Antibody-based Immunoassay Research Articles

Detection and partial characterization of norovirus among children with acute gastroenteritis in Lagos, Nigeria

Norovirus (NoV) has captured increasing attention as an agent of childhood diarrhea, but its incidence in developing countries such as Nigeria has been underreported. This study was conducted to investigate the role of NoVs in sporadic cases of acute diarrhea among hospitalized children. One hundred and eighty-eight (188) specimens comprising 161 diarrheic and 27 non-diarrheic stools were randomly selected from 668 stools previously screened for rotaviruses. These specimens were collected from children under 5 years of age who were hospitalized between November 2007 and May 2008 in Lagos, Nigeria. The specimens were examined for NoV antigen using monoclonal antibody-based enzyme immunoassay (EIA), and the positive specimens were further characterized for norovirus genogroups using reverse transcription-Polymerase chain reactions (RT-PCR) technique. NoV was detected in 60/161 (37.3%) of diarrhoeal and 1(3.7%) of non diarrhoeal children tested. Noroviruses were detected throughout the study period with most patients infected in age group 6 - 12 months. More males were infected with NoV than females however, the difference was not significant (χ2 = 0.983, p ≥ 0.05). Sixteen among the positive samples were amplified by PCR using specific primers, of these, 5 (31%) were typed to be GI, 8 (50%) were GII and three samples (19%) showed a mixed infection of norovirus GI and GII genogroups. This study confirmed the relevance of NoV as a causative agent of pediatric diarrhoea and reported norovirus genogroup GII as the predominant type in circulation in Lagos, Nigeria. Key words: Diarrhoea, norovirus, RT-PCR, children, Nigeria.

Hapten Synthesis and Polyclonal Antibody-Based Immunoassay Development for the Analysis of Forchlorfenuron in Kiwifruit

High-affinity polyclonal antibodies directed against the synthetic cytokinin forchlorfenuron (CPPU) were produced from three immunizing haptens with equivalent spacer arms located at different positions. A competitive immunoassay was developed with a limit of detection in buffer of 12.42 +/- 3.06 ng/L. In addition, the ability of the produced antibodies to recognize a set of synthetic CPPU analogues was studied. It was evidenced that the linker position had a strong impact on the specificity of the generated polyclonals, which were more sensitive to changes at moieties of the target analyte located furthest from the derivatization site of the immunogen. Finally, matrix effects of gold and green kiwifruit over assay parameters were evaluated. Excellent recoveries and low coefficients of variation were found with just a 100-fold dilution of the sample in buffer, hence indicating the effectiveness of the developed immunoassay as an analytical tool for monitoring this agrochemical in kiwifruit samples.

A highly sensitive and specific polyclonal antibody-based enzyme immunoassay for therapeutic monitoring and pharmacokinetic studies of atorvastatin

This study describes the development and validation of a highly sensitive and specific enzyme immunoassay (EIA) for therapeutic monitoring and pharmacokinetic studies of atorvastatin (ATR). The assay employs a polyclonal antibody that recognizes ATR with high specificity and affinity, and ATR conjugated to bovine serum albumin (ATR-BSA) immobilized onto microwell plates as a solid phase. The assay involved a competitive binding reaction between ATR and the immobilized ATR-BSA for the binding sites on a limiting amount of the anti-ATR antibody. The bound anti-ATR antibody was quantified with horseradish peroxidase-labeled anti-immunoglobulin secondary antibody and 3,3′,5,5′-tetramethylbenzidine as a substrate for the peroxidase enzyme. The concentration of ATR in the sample was quantified by its ability to inhibit the binding of the anti-ATR antibody to the immobilized ATR-BSA and subsequent color development in the assay wells. The conditions for the EIA were investigated and optimized for the determination of ATR in plasma samples. The limit of detection was 0.04 ng mL−1 and the effective working range at relative standard deviations (RSD) of ≤5% was 0.1–10 ng mL−1. Mean analytical recovery of ATR from spiked plasma was 99.3 ± 2.8%. The precision of the assay was satisfactory; RSD were 2.7–4.6 and 3.3–5.7% for intra- and inter-assay precision, respectively. The reliability of the EIA was confirmed by HPLC. The EIA is convenient, and one can analyze ∼ 200 samples per working day, facilitating the processing of large-number of samples of ATR.

Monoclonal antibody-based fluorescence polarisation immunoassay for nifursol in feed

Monoclonal antibody-based fluorescence polarisation immunoassay for nifursol in feed

A review of analytical methods for the determination of 5-fluorouracil in biological matrices

5-Fluorouracil (5-FU) is a cytostatic agent that has been widely used in the treatment of various solid tumours for more than 20 years, and is still considered to be among the most active antineoplastic agents in advanced colorectal cancer and malignancies of the head and neck. A large number of non-chromatographic and chromatographic methods for the quantitation of 5-FU, related prodrugs and their metabolites in biological matrices have been developed in the last 30 years to support preclinical and clinical studies. However, 5-FU monitoring has not been widely used, at least not in the USA, and certainly not outside the clinical research setting, given the absence of simple, fast and inexpensive testing methods for 5-FU monitoring. Recent developments with testing based on liquid chromatography-tandem mass spectrometry and a nanoparticle antibody-based immunoassay may facilitate routine monitoring of 5-FU in daily clinical practice. In this review the advantages and disadvantages of the bioanalytical methods developed and used for 5-FU, its metabolites and related prodrugs are discussed.

ALP Trends in Men and Women

Bone‐specific Alkaline phophatase (ALP)is found on the surface of osteoblasts and its levels reflect osteoblastic activity. The purpose of this study was to evaluate the concentrations of ALP in 81 men and women (n=20 and 61, respectively) and match these values to their duel x‐Ray absorptiometry (DEXA) findings. Subjects were identified in three age groups (20–50, 51–70 and >71 years of age for men and 18–40, 41–60 and > 60 years for women). ALP was measured in serum with a monoclonal antibody‐based immunoassay (Tandem®‐R Ostase®; Beckman Coulter). ALP values in men were highest in the youngest age category (14 mcg/L) and decreased to 12 mcg/L in the oldest (p<.098; ns). ALP values in women increased from 8 mcg/L in the youngest group to 12.2 mcg/L in the oldest group (p< .001) Our findings did not show a significant difference between the bone mineral density of our subjects. We did, however observe a inverse (and significant, p<.001) trend in the ALP, suggesting that the mechanisms of bone growth and loss may be gender specific.

Current antibody-based immunoassay algorithm failed to confirm three late-stage AIDS cases in China: case report

BackgroundImmunoassays composed of screening and confirmation are the established algorithm to confirm HIV infection in China, with a Western blot result as the final diagnosis.Case presentationIn this report, three late-stage AIDS patients were initially tested HIV antibody positive using multiple screening kits, but tested indeterminate using Western blot. HIV infection diagnosis was confirmed based on nucleic acid assays, clinic manifestations and epidemiological history. Case A was identified positive at 30 months, using Western blot, Case B at 8 months, and case C remained indeterminate until he died of Kaposi's sarcoma 4 months after HAART.ConclusionThe report indicates that current antibody-based testing algorithms may miss late-stage AIDS patients and therefore miss the opportunity for preventing these cases from further transmission. The report also implies that viral load assays is not easy to be universely applicated in developing country like China although it is helpful in diagnosing complicated cases of HIV infection, so the counselling before and after testing is imperative to the diagnosis of HIV infection and risk behavior survey on the examinee should be as detailed as possible.

Determination of Alt a 1 (Alternaria alternata) in poultry farms and a sawmill using ELISA

Farm and sawmill workers are exposed to high levels of allergenic fungi, such as Alternaria alternata, which are associated with respiratory diseases and asthma. The aim of this study was to measure the concentration of Alt a 1, a major allergen of A. alternata, in indoor dust samples collected in poultry farms and a sawmill using a monoclonal antibody-based enzyme immunoassay. A total of 45 dust samples were collected in poultry farms (30) and the sawmill (15) in Zagreb County (Croatia). The Alt a 1 allergen was detected in all dust samples (100%) collected in three poultry farms. The levels of Alt a 1 were in the range of 0.1–14 μg/g, and the median value was 0.37 μg/g. About 86% of dust samples contained Alt a 1 in the range of 0.1–1.0 μg/g. In the sawmill, no detectable level of Alt a 1 was found (limit of detection =0.12 μg/g). This study has shown that occupational exposure to Alt a 1 allergen in poultry farms deserves monitoring.

Pharmacokinetically Guided Dose Adjustment of 5-Fluorouracil: A Rational Approach to Improving Therapeutic Outcomes

Chemotherapy dosing of the fluoropyrimidine 5-fluorouracil (5-FU) is currently based on body surface area. However, body surface area-based dosing has been associated with clinically significant pharmacokinetic variability, and as such, dosing based on body surface area may be of limited use. The clinical activity of 5-FU is modest at standard doses, and in general, dosing is limited by the safety profile, with myelosuppression and gastrointestinal toxicity being the most commonly observed side effects. Various strategies have been developed to enhance the clinical activity of 5-FU, such as biochemical modulation, alterations in scheduling of administration, and the use of oral chemotherapy. Studies that have shown an association between plasma concentration with toxicity and clinical efficacy have shown that pharmacokinetically guided dose adjustments can substantially improve the therapeutic index of 5-FU treatment. These studies have shown that only 20%-30% of patients treated with a 5-FU-based regimen have 5-FU levels that are in the appropriate therapeutic range--approximately 40%-60% of patients are underdosed and 10%-20% of patients are overdosed. To date, 5-FU drug testing has not been widely used because of the lack of a simple, fast, and inexpensive method. Recent advances in testing based on liquid chromatography-mass spectroscopy and a nanoparticle antibody-based immunoassay for 5-FU may now allow for routine monitoring of 5-FU in clinical practice. We review the data on pharmacokinetically guided dose adjustment of 5-FU and discuss the potential of this approach to advance therapeutic outcomes.

Monoclonal Antibody-Based Immunoassay for the Detection of Maduramicin in Chicken Tissues

This paper reports on the preparation of a monoclonal antibody (Mab) against maduramicin (MD) and the development of a simple and sensitive ELISA for MD in chicken tissues. MD was conjugated to bovine serum albumin for immunogens and ovalbumin for coating antigens by mixed anhydride (MA) and active ester (AE) methods. Hybridoma cells were generated using spleen cells from a mouse immunized with MD-BSA conjugate. After screening with ELISA, the Mab with high anti-interference ability and high affinity was selected, and it exhibited negligible cross-reactivity with other usual-used polyether antibiotics. After optimization, the developed ELISA showed an IC50 value of 2.12 ± 0.46 ng/ml (n = 20). Chicken muscle and liver samples were extracted with methanol-8% NaCl solution (4:1) and then directly diluted with PBS-10% methanol for analysis. The recoveries of MD from spiked chicken tissues at levels of 40–480 µg/kg ranged from 81.3% to 91.3% with variation coefficients of 5.2–12.1%, and the detection limits were 6.5 µg/kg in muscle and 9.2 µg/kg in liver, respectively (n = 20).

Comparison of a monoclonal with a polyclonal antibody-based enzyme immunoassay stool test in diagnosing Helicobacter pylori infection after eradication therapy

Recently, a novel Helicobacter pylori stool antigen test (Testmate pylori antigen EIA) using monoclonal antibodies against H. pylori catalase has been developed commercially. This study assessed the diagnostic usefulness of the stool antigen test compared with a polyclonal enzyme immunoassay (HpSA test) after H. pylori eradication. A total of 150 patients with H. pylori infection were treated by triple therapy with PPI and amoxicillin with either clarithromycin or metronidazole. H. pylori stool antigen was tested 4 and 8 weeks after eradication. The outcome of H. pylori eradication was assessed by urea breath test (UBT) 8 weeks after the end of therapy. Discordant results were followed by endoscopic examination. Of 150 patients enrolled, H. pylori status was negative in 122 cases and positive in 28 cases, assessed by the 13C-UBT. On the other hand, the monoclonal stool antigen test results were negative in 126 cases and positive in 24. The polyclonal stool test results were negative in 126 cases and positive in 22. The overall sensitivity and specificity of the monoclonal stool antigen test were 91.6% (95% CI 85.9-97.3%) and 98.4% (95% CI 97.3-99.5%). The overall sensitivity and specificity of the polyclonal stool antigen test were 87.0% (95% CI 86.9-94.0%) and 97.5% (95% CI 96.1-98.9%). The new stool antigen test using monoclonal antibody is useful for the diagnosis of H. pylori eradication 4 weeks after the end of treatment.

Genome-wide association study of plasma lipoprotein(a) levels identifies multiple genes on chromosome 6q

Plasma lipoprotein(a) (Lp[a]) level is an independent risk factor of cardiovascular disease that is under strong genetic control. We conducted a genome-wide association study of plasma Lp(a) in 386 members of a founder population that adheres to a communal lifestyle, proscribes cigarette smoking, and prepares and eats meals communally. We identified associations with 77 single nucleotide polymorphisms (SNPs) spanning 12.5 Mb on chromosome 6q26-q27 that met criteria for genome-wide significance (P <or= 1.3 x 10(-7)) and were within or flanking nine genes, including LPA. We show that variation in at least six genes in addition to LPA are significantly associated with Lp(a) levels independent of each other and of the kringle IV repeat polymorphism in the LPA gene. One novel SNP in intron 37 of the LPA gene was also associated with Lp(a) levels and carotid artery disease number in unrelated Caucasians (P = 7.3 x 10(-12) and 0.024, respectively), also independent of kringle IV number. This study suggests a complex genetic architecture of Lp(a) levels that may involve multiple loci on chromosome 6q26-q27.

Determination of relative assay response factors for toxic chlorinated and brominated dioxins/furans using an enzyme immunoassay (EIA) and a chemically-activated luciferase gene expression cell bioassay (CALUX)

Determination of toxic activity requires knowledge of both the concentration and toxicity to evaluate the risk for adverse human health and environmental effects. A chemically-activated luciferase gene expression cell bioassay system (CALUX) and an antibody-based method enzyme immunoassay (EIA) were used to detect the dioxin-like response of several polybrominated, polychlorinated, and polybrominated/chlorinated dibenzo- p-dioxins/furans (PBDDs/Fs, PCDDs/Fs, and PBCDDs/Fs, respectively). It has been suggested that the biological activity of the brominated and mixed bromo/chloro compounds is similar to their chlorinated analogues (measured by binding to the Ah receptor). PBDD/F, PCDD/F, and PBCDD/F laboratory standards exhibited biological activity ranging over three orders of magnitude. The highest relative potency (REP) values from CALUX analysis, when compared to 2,3,7,8-TCDD, were 2,3,7,8-TBDD at 0.99 (± 0.07), 1,2,3,7,8-PeCDD at 0.69, and 2-Br-3,7,8-TriCDD at 0.72 (± 0.02). Cross-reactivities were calculated using EIA for several PBDDs/Fs and PBCDDs. The highest percent cross-reactivity was found for 2,3,7,8-TBDD at 138 (± 34%), and 2,3,7-TriBDD at 84 (± 36%).

Rotavirus-associated neonatal necrotizing enterocolitis

Purpose : This study aimed to test whether rotavirus-associated necrotizing enterocolitis (RV+NEC) produced different clinical findings or outcomes from those of non-rotavirus necrotizing enterocolitis (RV-NEC). Methods : Eight patients from the RV+NEC group and 22 patients from the RV-NEC group diagnosed with modified Bell stage II or higher NEC were selected for this study. Fecal specimens from all infants were tested for rotavirus infection using a monoclonal antibody-based enzyme immunoassay (EIA). Clinical, radiographic, and clinical outcome data were analyzed retrospectively. Results : RV+NEC infants had a significantly higher birth weight and were born at a significantly higher gestational age (33.53.3 weeks vs. 29.34.4 weeks; P=0.01). There were no differences in the occurrence of thrombocytopenia, mural gas, and pneumoperitoneum between the 2 groups. However, portal vein gas was more common in the RV+NEC group (88% vs. 9%; P

Determination of Alt a 1 (Alternaria alternata) in poultry farms and a sawmill using ELISA

Farm and sawmill workers are exposed to high levels of allergenic fungi, such as Alternaria alternata, which are associated with respiratory diseases and asthma. The aim of this study was to measure the concentration of Alt a 1, a major allergen of A. alternata, in indoor dust samples collected in poultry farms and a sawmill using a monoclonal antibody-based enzyme immunoassay. A total of 45 dust samples were collected in poultry farms (30) and the sawmill (15) in Zagreb County (Croatia). The Alt a 1 allergen was detected in all dust samples (100%) collected in three poultry farms. The levels of Alt a 1 were in the range of 0.1-14 microg/g, and the median value was 0.37 microg/g. About 86% of dust samples contained Alt a 1 in the range of 0.1-1.0 microg/g. In the sawmill, no detectable level of Alt a 1 was found (limit of detection =0.12 microg/g). This study has shown that occupational exposure to Alt a 1 allergen in poultry farms deserves monitoring.

Three Dimensional Quantitative Structure-Activity Relationships of Sulfonamides Binding Monoclonal Antibody by Comparative Molecular Field Analysis

AbstractThe three-dimensional quantitative structure-activity relationship (3D-QSAR) model of sulfonamide analogs binding a monoclonal antibody (MabSMR) produced against sulfamerazine, was carried out by comparative molecular field analysis (CoMFA). The affinities of MabSMR, expressed as Log10IC50, for 17 sulfonamide analogs were determined by competitive fluorescence polarization immunoassay (FPIA). Removal of two outliers from the initial set of 17 sulfonamide analogs improved the predictability of the models. The 3D-QSAR model of 15 sulfonamides resulted in q2cv values of 0.600, and r2 values of 0.995, respectively. This novel study combining FPIA with CoMFA demonstrates that multidisciplinary research can be used as a useful tool to investigate antigen-antibody interactions and provide information required for design of novel haptens, which may result in new antibodies with properties already optimized by an antibody-based immunoassay.

Development of a two-site enzyme immunoassay based on monoclonal antibodies to measure airborne exposure to (1→3)-β- d-glucan

(1→3)-β- d-glucan is found in cell walls of some fungi, bacteria and plants. It plays a crucial role in bioaerosol-induced inflammatory reactions. To estimate the level of airborne (1→3)-β- d-glucan exposure, a monoclonal antibody-based two-site enzyme immunoassay (mAb-EIA) was developed. The results obtained with the mAb-EIA were compared with the results of a Limulus amoebocyte lysate-based assay for (1→3)-β- d-glucan. Three mAbs produced by mouse immunization with bovine serum albumin-conjugated laminarin were enriched by in vitro production in a modular minifermenter and affinity purified. Two mAbs were selected for the development of a two-site EIA specific for (1→3)-β- d-glucan. Different polysaccharides, fungal and plant seed extracts, and airborne inhalable dust from workplaces (poultry farms, pig stables, grain storage houses, and a laboratory animal facilility) were sampled with portable pumps and measured with both the mAb-EIA and Glucatell ® assay. Using carboxymethylated curdlan as a standard, the mAb-EIA gave a steep dose-response curve for concentrations between 0.36–15 ng/ml. The mAb-EIA was specific for (1→3)-β- d-glucan and was sufficiently sensitive to detect (1→3)-β- d-glucan in airborne dust samples. In comparing the EIA results to the values obtained with the Glucatell ® assay, the correlation was found to be high (coefficient of correlation r 2 = 0.91), and the mean ratio of the values was 1.7. Depending on the dust source, either the Glucatell ® assay or the mAb-EIA gave higher results. The mAb-EIA is sensitive enough to detect (1→3)-β- d-glucan in airborne dust samples collected with portable pumps. Thus, the assay is suited for the investigation of the health effects induced by exposure to this class of biologically active molecules.

Simultaneous determination of sulphamerazine, sulphamethazine and sulphadiazine in honey and chicken muscle by a new monoclonal antibody-based fluorescence polarisation immunoassay

A new monoclonal antibody (Mab) against sulphamerazine (SMR) was produced and a fluorescence polarisation immunoassay (FPIA) based on the Mab was developed and optimized for the simultaneous qualitative screening of SMR, sulphamethazine (SMZ) and sulphadiazine (SDZ). The Mab, raised from mice immunized with SMR, was bound to bovine serum albumin (BSA) using glutaraldehyde as the coupling reagent. Fluorescein-labelled SMR and SMZ (tracer) were synthesized and purified by thin layer chromatography (TLC). Cross-reactivities below 3.6% were displayed in the optimized FPIA for another 14 sulphonamides when both tracers were employed. The limits of detection (LOD) were 0.9 ng g−1 for SMR, 2 ng g−1 for SMZ and 3.1 ng g−1 for SDZ. Analysis of SMR, SMZ and SDZ fortified chicken muscle and honey samples by the FPIA showed average recoveries of 86–131% with a standard deviation (SD) of 4.6–32. Comparative analyses of a SMZ-treated chicken muscle sample by both FPIA and high performance liquid chromatograph (HPLC) showed a good correlation (r = 0.9991). The study demonstrates the practical application of FPIA in screening chicken muscle and honey samples for sulphonamides residues.

Hapten Synthesis and Monoclonal Antibody-Based Immunoassay Development for Detection of the Fungicide Trifloxystrobin

High-affinity and selective monoclonal antibodies have been produced against the strobilurin fungicide trifloxystrobin. A battery of functionalized haptens has been synthesized, and conjugate-coated enzyme-linked immunosorbent assays following different procedures have been developed. On the one hand, a two-step conjugate-coated immunoassay was optimized using extended or short incubation times, with limits of detection of 0.10 ng/mL for the extended assay and 0.17 ng/mL for the rapid assay. On the other hand, an immunoassay in the conjugate-coated format was optimized following a procedure consisting of just one incubation step. This one-step assay had a limit of detection of 0.21 ng/mL. All of these assays showed detection limits for trifloxystrobin in the low parts per billion range, well below the common maximum residue limits for this pesticide in foodstuffs (50 microg/kg).

Hapten Synthesis and Monoclonal Antibody-Based Immunoassay Development for the Detection of the Fungicide Kresoxim-methyl

Strobilurin fungicides have been increasingly used for fungus pest control since they were introduced in 1996. For pesticide residue detection, immunoassays constitute nowadays a valuable approach. This paper describes the synthesis of functionalized haptens of kresoxim-methyl, the production of monoclonal antibodies, and the development of enzyme-linked immunosorbent assays. On the one hand, a two-step conjugate-coated immunoassay was optimized using extended or short incubation times, with limits of detection of 0.4 ng/mL for the extended assay and 0.3 ng/mL for the rapid assay. On the other hand, an immunoassay was optimized following a procedure consisting of just one incubation step. This one-step assay had a limit of detection of 0.4 ng/mL. All of these assays showed a similar performance, with sensitivities well below common maximum residue limits for this pesticide (50 microg/kg) and lower than the detection limits of the usual chromatographic detection methods.