %. The sensitivity of the developed method was estimated at 1985.2 ng/mL of neutralizing anti-trastuzumab antibodies.Conclusion. The obtained results allowed us to apply the developed ELISA method for the neutralizing anti-trastuzumab antibodies determination in human blood serum during trastuzumab immunogenicity assessment in bioequivalence clinical trials.

A neutralizing bispecific single-chain antibody against SARS-CoV-2 Omicron variant produced based on CR3022.

The constantly mutating SARS-CoV-2 has been infected an increasing number of people, hence the safe and efficacious treatment are urgently needed to combat the COVID-19 pandemic. Currently, neutralizing antibodies (Nabs), targeting the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein are potentially effective therapeutics against COVID-19. As a new form of antibody, bispecific single chain antibodies (BscAbs) can be easily expressed in E. coli and exhibits broad-spectrum antiviral activity. In this study, we constructed two BscAbs 16-29, 16-3022 and three single chain variable fragments (scFv) S1-16, S2-29 and S3022 as a comparison to explore their antiviral activity against SARS-CoV-2. The affinity of the five antibodies was characterized by ELISA and SPR and the neutralizing activity of them was analyzed using pseudovirus or authentic virus neutralization assay. Bioinformatics and competitive ELISA methods were used to identify different epitopes on RBD. Our results revealed the potent neutralizing activity of two BscAbs 16-29 and 16-3022 against SARS-CoV-2 original strain and Omicron variant infection. In addition, we also found that SARS-CoV RBD-targeted scFv S3022 could play a synergistic role with other SARS-CoV-2 RBD-targeted antibodies to enhance neutralizing activity in the form of a BscAb or in cocktail therapies. This innovative approach offers a promising avenue for the development of subsequent antibody therapies against SARSCoV-2. Combining the advantages of cocktails and single-molecule strategies, BscAb therapy has the potential to be developed as an effective immunotherapeutic for clinical use to mitigate the ongoing pandemic.

The survey of prolamin and gluten content in beers produced in Central Europe – The biochemical characterization of immunoreactive protein fractions

Prolamins (gliadin-related proteins) are cereal proteins which are a source of polypeptides that are toxic to people showing intolerance or allergy to gluten. Therefore, serious safety concerns regarding occasional and routine beer consumption emerge due to the presence of immunoreactive amino acid sequences of cereal origin that remain in the final product. The aim of the study was to investigate the presence of prolamins and gluten in samples of beers (72 commercially available beverages) produced in Central Europe. A competitive ELISA and immunoblotting methods were applied to analyze the presence and concentration of immunoreactive proteins/peptides. Gluten level in the studied beers ranged from 10 to over 6000 mg/kg. Approximately 30% beers could be regarded as gluten-free, around 40% were very low gluten and only 25% beers contained considerable amounts of gluten. The highest level of prolamin content was detected in wheat beers. We demonstrated that these high concentrations of prolamins are probably derived from residues of non-degraded wheat gliadins.

Structural Property, Immunoreactivity and Gastric Digestion Characteristics of Glycated Parvalbumin from Mandarin Fish (Siniperca chuaisi) during Microwave-Assisted Maillard Reaction.

This study was aimed to investigate the structural and immunological properties of parvalbumin from mandarin fish during the Maillard reaction. The microwave-assisted the Maillard reaction was optimized by orthogonal designed experiments. The results showed that the type of sugar and heating time had a significant effect on the Maillard reaction (p < 0.05). The SDS-PAGE analysis displayed that the molecular weight of parvalbumin in mandarin fish changed after being glycated with the Maillard reaction. The glycated parvalbumin was analyzed by Nano-LC-MS/MS and eleven glycation sites as well as five glycation groups were identified. By using the indirect competitive ELISA method, it was found that microwave heating gave a higher desensitization ability of mandarin fish parvalbumin than induction cooker did. In vitro gastric digestion experiments showed that microwave-heated parvalbumin was proved to be digested more easily than that cooked by induction cookers. The microwave-assisted Maillard reaction modified the structure of parvalbumin and reduced the immunoreactivity of parvalbumin of mandarin fish.

Diagnostic Performance of Competitive ELISA and Western Blot Methods for the Detection of Antibodies against Theileria equi and Babesia caballi.

Theileria equi (T. equi) and Babesia caballi (B. caballi) are the causative pathogens of Equine piroplasmosis (EP), a disease that has brought huge economic losses and great restrictions to the global equine industry. Rapid and accurate diagnostic methods are critical for the effective monitoring of the disease. In this study, we developed novel competitive ELISA methods and western blot assays based on the EMA1 or Bc48 proteins to detect antibodies against T. equi or B. caballi, respectively. In the novel cELISA, horseradish peroxidase (HRP)-labeled monoclonal antibodies are used in place of enzyme-conjugated secondary antibodies, in order to speed up the entire procedure. These methods have high sensitivity and no cross-reactivity with antibodies against other equine diseases. In the newly developed western blot assays, we optimized the dilution of T. equi or B. caballi positive serum samples to 1:200. Compared with the commercially available kit, both the novel cELISA assay and the western blot assay showed high coincidence rates in detecting antibodies against T. equi and B. caballi. Taken together, the novel cELISA and the western blot assays for detecting antibodies against T. equi or B. caballi have the potential to rapidly test for T. equi or B. caballi and to contribute to the surveillance and control of this disease.

Development of an indirect competitive ELISA method based on ORF2 detecting the antibodies of novel goose astrovirus

Goose astrovirus (GAstV) characterized by articular and visceral gout, is an emerging pathogen with a wide distribution on mainland China, leading to serious economic losses in the goose-raising industry. Because vaccines to prevent GAstV infections are not available currently, early diagnosis is critical when treating symptomatic geese and in preventing GAstV transmission. In this context, a highly sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) based on a monoclonal antibody (mAb) to detect GAstV-specific antibodies from geese was developed, and the detections were optimized. A series of experiments proved that the ic-ELISA shows excellent diagnostic performance and discriminatory power with high sensitivity and specificity. The ic-ELISA for GAstV detection was applied on 67 field serum samples, and comparing the detection results with the virus neutralization test verified the accuracy of the ic-ELISA. The correlation coefficient between the ic-ELISA and the virus neutralization test was 80%, demonstrating the proposed ic-ELISA method could be a useful and effective tool for the diagnostic, serological epidemiological investigation and immune monitoring of the GAstV in goose-producing regions.

Lysozyme quantity and quality in relation with early childhood caries: A longitudinal study

Context: Several studies have reported the relationship between lysozyme quantity (concentration) and quality (activity) with early childhood caries (ECC), but the results remain controversial. These are controversial, probably due to the cross-sectional design used in previous studies. Aims: To analyze the relation between lysozyme quantity and quality with ECC in two years old children. Methods: An observational analytic with a cohort study for nine months, with a total sample of 68 caries-free children aged 9-24 months old, selected by purposive sampling from Integrated Healthcare Center in Medan, Indonesia. The subject must have at least two primary upper incisors that have erupted. Lysozyme examination was carried out three times, at the beginning of the month, the third and the ninth month. ECC examination was assessed using the American Association of Pediatric Dentistry criteria. Lysozyme concentration was measured using a competitive ELISA method with human lysozyme C (Fine Test). Then lysozyme reading was done at an absorbance of 450 nm using a microplate reader. Lysozyme activity was assessed using the Lysozyme Detection Kit (Sigma-Aldrich and ready by spectrophotometer. Data analysis was processed with an unpaired t-test, Mann-Whitney test, and longitudinal analysis using a generalized estimating equation (linear) test with a significant value, p&lt;0.05. Results: This showed that there was not any relationship found between lysozyme concentration and ECC (p&gt;0.05). However, there was a relationship between lysozyme activity and ECC (p=o.oo8). Conclusions: ECC children had higher lysozyme activity when compared to caries-free children.

Regulation of Stanniocalcin Secretion by Calcium and PTHrP in Gilthead Seabream (Sparus aurata).

Simple SummaryCalcium regulation in body fluids is a fundamental process in vertebrates, which is exerted by a plethora of hormones. Stanniocalcin (STC) is a hypocalcemic hormone ubiquitously expressed in tetrapods; in bony fishes, it is produced mainly by specific glands called the corpuscles of Stannius. The present study described an ELISA method for the analysis of fish STC. Moreover, it also develops a methodology for ex vivo cultures of Stannius corpuscles of gilthead seabream (Sparus aurata). The results show a direct control of the production of STC by one calcium and two PTHrP (parathyroid-related protein, a hypercalcemic hormone) receptors. This study highlights the tight control of circulating calcium in vertebrates and shows the complexity of the processes involved.Calcium balance is of paramount importance for vertebrates. In fish, the endocrine modulators of calcium homeostasis include the stanniocalcin (STC), and some members of the parathyroid hormone (PTH) family, such as the PTH-related protein (PTHrP), acting as antagonists. STC is ubiquitously expressed in higher vertebrates. In turn, bony fish exhibit specific STC-producing glands named the corpuscles of Stannius (CS). Previous studies pointed to a calcium-sensing receptor (CaSR) involvement in the secretion of STC, but little is known of the involvement of other putative regulators. The CS provides a unique model to deepen the study of STC secretion. We developed an ex vivo assay to culture CS of fish and a competitive ELISA method to measure STC concentrations. As expected, STC released from the CS responds to CaSR stimulation by calcium, calcimimetics, and calcilytic drugs. Moreover, we uncover the presence (by PCR) of two PTHrP receptors in the CS, e.g., PTH1R and PTH3R. Thus, ex vivo incubations revealed a dose-response inhibition of STC secretion in response to PTHrP at basal Ca2+ concentrations. This inhibition is achieved through specific and reversible second messenger pathways (transmembrane adenylyl cyclases and phospholipase C), as the use of specific inhibitors highlights. Together, these results provide evidence for endocrine modulation between two antagonist hormones, STC and PTHrP.

Serum Hepcidin Level among Chronic Kidney Disease Subjects Accessing Healthcare in Braithwaite Memorial Specialist Hospital Port Harcourt

Hepcidin is the major controller of systemic iron homeostasis and the role of the kidney in regulating hepcidin level is vital in the whole process of iron and hepcidin relationship. This study was aimed at evaluating serum Hepcidin level among Chronic Kidney Disease subjects accessing Healthcare in BMSH Port Harcourt Metropolis. The study was conducted in Port Harcourt at Braithwaite Memorial Specialist Hospital among 55 CKD subjects and 33 normal individuals making up the control group. Subjects were selected randomly and 5mls of blood was collected in plain bottle using venipuncture technique for laboratory assessment of hepcidin. Hepcidin was assayed using competitive ELISA method. T-test was used to compare the mean difference oh hepcidin between both groups. Result showed that there was a significant difference in hepcidin level between CKD and control groups; 52.00±36.00ng/ml for CKD group and 16.00±13.00ng/ml for control group, p&lt;0.05. This study has shown that CKD has a significant impact in hepcidin level blood and consequently on iron regulation.

EVALUATION OF SALIVARY COTININE LEVEL IN SECOND-HAND SMOKERS - A CASE CONTROL STUDY

Understanding SHS exposure is important in measuring and preventing exposure. Estimation of the Cotinine values help in biochemical validation and cessation outcomes. The biochemically estimated cotinine levels is found to be an indicator, second hand smoke exposure or use of therapeutic nicotine. AIMS:To evaluate salivary cotinine level in second-hand smokers MATERIALS AND METHODS : 78 study subjects divided into 2 groups (Group 1 and Group 2) of 39 each. The saliva samples were collected from subjects who had no previous history of tobacco smoking and subjects who are second-hand smokers. Their cotinine contents were measured using the competitive ELISAmethod according to the standard curve. STATISTICALANALYSIS: Data analysed by using descriptive analysis. Independent t-test was used to compare the cotinine concentration between the groups. RESULTS:The mean salivary cotinine level in groups (Group 1 and Group 2) was found to be 20.37 ng/ml and 6.78 ng/ml respectively.. CONCLUSIONS:salivary cotinine level was signicantly high in second hand smokers compared to nonsmokers.

Screening of monoclonal antibodies against specific phosphonylation sites and analysis of serum samples exposed to soman and VX using an indirect competitive enzyme-linked immunosorbent assay.

Organophosphorus nerve agents (OPNAs) covalently bind to tyrosine 411 of human serum albumin (HSA) and the formed adducts are stable biomarkers of OPNA exposure. The detection of these adducts has been limited to mass spectrometry techniques combined with protein digestion. Here, we developed indirect competitive ELISA (icELISA) methods to verify OPNA exposure by the detection of OPNA-phosphonylated adducts at tyrosine 411 residue (OPNA-HSA adducts), in which monoclonal antibodies (mAbs) against phosphonylation sites at tyrosine 411 were introduced. The two mAbs were prepared by the fourth generation of rabbit mAb technology using the phosphonylated peptides of LVRY(GD or VX)TKKVPQC as the haptens. These mAbs were screened using our developed competitive ELISA method and then selected based on their individual affinity and selectivity. As a result, we obtained two mAbs that recognized the HSA Tyr 411 adduct of GD (mAb-5G2) or VX (mAb-12B9), respectively. They shared the highest affinity exhibiting a Kd value of about 10-6mol/L of the OPNA exposure concentration. They also had remarkable selectivity, which could especially recognize their individual OPNA-HSA adducts in a native state but did not recognize other OPNA-HSAs and unadducted HSAs. Especially for mAb-12B9, it could clearly distinguish VX-HSA and GB-HSA between which there was only one alkyl difference in their phosphonyl portion of the adducted sites. The two mAbs were then used to build the icELISA method for analysis of the serum samples exposed to OPNA. It was found that the detectable lowest GD- and VX-exposed concentrations in serum samples were respectively 1.0 × 10-6mol/L and 10.0 × 10-6mol/L. This study provides one novel approach and strategy for the retrospective detection of OPNA exposure, and the two mAbs have great potential to be extended for point-of-care testing of OPNA intoxication.