Enzyme-linked Immunosorbent Assay For Determination Research Articles

Development of a monoclonal antibody-based enzyme-linked immunosorbent assay for determination of vomitoxin (DON) in Coicis Semen

Coicis Semen is a common bulk medicinal material used for both medicine and food, which has the effect of promoting diuresis, draining dampness, invigorating the spleen and checking diarrhea. It is derived from Coix lacryma-jobi var. ma-yuen of the family Poaceae, and is easily contaminated by fungi such as Fusarium graminearum and F. flavum due to climate reasons to produce vomitoxin. The guiding principles for determination of vomitoxin in Chinese medicinal materials in Chinese Pharmacopoeia are mainly HPLC and LC-MS, which have long detection period and are time-consuming and laborious, and thus cannot meet the requirements of on-site quality inspection of drugs. The complete antigen of vomitoxin-protein was obtained by chemical derivatization of vomitoxin. The monoclonal antibody against vomitoxin was prepared by classic monoclonal antibody preparation technology, and enzyme-linked immunosorbent assay(ELISA) method for detection of vomitoxin in Coicis Semen was established through methodological investigation. The IC_(50) based on the ELISA for vomitoxin in Coicis Semen was 3.88 μg·L~(-1), and the average recoveries and the RSD were 77.32%-93.73% and 4.6%-9.7%, respectively. With the established ELISA method, the vomitoxin residue in 14 batches of Coicis Semen samples were determined and validated by LC-MS, and the correlation between the two assays was found to be 0.997 8, indicating that the established ELISA method could be used for quantitative determination of vomitoxin residue in Coicis Semen and could achieve the rapid quantitative determination of the vomitoxin residue.

Development of a Highly Sensitive Biotin-Streptavidin Amplified Enzyme-Linked Immunosorbent Assay for Determination of Progesterone in Milk Samples

Development of a Highly Sensitive Biotin-Streptavidin Amplified Enzyme-Linked Immunosorbent Assay for Determination of Progesterone in Milk Samples

A quality control system for ligand-binding assay of plasma renin activity: Proof-of-concept within a pharmacodynamic study

While the role of plasma renin activity (PRA) in heart failure has been widely studied in adults, comprehensive data on pediatric heart failure remain lacking. This drawback is increasingly being addressed by academic research. Nevertheless, such pediatric investigations are commonly conducted only once due to ethical constraints. Therefore, the quality of bioanalytical data must be ensured to acquire meaningful insights into maturing humoral parameters. However, appropriate post-validation assessment of bioanalytical runs is currently underrepresented by regulatory guidance. Thus, for applications in an academic environment, an easy-to-handle six-step bioanalytical quality control system was designed based on regulatory guidelines (e.g. U.S. Food and Drug Administration) combined with international recommendations (e.g. Clinical and Laboratory Standards Institute) and current scientific discussion. Its applicability to an enzyme-linked immunosorbent assay for determination of PRA was investigated within three pediatric trials of the EU-funded “Labeling of Enalapril in Neonates up to Adolescents” project. This quality control system identified 15 % bioanalytical runs as non-compliant to the predefined specifications and ensured the reliable quantification of 940 pharmacodynamic samples. The inter-run assessment of quality controls was able to demonstrate the comparability of the study results. Furthermore, 86 % of incurred sample reanalysis pairs complied with regulatory requirements (>67 %), thus underlining the long-term reproducibility of the utilized ligand-binding assay. Successful participation in interlaboratory testing confirmed the accuracy of the applied method throughout the entire study period. Further investigations showed no notable differences between the five applied lots of the PRA assay. The applicability of this quality control system was proven in an academic environment and ensured reliable results for PRA over the entire 24-month study period.

Development of a direct competitive enzyme-linked immunosorbent assay for determination of the fungicide mepanipyrim and its metabolite.

A direct competitive enzyme-linked immunosorbent assay (dc-ELISA) was developed for determination of anilinopyrimidine fungicide mepanipyrim in vegetables. Two derivatives of mepanipyrim and mepanipyrim propanol type metabolite which carried carboxy acid were synthesized and conjugated with keyhole limpet hemocyanin. BALB/c mice were immunized to prepare anti-mepanipyrim monoclonal antibodies (MoAbs) by obtained conjugates. The dc-ELISAs based on the prepared MoAbs, MPP107 and MPP204, showed working ranges between 0.12 and 1.8 ng/mL with mepanipyrim for MPP107, 0.12 and 2.4 ng/mL with mepanipyrim for MPP204, and 0.2 ng/mL and 5.7 ng/mL with the mepanipyrim propanol type for MPP204. The dc-ELISAs showed the sufficient sensitivity to determine the mepanipyrim residues for the MRLs of 1-15 mg/kg among the majority of vegetables and fruits in Japan. Recovery and/or correlation results from HPLC suggested that the dc-ELISAs would be applicable to the residue analysis of mepanipyrim and its propanol type in vegetables.

QuEChERS extraction followed by enzyme-linked immunosorbent assay for determination of deoxynivalenol and zearalenone in cereals

ABSTRACTA simple, rapid and cost-effective QuEChERS method for the determination of ZEN and DON by ELISA in cereals was developed. The single factor experiment was used to find the optimal QuEChERS parameters (extraction solvent, extraction method, extraction time, the QuEChERS composition, sorbent and clarification time) and extracts were analysed by ELISA. The optimised method was validated for selectivity, linearity, limits of detection, matrix effect and precision. The recovery values of ZEN and DON by ELISA were compared with a standard HPLC method and the results showed good coefficient with HPLC, which proved to be suitable for the screening of cereal samples for the presence of ZEN or DON. This is the first report of the determination of ZEN and DON in cereals after QuEChERS extraction combined with the enzyme-linked immunosorbent assay method.

Goldmag-based enzyme-linked immunosorbent assay for determination of α-lactalbumin in milk

ABSTRACTIn this work, a goldmag-based enzyme-linked immunosorbent assay was developed for determination of α-lactalbumin (α-LA) in milk. The magnetic nanoparticle functionalized with polyetherimide was synthesized by one-pot method and coated with two layers of gold nanoparticles on the surface to synthesize goldmag nanoparticles. Anti-α-LA monoclonal antibody, prepared by hybridoma cell lines via cell fusion, was then bound to this goldmag nanoparticle to develop a capture nanoprobe. The results showed that this developed immunoassay had a good linear range of 2.33–127.1 ng/mL with IC50 of 17.2 ng/mL. Besides, recovery rates for α-LA in four commercial milks were from 86.7% to 109.8%. The coefficients of variation in intra-assay and inter-assay were 3.9–6.8% and 5.5–9.8%, separately, which could meet the requirements to quantify α-LA content in milk. This goldmag-based immunoassay might have considerable potentials in the detection of food allergies.

Development of Enzyme-Linked Immunosorbent Assay for Determination of Boldenone in Dietary Supplements

There is an increasing interest in the investigation and implementation of methods for the analysis of anabolic steroids in dietary supplements. In this study, a competitive indirect enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of boldenone. For this purpose, an antiserum against boldenone was raised in a rabbit using boldenone-17-hemisuccinate-bovine serum albumin as an immunization conjugate. Based on the ELISA standard curve, the detection limit was as low as 0.014 ± 0.007 ng mL−1 with the IC50 value of 0.293 ± 0.084 ng mL−1 and linear working range of 0.065–1.529 ng mL−1. The intra- and inter-assay variations were found to be satisfactory. Relative standard deviations were calculated in the range of 3.8–10.5 and 7.3–12.9 %. The developed ELISA was applied in the analysis of extracts obtained from spiked samples of dietary supplements. Ethanol extracts were applied into the immunoassay without a cleanup procedure (only diluted) to minimize the effect of the matrix. Recoveries from spiked samples were from 86.0 to 115.7 %. An excellent correlation between ELISA and UHPLC-MS/MS was obtained with the linear equation of y = 1.0256 x − 0.7772 (R 2 = 0.9999). This ELISA as proposed here could be successfully applied for the simple monitoring of boldenone in dietary supplements.

A new approach of indirect enzyme-linked immunosorbent assay for determination of D-glutamic acid through in situ conjugation

We propose a new approach of an indirect enzyme-linked immunosorbent assay (ELISA) for determination of D-glutamic acid (D-Glu) using a monoclonal antibody against D-glutamic acid (D-Glu-MAb), which recognizes D-Glu-glutaraldehyde (GA) molecule but not D-Glu molecule. Human serum albumin (HSA) was coated on an immunoplate and reacted with D-Glu via GA to produce D-Glu-GA-HSA conjugates in situ in the well to be recognized by D-Glu-MAb, which enabled the development of an indirect ELISA for the determination of free D-Glu. In this indirect ELISA, D-Glu can be specifically detected with limit of detection of 7.81 μg/mL. Since anti-conjugate antibodies are often produced, even though anti-hapten antibodies are desired, this new approach could be very useful as an application of anti-conjugate antibodies to the development of quantitative analysis for detecting hapten.

Broad specific enzyme-linked immunosorbent assay for determination of residual phenothiazine drugs in swine tissues

In this study, a novel generic hapten of phenothiazine drugs was synthesized by derivatization of 2-chlorophenothiazine with sodium bromoacetate. Then the hapten was coupled to bovine serum albumin for production of the monoclonal antibody. Results showed that the obtained monoclonal antibody recognized five phenothiazine drugs simultaneously: chlorpromazine, promethazine, acepromazine, perphenazine, and fluphenazine. After evaluation of different coating antigens, a heterologous competitive indirect enzyme-linked immunosorbent assay was developed to determine the residues of the five phenothiazine drugs in swine tissues (muscle, liver, and kidney). The cross-reactivities to the five analytes were in the range of 71 to 98%, and the limits of detection were in the range of 0.2 to 0.4ng/ml, depending on the drug. Their recoveries from the fortified blank samples were in the range of 73.8 to 96.2%, with coefficients of variation in the range of 4.1 to 14.3%. This is the first study reporting a broad specific immunoassay for multi-determination of the residues of five phenothiazine drugs in animal-derived foods.

Development of a single-chain variable fragment antibody-based enzyme-linked immunosorbent assay for determination of fumonisin B1in corn samples

Fumonisin B1 (FB1 ) is a cancer-promoting mycotoxin produced by Fusarium species and one of the major food-borne toxins in corn and corn products. The objective of this study was to produce a single-chain variable fragment (scFv) antibody for determination of FB1 in corn samples. Anti-FB1 monoclonal antibodies were obtained via the hybridoma technique. Specific heavy- and light-chain variable fragments were amplified with degenerate primers and constructed into scFv antibody fragments by splice overlap extension polymerase chain reaction with linker sequences. The resulting scFv DNA fragments were cloned into the phagemid pHEN1for selection and identification of functional scFv fragment by phage display. Prokaryotic expression vector pET22b-scFv was constructed to prepare anti-FB1 scFv antibody for establishment of indirect competitive ELISA. The detection capability (CCβ) of the scFv-based ELISA was 15.00 µg kg(-1), and the limit of detection was 8.32 µg kg(-1). The recoveries and coefficients of variation were 86.74-107.34% and 9.72-14.03%, respectively. In addition, the determined results of 30 naturally contaminated corn samples by the scFv-based ELISA are in agreement with the findings of high-performance liquid chromatography (R(2) = 0.97). This scFv-based ELISA could be used as an efficient screening method for routine monitoring the residues FB1 in corn samples.

An Enzyme-Linked Immunosorbent Assay for Determination of Dicyclanil in Animal Tissue

Dicyclanil is a toxic, pyrimidine-derived insect growth regulator used in veterinary medicine for the prevention of myiasis. A competitive indirect enzyme-linked immunosorbent assay was developed firstly for the determination of dicyclanil in animal tissue. The antigen was prepared using the glutaraldehyde method and polyclonal antibodies were obtained by immunizing rabbits with a dicyclanil-bovine serum albumin conjugate. The antibody obtained was specific for dicyclanil with an IC50 of 9 ng/mL. Recoveries from sheep tissue and liquid milk were in the range of 52.5–72.4%, with coefficients of variation between 4.7% and 11.9%. The results were further confirmed by high performance liquid chromatography–tandem mass spectrometry.

Development of enzyme-linked immunosorbent assay for determination of polybrominated diphenyl ether BDE-121

Our interests are in the development of immunoassay-based fast scanning methods for persistent organic pollutants. To develop the immunoassay method of polybrominated diphenyl ether (PBDE), a model compound of PBDE, 2,3′,4,5′,6-pentabromodiphenylether (BDE-121), has been chosen to develop its antibody and the competitive indirect enzyme-linked immunosorbent assay (ELISA) is developed. The hapten of BDE-121 containing reactive carboxylic acid was synthesized and conjugated to carrier proteins (bovine serum albumin [BSA] and ovalbumin [OVA]). Anti-BDE-121 polyclonal antibody was then developed in rabbits as a result of immunization with the BDE-121–BSA conjugate. The optimal amount of coating antigen BDE-121–OVA conjugate and the dilution of antiserum needed in the ELISA were determined with the checkerboard method, and the effects of the properties of PBST (phosphate-buffered saline and Tween 20) buffer (pH and salt concentration) and chemical solvent (types and concentrations) on the ELISA were investigated to achieve a rapid robust assay with high sensitivity. Under the optimized conditions, the developed indirect ELISA shows a linear detection range from 1.74 to 84.1ng/ml, with an IC50 value of 8.07ng/ml and a detection limit of 0.644ng/ml. In total, 11 kinds of compounds were tested for calculating the cross-reactivity, which was less than 8% for nearly all of them. Real samples were analyzed by the proposed immunoassay and gas chromatography/mass spectrometry (GC/MS).

Development of an enzyme-linked immunosorbent assay for determination of iriflophenone 3-C-β-D-glucoside from Aquilaria spp. using a specific polyclonal antibody

Aquilaria species (agarwood) are cultivated in Asian countries for the source of agarwood oil and endangered due to the scent industry. The leaves of Aquilaria spp. are used ethnomedically to treat anti-inflammatory conditions, diabetes, and as laxative [1 – 3]. The active compounds in Aquilaria spp. leaves are benzophenone glycosides including iriflophenone 3-C-β-D-glucoside (IP3G), iriflophenone 2-O-α-L-rhamnopyranoside and iriflophenone 3,5-C-β-D-diglucopyranoside [4]. Among them, IP3G is major active compound found in Aquilaria spp. leaves. Thus, a reliable and sensitive analysis method is necessary for quality control of the plant raw material and product. An enzyme-linked immunosorbent assay (ELISA) was developed using polyclonal antibody (PAb) against IP3G. The indirect competitive ELISA using anti-IP3G PAb was high specific for IP3G. There were low cross-reactivity with iriflophenone 3,5-C-β-D-diglucopyranoside (13%), genkwanin 5-O-β-primeveroside (3.55%) and no cross reactivity found in other compounds. The range of ELISA assay extends from 100 – 1,560 ng/ml with coefficient of variation (CV) 1.19 – 2.07% for intra-assay and 3.76 – 5.70% for inter-assay precision levels. The percentages of recovery in the range of 96.0 – 99.0% with CV of 4.50 – 5.32%, which indicated that the method is accurate in agarwood samples. The correlation between the result from the ELISA and HPLC had a coefficient of determination (r 2) of 0.9321. The developed immunoassays using the PAb against IP3G are simple, reliable, sensitive and cost-effective assay for quality control of agarwood raw material and product.

Novel hapten synthesis for antibody production and development of an enzyme-linked immunosorbent assay for determination of furaltadone metabolite 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ)

A heterologous competitive indirect enzyme-linked immunosorbent assay (ciELISA) for the determination of the furaltadone metabolite 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ) was developed. AMOZ was derivatised with 2-(4-formylphenoxy) acetic acid or 2-(3-formylphenoxy) acetic acid to obtain two novel immunizing haptens. The ability of these haptens in producing specific polyclonal antibodies against the nitrophenyl derivative of AMOZ (NPAMOZ) was compared with that of traditional immunizing haptens (derivatised AMOZ with 3-carboxybenzaldehyle or 4-carboxybenzaldehyle). The results indicated that the novel immunizing haptens were able to produce antibodies with almost a two-fold improvement in sensitivity of the ciELISA for NPAMOZ in comparison with the existing antibody based ELISAs. The differences in sensitivity were explained by the molecular modeling of the lowest energy conformations of NPAMOZ and the haptens. Another novel hapten, derivatised AMOZ with 2-oxoacetic acid, was synthesized and used as a heterologous coating hapten. The results showed that this strategy of using only a partial structure of the target molecule as the coating hapten was able to obtain a two to three-fold improvement in sensitivity. This study provided a modern approach for the development of an immunoassay with improved sensitivity for the metabolites of nitrofuran antibiotics.

Computer-Aided Molecular Modeling Study on Antibody Recognition of Small Molecules: An Immunoassay for Triazine Herbicides

Most immunoassays for determination of small molecules are still designed on the basis of the "trial and error" method, due to the lack of understanding of antibody recognition. In the present study, we developed a heterologous indirect competitive enzyme-linked immunosorbent assay for determination of triazine herbicides, with limits of detection for 11 triazines ranging from 0.05 to 29.4 μg/L. Mechanisms of the antigen-antibody interaction were studied by computer-aided molecular modeling (CAMM)-based quantitative structure-activity relationship analyses. Co-effects of the analytes' substructural hydrophobic, electrostatic, and steric fields on antibody recognition were further revealed. Hydrophobicity of the antigens was demonstrated to have the most important impact. Even less exposed substituents provided hydrophobic force to the antigen-antibody interaction. Dislocated orientation of analyte functional groups could lead to steric hindrance and hydrophobic misleading of antibody recognition. This may happen even when the antigens contained the same substituent as the hapten. Frontier orbital energies also affect the reaction significantly. This study highlights of the power of CAMM-based analyses, providing insights into antibody recognition of small molecules.

Development of a Capillary Electrophoresis-Based Enzyme Immunoassay with Electrochemical Detection for the Determination of Okadaic Acid and Dinophysistoxin2 in Shellfish Samples

A capillary electrophoresis-based enzyme immunoassay (CE-EIA) with electrochemical (EC) detection system was developed for the determination of two diarrheic shellfish poisoning (DSP) toxins okadaic acid (OA) and dinophysistoxin2 (DTX2). In this method, after the competitive immunoreaction in liquid phase, the horseradish peroxidase (HRP)-labeled antigen (Ag*) and the bound enzyme-labeled complex (Ag*-Ab) were separated and then the system of HRP catalyzing H2O2/o-aminophenol (OAP) reaction was adopted. The limit of detection (S/N = 3) was determined to be 0.05 and 0.07 ng/mL for OA and DTX2, respectively. The total analysis time was less than 40 min. The developed CE-EIA with EC detection system was capable of quantitatively detecting OA and DTX2 contents in the tested contaminated samples, and the results were compared with the same samples analyzed through enzyme-linked immunosorbent assay (ELISA). Consistent results between CE-EIA with EC detection and ELISA were found in most of the tested samples. The proposed system appeared to be more sensitive and faster than ELISA for determination of OA and DTX2 in shellfish meat extracts. Real shellfish samples were validated in recovery test, and the recoveries tested by the proposed method were 91.7–108.3% and 95.2–112.5% for OA and DTX2, respectively. The CE-EIA with EC detection provides a valid and sensitive analytical approach, not previously available, for the determination of OA and DTX2 in shellfish samples.

Development of a highly sensitive and specific monoclonal antibody-based enzyme-linked immunosorbent assay for determination of doxycycline in chicken muscle, liver and egg

A modified indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) method was developed using a highly sensitive and specific monoclonal antibody (McAb) to determine doxycycline (DC) residues in chicken tissues and egg. The McAb against DC was produced by hybridoma technique and a modified ic-ELISA was characterised in terms of sensitivity, specificity, precision and accuracy. At optimal experimental conditions, the standard curve was constructed at concentrations ranged from 0.01 to 100 ng/ml. The IC(50) value was 1.32±0.18 ng/ml. The limit of detection was 0.14±0.02 ng/g. The recoveries of DC from spiked chicken liver, muscle, and egg at levels of 50-600 ng/g were 84.6-85.5%, 88.2-89.1%, and 84.4-89.3%, respectively. The coefficient variations (CVs) were 5.1-9.3%, 3.7-11.3%, and 4.7-9.8%, respectively. Linear regression analysis showed good correlation, with r(2) values 0.9909 for chicken liver and 0.9916 for chicken muscle.

Development of an enzyme-linked immunosorbent assay for the determination of 5-hydroxymethyl-2-furfural in food

5-Hydroxymethyl-2-furfural (5-HMF) is considered to be an excellent indicator of quality deterioration due to excessive heating or storage for a wide range of carbohydrate-containing foods. To facilitate its analysis, a highly selective and sensitive enzyme-linked immunosorbent assay for determination of 5-HMF in food has been developed. A specific polyclonal antibody was produced against a conjugate of 5-HMF coupled to bovine serum albumin. The IC(50) and limit of method detection were 0.15 ± 0.012 mg L(-1) and 0.02 ± 0.002 mg L(-1), respectively. The proposed method was applied to detect 5-HMF in French mini bread, potato chips, French soft bread, and wheat chicken nuggets with recoveries ranging from 84.07 to 97.09% and relative standard deviation (n = 3) below 8.65% in all samples. The quantitative results were in good agreement with those obtained by the high-performance liquid chromatography method, which suggests that the method developed will be very useful for monitoring 5-HMF in food samples.

Development of an enzyme-linked immunosorbent assay for determination of pretilachlor in water and soil

An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) has been developed for detection of pretilachlor in water and soil. An immunogen was prepared from haptens of pretilachlor conjugated to bovine serum albumin(BSA). The specific polyclonal antibodies were obtained by immunizing New Zealand white rabbits. The influence of parameters including concentrations of methanol, ionic strength and pH values were optimized to improve the sensitivity of the assay. The optimized ELISA was shown to have a high sensitivity and specificity for pretilachlor. Under optimal conditions, the ELISA has demonstrated an 50% inhibitory concentration (IC 50) value of 0.0359 mg/L with a limit of detection (LOD, IC 10) of 6.9 ng/L. The cross-reactivities to some analogs of pretilachlor (acetochlor, butachlor, metazachlor and metalaxyl) were below 1.5%. The average recoveries of pretilachlor from distilled water, tap water, paddy water and soil were in the range of 77.0–115.2% between 0.005 and 5.0 mg/L. The results of ELISA for spiked samples were confirmed by GC–ECD with a high correlation coefficient of 0.9950( n=11). Thus, the ELISA proven to be a sensitive, specific, inexpensive and quantitative tool for detection of pretilachlor from four kinds of spiked samples.

Enzyme-linked immunosorbent assay for determination of cyclosporin a in the whole blood

Enzyme-linked immunosorbent assay for determination of cyclosporin a in the whole blood