Polyclonal Antibody-based Enzyme-linked Immunosorbent Assay Research Articles

Polyclonal antibody-based indirect competitive enzyme-linked immunosorbent assay for screening of paclobutrazol in fruits

Paclobutrazol (PBZ) is a plant growth regulator (PGR) widely used in fruit and vegetable cultivation. However, due to the severe toxicity of PBZ, a sub-ppm level maximum residue limit (MRL) was established worldwide. Therefore, it is significant to propose a rapid, sensitive and high throughput screening method for monitoring the PBZ residues in foods. In this study, a simple and sensitive indirect competitive Enzyme-linked immunosorbent assay (icELISA) was established for PBZ detection in fruits basing polyclonal antibody. For both economy and pollution prevention, a microwave-solvent-free method was used to synthesize the PBZ hapten with high efficiency. The detection conditions, such as coating antigen concentration, antibody concentration, organic reagent concentration, ionic strength and pH, were optimized. Under the optimized conditions, this method showed high sensitivity and specificity. The detection range is 1.27-138.23 ng/mL, half-maximum inhibition concentration (IC50) is 13.26 ng/mL, and the IC20 was lower than the reported ELISAs for PBZ. Additionally, this method had high accuracy and precision. The recoveries were ranged from 88.78% to 96.80% in PBZ spiked apple samples with RSD below 4%. All the results showed that the polyclonal antibody based icELISA could be useful for PBZ screening in fruit samples.

Development of a polyclonal antibody-based indirect competitive ELISA for determination of sterigmatocystin in wheat and corn flours

ABSTRACTSterigmatocystin (STC) is a toxic secondary metabolite produced by more than 50 fungal species, including Aspergillus flavus, A. parasiticus, A. nidulans, and A. versicolor. The Joint FAO/WHO Expert Committee on Food Additives concluded that sterigmatocystin is genotoxic and carcinogenic with the critical effect determined to be carcinogenicity. The present study describes a simple method to prepare hapten and immunogens in order to generate polyclonal antibodies against this metabolite. We developed a sensitive and specific polyclonal antibody-based competitive indirect enzyme-linked immunosorbent assay (ciELISA) for monitoring STC in wheat and corn flours without the need for derivatisation of STC or clean-up of samples by immunoaffinity chromatography for quantification. The half inhibitory concentration (IC50) of the established method was 4.52 ± 0.81 ng mL−1, with the limit of detection (IC10) being 0.19 ± 0.04 ng mL−1 in wheat and corn flour matrices with the coefficient of variation of less than 22%.The assay was very specific to STC and showed no cross-reactivity with its analogue structures. The ELISA allowed for up to 5% methanol without significant influence on the IC50 value. Validation of the assay was performed by spiking STC into a blank flour matrix and the recoveries were in the range of 75.3 % to 104.5 % with a coefficient of variation less than 15%. A small retail survey was conducted by purchasing wheat (n = 8) and corn flours (n = 2) from local grocery stores. All of these retail samples were negative for STC using the developed ELISA method and were confirmed by LC-MS/MS. We demonstrated a rapid, simple, and reliable method for screening STC in wheat and corn flours.

Rapid screening of flonicamid residues in environmental and agricultural samples by a sensitive enzyme immunoassay

A fast and sensitive polyclonal antibody-based enzyme-linked immunosorbent assay (ELISA) was developed for the analysis of flonicamid in environmental and agricultural samples. Two haptens of flonicamid differing in spacer arm length were synthesized and conjugated to proteins to be used as immunogens for the production of polyclonal antibodies. To obtain most sensitive combination of antibody/coating antigen, two antibodies were separately screened by homologous and heterologous assays. After optimization, the flonicamid ELISA showed that the 50% inhibitory concentration (IC50 value) was 3.86mgL−1, and the limit of detection (IC20 value) was 0.032mgL−1. There was no cross-reactivity to similar tested compounds. The recoveries obtained after the addition of standard flonicamid to the samples, including water, soil, carrot, apple and tomato, ranged from 79.3% to 116.4%. Moreover, the results of the ELISA for the spiked samples were largely consistent with the gas chromatography (R2=0.9891). The data showed that the proposed ELISA is an alternative tool for rapid, sensitive and accurate monitoring of flonicamid in environmental and agricultural samples.

A sensitive monoclonal antibody-based enzyme-linked immunosorbent assay for the detection of bifenthrin in a chemical soil barrier

A sensitive monoclonal antibody-based enzyme monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) was developed to detect bifenthrin in a chemical soil barrier for termite control. LBc ((2-methyl[1,1-biphenyl]-3-methoxy) carbonyl propionic acid) was conjugated to bovine serum albumin (BSA) for producing monoclonal antibody. After optimization, the IC50 and limit of detection (LOD, IC10) were 0.05mgL−1 and 0.004mgL−1, respectively. The sensitivity was improved 40-fold compared to polyclonal antibody-based ELISA reported earlier. No cross-reactivity was measured for the other analogues such as cyhalothrin, cyhalothric acid, cypermethrin, deltamethrin, fenpropathrin, fenvalerate, ethofenprox and tetramethrin except 2-methyl-3-biphenylmethyanol. Spiked recoveries were between 83.5% and 104.7% for the detection of bifenthrin in loess, red soil and black soil. All the relative standard deviations (RSDs) were less than or equal to 15.0%. Moreover, the ELISA for authentic samples showed reliability and high correlation with gas chromatography. The developed ELISA is an alternative tool for simple, sensitive and accurate monitoring of bifenthrin in chemical soil barriers.

Development of enzyme-linked immunosorbent assay (ELISA) for glutathione S-transferase (GST-S) protein in the intertidal copepod Tigriopus japonicus and its application for environmental monitoring

To utilize the GST-S protein as a useful biomarker for environmental contamination, we developed a polyclonal antibody-based enzyme-linked immunosorbent assay (ELISA) in the intertidal copepod Tigriopus japonicus. Two polyclonal antibodies, TJ-GST-S1 and TJ-GST-S2, were raised against two TJ-GST-S synthetic peptides. Also a recombinant TJ-GST-S protein was purified as a standard for ELISA development. Each polyclonal antibody was tested by Western blot analysis and indirect ELISA. Of two polyclonal antibodies, TJ-GST-S2 ELISA was further employed due to its wide range of detection and the limit of specificity compared to those of TJ-GST-S1 ELISA system. After exposure to 4 metals (Ag, As, Cd, and Cu) to T. japonicus, the amount of TJ-GST-S protein was significantly elevated in a concentration-dependent manner. Also, TJ-GST-S protein was upregulated at relative high concentrations of B[α]P, PCB, and TBT. In this paper, we suggest that T. japonicas ELISA for TJ-GST-S2 is useful as a potential indicator system for marine contaminants.

Highly selective and sensitive determination of deoxymiroestrol using a polyclonal antibody-based enzyme-linked immunosorbent assay

Pueraria candollei-associated products are of interest to worldwide consumers for their rejuvenating and cosmetic purposes. In addition, clinical trials have supported the beneficial effects of P. candollei on the alleviation of menopausal symptoms. Deoxymiroestrol, which was reported as the most potent phytoestrogen in the tuberous root of P. candollei, exhibited potential as a biomarker of P. candollei-derived samples and products. A polyclonal antibody-based enzyme-linked immunosorbent assay (ELISA) was developed for deoxymiroestrol determination. The raised antibody bound specifically to deoxymiroestrol with very low cross reactivities of 1.26 and 0.42% to structurally related miroestrol and isomiroestrol, respectively. The linear range was 0.73–3000.00ngmL−1, and the coefficients of variation for both the intra- and inter-plate determinations were less than 5%. In samples spiked with a known amount of deoxymiroestrol, the recoveries were 99.82–102.58% in P. candollei samples and 98.07–106.33% in its products samples. In comparison with other analytical methods, the validated ELISA was comparable to published HPLC-UV methods for samples with high deoxymiroestrol content (R2=0.9993). Furthermore, ELISA can be used for samples with deoxymiroestrol concentrations that are too small to detect by HPLC and for conditions when other chemicals cause interference with chromatographic analysis. For the P. candollei-derived products, the preparations contained 0.154–10.998µgg−1 dry wt. Our ELISA successfully measured deoxymiroestrol content with high sensitivity, selectivity, accuracy and rapidity. Therefore, this ELISA showed potential for dosage standardization of P. candollei-associated samples.

Development of a polyclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for detection of Sunset Yellow FCF in food samples

Sunset Yellow FCF is widely used as food additives to make foods more attractive. Due to its abuse and potential risk to human health, Sunset Yellow FCF is precisely limited to use in food. To monitor the illegal use of Sunset Yellow FCF, a polyclonal antibody-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) with satisfactory sensitivity and specificity was developed. A carboxyl group was introduced to Sunset Yellow FCF, then the modified hapten was coupled with carrier proteins to synthesize the immunogen and coating antigen. The IC50 value of 0.52ngmL−1 and detection limit of 25pgmL−1 (in buffer) were achieved by this method. The cross-reactivity values of the antibodies with six structurally related colorants were less than 1.5%, indicating the high selectivity. Three kinds of food samples (beverage, dried beancurd, braised pork) and serum were chosen to evaluate the application of the immunoassay in real systems. The limits of detection (LOD) in the above three food samples were 0.12, 0.04 and 1.11, respectively (mean+3SD). The recovery (94%–106%), intra-assay (<5%) and inter-assay (<12%) coefficients of variation in foods and serum samples were also acceptable. The results suggest that this ELISA method is a specific, sensitive and simple method for the determination of Sunset Yellow FCF additives.

Highly sensitive and specific detection of neonicotinoid insecticide imidacloprid in environmental and food samples by a polyclonal antibody‐based enzyme‐linked immunosorbent assay

Imidacloprid is one of the main neonicotinoid insecticides widely used in agriculture owing its broad spectrum of activity and low bioaccumulation. However, imidacloprid is toxic to honey bees and other beneficial organisms, and its residues may occur in environmental and food samples, posing a potential hazard to consumers. In this study the imidacloprid derivative bearing a three-atom length spacer was synthesized and coupled to carrier proteins. Highly sensitive and specific polyclonal antibodies against imidacloprid were successfully produced and the polyclonal antibody-based enzyme-linked immunosorbent assay (pAb-ELISA) was developed. The ELISA standard curve was constructed within the concentration range 0.1-100 ng mL(-1). The IC(50) value for nine standard curves was in the range 1.2-3.0 ng mL(-1) and the limit of detection was 0.03-0.16 ng mL(-1). The sensitivity of the assay was one order of magnitude higher than that in most published papers. There was almost no cross-reactivity of the antibody with four structurally related compounds (acetamiprid, nicotine, clothianidin and nitenpyram) and six other compounds, indicating that the assay displays not only high sensitivity but also high specificity. No detectable imidacloprid was found in 11 collected environmental and food samples by the assay. For imidacloprid-spiked samples, acceptable recoveries of 73.4-94.4% and intra-assay coefficients of variation of 2.2-12.8% were obtained. The assay was also validated with high-performance liquid chromatography (HPLC) and a good correlation of ELISA with HPLC was achieved. The proposed ELISA provides a sensitive, specific, simple and cost-effective quantitative/screening method for detecting imidacloprid in environmental and food samples.

Utility of Fecal Lactoferrin in Identifying Crohn Disease Activity in Children

Fecal lactoferrin (FL) is a noninvasive biomarker that is elevated in Crohn disease (CD) compared to irritable bowel syndrome. The purpose of this study was to evaluate FL in identifying children with active versus inactive CD. Fresh stool samples were collected from children with CD scheduled for endoscopy or a clinic visit, and from new outpatients who were scheduled for colonoscopy. FL was determined using a polyclonal antibody-based enzyme-linked immunosorbent assay. Physical global assessment, endoscopic findings, erythrocyte sedimentation rate (ESR), and the Pediatric CD Activity Index (PCDAI) were recorded for patients with CD. The PCDAI scores symptoms, laboratory parameters, physical examination, and extraintestinal manifestations. A score of ≤10 is inactive disease, 11 to 30 is mild active, and ≤31 is moderate to severe active. Of 101 study patients (4- to 20-year-old, 66 boys), 31 had active CD, 23 had inactive CD, and 37 had noninflammatory bowel disease (non-IBD) conditions. Four patients with ulcerative colitis and 6 patients with polyposis were excluded from analysis. FL was significantly elevated in CD versus non-IBD (P < 0.001) and in active versus inactive CD (P < 0.001). The PCDAI and ESR were higher in active CD than in inactive CD (both P < 0.001). Using an FL cutoff of 7.25 μg/g, FL has 100% sensitivity and 100% negative predictive value in detecting active CD. Using an FL cutoff level of 60 μg/g, FL had 84% sensitivity, 74% specificity, 81% positive predictive value, and 77% negative predictive value for detecting active CD. FL is a promising biomarker of active CD and may be more practical to use when it is not feasible to obtain all of the necessary clinical information for the PCDAI.

A sensitive and specific enzyme-linked immunosorbent assay for the detection of lincomycin in food samples

Lincomycin (LIN) is an antibiotic widely used in veterinary medicine to cure infections caused by Gram-positive pathogens. Although the toxicity of LIN is not serious, it will cause adverse effects in humans, such as pseudomembranous enteritis and bacterial resistance. In this study, for the preparation of a LIN derivative, a novel modification method was adopted. The LIN derivative modified at 2-position with a carboxylic group at the end of the spacer was synthesised and coupled to carrier proteins. A LIN polyclonal antibody-based enzyme-linked immunosorbent assay (ELISA) was developed and characterised. The ELISA standard curve was constructed with concentrations of 0.1-1000 ng mL(-1). The IC(50) value for nine standard curves was in the range 23.7-29.3 ng mL(-1) and the limit of detection at a signal-to-noise ratio of 3 was 0.15-0.98 ng mL(-1). The cross-reactivity value of the LIN antibody with clindamycin hydrochloride, a homologue of LIN with similar molecular structure, was 18.9%, while less than 0.1% cross-reactivity was found with seven other compounds. For LIN-spiked food samples, the recoveries and relative standard deviation (RSD) were 76.6-117.6% and 1.7-34.6%, respectively. The proposed ELISA can be utilised as a sensitive and specific analytical tool for the detection of LIN in food samples.

This Month in Gastroenterology

This Month in Gastroenterology

A highly sensitive and specific polyclonal antibody-based enzyme-linked immunosorbent assay for detection of antibiotic olaquindox in animal feed samples

The use of olaquindox (OLA) as an additive in animal feedstuffs has been prohibited in the European Union and many other countries. In this study, a highly sensitive and specific indirect competitive enzyme-linked immunosorbent assay (ELISA) for determination of OLA in animal feed samples was developed. OLA was activated by N'N-carbonyldiimidazole and coupled with bovine serum albumin (BSA) and ovalbumin (OVA). It was found that the sensitivity and specificity of the two antisera were very similar, with the IC(50) values of 16 ng mL(-1) and 19 ng mL(-1), respectively. Cross-reactivity was less than 35% for four structurally related compounds and no recognition of five other antibiotics was observed. The better antiserum I was selected for further experiments, for example testing stability, solvent effect, accuracy, and precision. The IC(50) value for eight standard curves was in the range 12-18 ng mL(-1) and the LOD at a signal-to-noise ratio of 3 (S/N = 3) was 0.31 +/- 0.11 ng mL(-1). The ELISA tolerated 5% methanol without significant influence on IC(50) value. The recoveries of spiked OLA in five different animal feed types including auxin, pig complex feed, fish complex feed, broiler concentrated feed, and pig premix feed were in the range 88.3-119.0% and the intra-assay relative standard deviation (RSD) was within 4.7-33.5% (n = 3). The ELISA for unspiked feed samples was confirmed by high-performance liquid chromatography (HPLC), with a high correlation coefficient of 0.9862 (n = 5). The proposed ELISA could be a feasible quantitative/screening method for OLA analysis in feed samples with the properties of high sensitivity, specificity, simplicity of sample pretreatment, high sample throughput, and low expense.

Ascitic Fluid Lactoferrin for Diagnosis of Spontaneous Bacterial Peritonitis

The diagnosis of spontaneous bacterial peritonitis (SBP) is based on a manual count of ascitic fluid polymorphonuclear cells (PMNs). This procedure is operator-dependent and lysis of PMNs during transport to the laboratory may lead to false-negative results. Furthermore, ascitic fluid culture is insensitive and leads to delays in diagnosis. The aim of this study was to assess the utility of ascitic fluid lactoferrin (AFLAC) for the diagnosis of SBP and to identify a cut-off level that can be used for future development of a rapid bedside test. A total of 218 consecutive ascites samples from 148 patients (1-8 samples per patient) with cirrhosis at 2 tertiary care medical centers were examined for PMN count, bedside culture, and lactoferrin concentration. AFLAC concentrations were determined using a polyclonal antibody-based enzyme-linked immunosorbent assay. An ascitic fluid PMN count of 250 cells/mL or greater with or without a positive culture was used for diagnosis of SBP. Twenty-two (10.1%) samples fulfilled diagnostic criteria for SBP. Samples with SBP had a significantly higher lactoferrin concentration (median, 3744 ng/mL; 25th-75th percentiles [P25-P75], 788-9617) compared with non-SBP samples (median, 31 ng/mL; P25-P75, 12-67; P < .001). By using a cut-off level of 242 ng/mL, the sensitivity and specificity of the assay for diagnosis of SBP were 95.5% and 97%, respectively. The area under the receiver operating characteristic curve was 0.98. AFLAC can serve as a sensitive and specific test for diagnosis of SBP. Qualitative bedside assays for the measurement of AFLAC can be developed easily and may serve as a rapid and reliable screening tool for SBP in patients with cirrhosis.

The Importance of Antibody Cross-Reactivity for the Development of Environmental Immunoassays for Alternaria Alternata

Alternaria alternata is one of the most common fungi indoors and outdoors and exposure to the fungus has been linked to the severity of asthma and the pathophysiology of chronic rhinosinusitis. A recent national survey of house dust samples concluded that 95-99% of American homes contained detectable amounts of Alternaria antigens when analyzed with a polyclonal antibody-based enzyme-linked immunosorbent assay (ELISA). Polyclonal antibodies (pAbs) can lack assay specificity and in this study we investigated the cross-reactivity of the pAbs to other common fungal contaminants. Reactivity to 24 fungal species typically found in indoor environments was analyzed by inhibition ELISA. The pAbs were also tested by immunoblotting and Halogen immunoassay (HIA) for a subgroup of fungi. Spores of 6 fungi including species of Alternaria, Ulocladium, Stemphylium, Epicoccum, Drechslera and Exserohilum were found to react with the pAbs more strongly than A. alternata when tested by ELISA. Six other fungi reacted in the ELISA at a lower level and 11 fungal species including several Penicillium, Aspergillus, Fusarium and Cladosporium species were not found to inhibit pAb binding. The immunoblots and the HIA staining confirmed that the pAbs react with numerous fungi commonly found in US homes. Due to extensive cross-reactivity, the ELISA prevalence data previously reported for A. alternata should be considered to represent fungal-specific antigens rather than be interpreted as exclusive contamination with A. alternata alone. The characterization of antibody specificity before application is critical to avoid analytical bias and ambiguities in data interpretation.

Development of polyclonal ELISA for the N-methylcarbamate insecticide bendiocarb

A polyclonal antibody-based enzyme-linked immunosorbent assay (ELISA) method was developed for the N-methylcarbamate insecticide bendiocarb (2,2-dimethyl-1,3-benzodioxol-4-yl methylcarbamate). Two novel haptens having dimethylbenzodioxyl and dimethylbenzofuranyl groups connected to oxyacetyl-γ-aminobutanoic acid and oxyacetyl-β-alanine spacer arm respectively were synthesised. The first hapten was conjugated to carrier proteins to make antigens that were used to raise polyclonal antibodies in rabbits. The antibodies specifically recognised bendiocarb and its metabolite 2,2-dimethyl-1,3-benzodiox-4-ol with an IC50 value of 9 ppb (ng ml−1). The assay was standardised using the competitive ELISA format at 0.0625 µg antibody concentration and at 1/10k pesticide-HRP dilution. Matrix effect studies were carried out in four vegetable and cereal food samples. Matrix effect elimination in cabbage, cauliflower and rice was achieved by simple dilution of the extract. Five different approaches were attempted to achieve matrix clean up in paddy rice. C-18 column and gel permeation column chromatography (GPC) helped in the matrix removal. The spike and recovery studies for all the four food samples gave a recovery in the range of 75–95%, thus indicating the efficiency of the matrix elimination procedures developed.

Fecal pancreatic elastase: a reproducible marker for severe exocrine pancreatic insufficiency

In order to apply fecal pancreatic elastase for follow-up of exocrine pancreatic function in chronic pancreatitis and cystic fibrosis, we examined the sensitivity, specificity, and long-term variability of a new polyclonal antibody-based enzyme-linked immunosorbent assay (ELISA). Patients with definite chronic pancreatitis (n = 23), probable or possible chronic pancreatitis (n = 14), autoimmune pancreatitis (n = 7), or acute pancreatitis (n = 11), and 51 healthy subjects and 11 healthy infants participated in this study. Pancreatic function was graded as normal (n = 3), mild (n = 18), moderate (n = 9), or severe (n = 18) exocrine insufficiency on the basis of secretin tests. Fecal pancreatic elastase was measured by a new ELISA. Fecal pancreatic elastase concentration in control subjects varied widely, with a median of 478 microg/g. The specificity of this test was 90.2% with a cutoff value of >200 microg/g. The sensitivities were 60.9% for detecting definite chronic pancreatitis, 76.5% for calcifying pancreatitis, 71.4% for autoimmune pancreatitis, and 7.1% for probable or possible chronic pancreatitis. The sensitivities were 16.7% for mild, 12.5% for moderate, and 72.2% for severe exocrine pancreatic insufficiency. Forty patients were reexamined after a median interval of 347 days. The fecal pancreatic elastase levels between the first and second tests were not significantly different. Two infants, 4.5 and 5 months old, had abnormally low values, but after a median of 304 days all infants showed normal levels (median, 444 microg/g). Fecal pancreatic elastase is a reproducible marker for severe exocrine pancreatic insufficiency. This test is valuable for longitudinal follow-up of exocrine pancreatic function.

Urinary type II collagen helical peptide (HELIX‐II) as a new biochemical marker of cartilage degradation in patients with osteoarthritis and rheumatoid arthritis

Type II collagen, which consists of a large helical domain and telopeptides at each end, is the most abundant protein of cartilage matrix. The aim of this study was to develop a biochemical marker reflecting the degradation of the helical region of type II collagen and to evaluate its clinical performance in patients with osteoarthritis (OA) and rheumatoid arthritis (RA). We developed a competitive polyclonal antibody-based enzyme-linked immunosorbent assay (ELISA) using the 622-632 peptide derived from the sequence of the alpha1 chain of human type II collagen (HELIX-II) as immunogen and standard. We measured urinary levels of HELIX-II peptide and C-terminal crosslinking telopeptide of type II collagen (CTX-II) in 90 patients with knee OA (73% women; mean +/- SD age 63.0 +/- 8.0 years, mean +/- SD disease duration 6.1 +/- 6.8 years), 89 patients with early RA (disease duration </=3 years) (79% women; mean +/- SD age 48.7 +/- 11.6 years), 25 patients with Paget's disease of bone (HELIX-II only), and 162 healthy controls. In RA patients, we investigated the relationships between baseline urinary HELIX-II and CTX-II levels and the progression of joint destruction as measured by the changes in the total Sharp score (average from 2 independent readers) over 1 year. The intraassay and interassay variations of the HELIX-II ELISA were lower than 13% and 15%, respectively. The HELIX-II ELISA showed no significant cross-reactivity with human intact or denatured type II collagen, with the homologous peptides from human type I or type III collagens, or with HELIX-II peptides elongated (by 1 amino acid) or shortened (by 1 or 2 amino acids) at the C-terminal end, indicating that the HELIX-II ELISA recognized a neoepitope from the alpha1 chain of type II collagen. Median urinary HELIX-II levels were increased in patients with knee OA (by 56%; P < 0.0001) or early RA (by 123%; P < 0.0001) compared with those in age- and sex-matched healthy controls. Baseline urinary HELIX-II levels in the highest tertile were associated with an increased risk of radiographic progression in RA (increase in the total Sharp score >/=0.5 units/year), with an odds ratio (OR) of 5.9 (95% confidence interval [95% CI] 2.0-17.2) after adjustment for serum C-reactive protein (CRP) levels and baseline joint damage. Patients with increased levels of both urinary HELIX-II and CTX-II had the highest risk of progression (OR 17.5 [95% CI 3.1-99]). The HELIX-II ELISA is specific for type II collagen degradation, has adequate technical performance, and can distinguish patients with knee OA or RA from healthy controls. Elevated HELIX-II levels are associated with increased risk of radiographic progression in RA independently of CRP levels, baseline joint damage, and urinary CTX-II levels. The HELIX-II ELISA should be useful for the clinical investigation of patients with arthritis and for identifying RA patients at higher risk of progression.

Development and optimization of an indirect enzyme-linked immunosorbent assay for 4-nitrophenol. Application to the analysis of certified water samples

A polyclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for the detection of 4-nitrophenol (4-NP) was developed, optimized and validated for the analysis of this polar compound in water samples. The concentrations of the immunoreagents were selected to provide the highest sensitivity on an indirect ELISA format. Moreover, the influence of several physico-chemical parameters, such as incubation time, ionic strength, detergent concentration and pH were also studied. The IC 50 and limit of detection (LOD) of the optimized assay As34/3-BSA (antiserum 34 and hapten 3 conjugated to bovine serum albumin) were 9.32 and 0.61 μg l −1, respectively. The specificity of this immunoassay was also evaluated by using eleven phenolic compounds, four different structurally related organophosphorus pesticides and a surfactant. Two kinds of sample matrices (Milli-Q and river water) were evaluated for possible interferences. A good correlation between the analysis by conventional techniques and by immunoassay was obtained, when five certified water samples were analyzed.

Development of Improved Immunoassay and HPLC Methods for the Analysis of Chlorodiamino-s-triazine in Environmental Samples

An enzyme-linked immunosorbent assay (ELISA) for the analysis of chlorodiamino-s-triazine (CAAT) was developed using specific rabbit polyclonal antibodies. The assay was sensitive in the submicromolar range. The method was more sensitive and specific than previously reported mouse polyclonal antibody-based ELISAs. A competitive inhibition haptenated enzyme ELISA was optimized and validated which showed that the method was accurate, precise, and reproducible. A new HPLC method was developed to resolve polar s-triazine metabolites with improvements in run time and solvent use. The ELISA method was more sensitive than the HPLC method for detecting CAAT in soil and ground water samples. Solid-phase extraction was evaluated in an attempt to further increase the sensitivity of the ELISA method. However, nonspecific interferences were observed from various types of strong cation exchange columns from different manufacturers. This made it difficult to couple a solid-phase extraction technique with the ELISA. Keyw...

ELISA screening of staphylococcal enterotoxins by means of a specially developed test kit

The development of a new test kit for the detection of staphylococcal enterotoxins is described. This polyclonal antibody-based enzyme-linked immunosorbent assay (ELISA) is designed for qualitative and quantitative rapid detection of toxin serotypes A, B, C (subgroup 1), D and E in one test system. Results are obtained in 60 min. The lower limit of detection is 0.5 ng/ml; the precision is 90%.