Development of Monoclonal Antibody-based Enzyme-linked Immunosorbent Assay to the Estrogen Diethylstilbestrol

Diethylstilbestrol （DES） is a synthetic estrogen that has ever been used worldwide. Polyclonal antibodies （PAbs） were used in immunoassay for detection of DES residues in environmental and agricultural samples in previous paper. In this paper, an indirect competitive enzyme-linked immunosorbent assay （icELISA） was developed based on monoclonal antibody （MAb） for the determination of diethylstilbestrol. Mono-o-carboxypropyldiethylstilbestrol （DES-CP） and mono-o-carboxymethyldiethylstilbestrol （DES-CME） were synthesized to be haptens. DES-CP was coupled to bovine serum albumin （BSA） to be an immunogen in BALB/c female mouse for MAb production. The MAb was characterized for specificity and affinity to DES in icELISA. Under the optimum condition, the icELISA showed an ICs0 of 9.8 ng/mL, the limit of detection （IC20） of 2.3 ng/mL and a working range of 2-42 ng/mL. Hexestrol and dienestrol exhibited cross-reactivity values were 44% and 27%, respectively. Cross-reactivity of natural estrogen 17β-estradiol was less than 0.1%. The influences of some factors such as salt concentration, pH and organic solvent concentration on the assay were evaluated. The concentrations of DES in the fortified water samples determined by the assay were correlated well with the fortification levels. The results were conf＇m＇ned with analysis by HPLC.     

Post-column reaction detection of biotin in human plasma ultrafiltrate based on laser-induced fluorescence energy transfer in the far-red spectral region

High performance liquid chromatography followed by post-column reaction detection in the far-red spectral region provides added sensitivity and selectivity. A homogeneous fluorescence energy transfer assay in the competitive mode based on the binding of biotin and streptavidin was developed as an on-line post-column reaction detection system. The labels used for energy transfer were R-Phycoerythrin conjugated to biotin and Cyanine 5 labeled with streptavidin. The energy transfer peak was measured at 670 nm and excitation was achieved using the 488 nm line of an argon ion laser. The biotin concentration in plasma ultrafiltrate ranged from 0.024 to 6.12 ng/mL (n = 6). The precision of the two controls, 0.24 and 2. 44 ng/mL, was found to be 18.70% and 9.92% relative standard deviation respectively. Accuracy was 10.47% and 1.95% difference from spiked, respectively (n = 6). The limit of detection was 21.70 pg/mL (8.90 x 10(-11)M) calculated based on a factor of 2x the standard deviation of the blank (n = 6). The correlation coefficient for the calibration curve was found to be 0.9995. Recovery from plasma ultrafiltrate at 2.44 ng/mL was 103.40% (n = 6). Detection selectivity was indicated by the absence of background fluorescence in six different plasma samples collected from six individual donors. Endogenous levels were detected in two of the six pools of plasma ultrafiltrates.     

A sensitive solid-phase fluoroimmunoassay for detection of opiates in urine

An automated flow fluorometer designed for kinetic binding analysis was adapted to develop a solid-phase competitive fluoroimmunoassay for urinalysis of opiates. The solid phase consisted of polymer beads coated with commercial monoclonal antibodies (MAbs) raised against morphine. Fluorescein-conjugated morphine (FL-MOR) was used as the fluorescein-labeled hapten. The dissociation equilibrium constant (K _D ) for the binding of FL-MOR to the anti-MOR MAb was 0.23 nM. The binding of FL-MOR to the anti-MOR MAb reached steady state within minutes and was displaced effectively by morphine and other opiates. Morphine-3-glucuronide (M3G), the major urinary metabolite of heroin and morphine, competed effectively with FL-MOR in a concentration-dependent manner for binding to the antimorphine MAb and was therefore used to construct the calibration curve. The sensitivity of the assay was 0.2 ng/mL for M3G. The assay was effective at concentrations of M3G from 0.2 to 50 ng/mL, with an IC₅₀ of 2 ng/mL. Other opiates and heroin metabolites that showed >50% crossreactivity when present at 1 μg/mL included codeine, morphine-6-glucuronide, and oxycodone. Methadone showed very low crossreactivity (<5%), which is a benefit for testing in patients being treated for opiate addictions. The high sensitivity of the assay and the relatively high cutoff value for positive opiate tests allows very small sample volumes (e.g., in saliva or sweat) to be analyzed. A double-blind comparison using 205 clinical urine samples showed good agreement between this single-step competitive assay and a commercially performed enzyme multiplied immunoassay technique for the detection of opiates and benzoylecgonine (a metabolite of cocaine).     

Development of an Indirect Competitive ELISA Based on Polyclonal Antibody for the Detection of Diethylstilbestrol in Water Samples

An indirect competitive enzyme-linked immunosorbent assay （icELISA） based on polyclonal antibody for the estrogen diethylstilbestrol （DES） was developed. With this aim, two different haptens mono-O-3-carboxypropyldiethylstilbestrol （DES-CP） and mono-O-carboxymethyldiethylstilbestrol （DES-CM） with carboxylic group that preserve the molecular structure character of diethylstilbestrol were synthesized. The haptens were conjugated with the carder proteins bovine serum albumin （BSA） by mixed-anhydride method for immunogen and conjugated with ovalbumin （OVA） by active ester method for coating antigen. Polyclonal antibodies for diethylstilbestrol were raised by immunizing mice with immune antigen DES-CP-BSA. Under optimized system, the lowest limit of detection （LLD） of diethylstilbestrol was 0.01 ng/mL, and IC50= 1.02 ng/mL. Its analogs were tested and no obvious cross-reactivity was found to anti-diethylstilbestrol antibody. DES-fortified water samples were determined by simple dilution to diminish the matrix effect. The comparison between the amount of DES estimated by ELISA and the amount added indicates good agreement for all water samples tested, with mean recovery values ranging from 86% to 120.2%.     

Optimization and validation of an enzyme immunoassay for the insect growth regulator fenoxycarb

A competitive enzyme-linked immunosorbent assay (ELISA) was developed for the quantitative detection of the insect growth regulator fenoxycarb. Polyclonal rabbit antisera, raised against protein conjugates of four haptenic derivatives of fenoxycarb, were utilized in immobilized antigen-based, competitive immunoassays. With ELISA systems that were both hapten- and carrier-heterologous, most antiserum titers fell in the range of 1:1000-1:30,000. Assay conditions, including concentrations of antisera and coating antigens, were optimized. The effect of pH, organic solvents, and various blocking agents was also investigated. A hapten-homologous and two hapten-heterologous indirect ELISAs allowed fenoxycarb determination in the range of 0.1-85 ng ml⁻¹ with apparent IC₅₀ values of 1.2-2.8 ng ml⁻¹. Cross-reactivities with a number of compounds (e.g. pesticides of related structure, hapten synthesis intermediates, fenoxycarb metabolite, photodegradation products) were determined, and the assay proved highly selective for fenoxycarb. In particular, no significant interference was found with selected pyrethroid and juvenile hormone analog insecticides, phenoxyacetic acid herbicides, and photodegradation products of fenoxycarb. Using spiked water samples, assay performance was validated by SPME/GC-MS.     

Trace analysis of diethylstilbestrol, dienestrol and hexestrol in environmental water by Nylon 6 nanofibers mat-based solid-phase extraction coupled with liquid chromatography-mass spectrometry

A simple, rapid and sensitive method for the determination of diethylstilbestrol (DES), dienestrol (DE) and hexestrol (HEX) was developed by using the Nylon 6 nanofibers mat-based solid-phase extraction (SPE) coupled with liquid chromatography-tandem mass spectrometry (LC-MS). These estrogens were separated within 8 min by LC using an ODS column and methanol/water (80/20, v/v) at a flow rate of 1.0 mL min(-1). Electrospray ionization conditions in the negative ion mode were optimized for MS detection of the estrogens. Under the optimum SPE conditions, all target analytes in 50 mL environmental water samples can be completely extracted by 1.5 mg Nylon 6 nanofibers mat at flow rate of 3.0 mL min(-1) and easily eluted by passage of 500 μL mobile phase. By using the novel SPE-LC/MS method, good linearity of the calibration curve (r(2) ≥ 0.9992) was obtained in the concentration range from 0.10 ng L(-1) to 1.0 mg L(-1) (except for DE which was 0.20 ng L(-1) to 1.0 mg L(-1)) for all analytes examined. The limits of detection (S/N = 3) of the three estrogens ranged from 0.05 ng L(-1) to 0.10 ng L(-1). This method was applied successfully to the analysis of environmental water samples without any other pretreatment and interference peaks. Several water samples were collected from Jinchuan River and Xuanwu Lake, and in Jinchuan River water DES was detected at 0.13 ng L(-1). The recoveries of estrogens spiked into tap water were above 98.2%, and the relative standard deviations were below 4.78%.     

Fast and sensitive dye-sensor based on fluorescein/reduced graphene oxide complex

We report on a fast, sensitive, label-free, and general dye-sensor platform for synthetic organic dyes detection by competitive adsorption on reduced graphene oxide (rGO) against a fluorescent dye (FD). Fluorescein (Fl) as fluorescence indicator and a cationic dye methylene blue (MB) as model analyte were employed to investigate the analytical feature of this assay platform. An anionic dye sunset yellow FCF (SY) was chosen as a comparison analyte to test the generality of this strategy. Results show that rGO can bind Fl and quench the fluorescence by fluorescence resonance energy transfer (FRET), while MB can displace Fl quickly from the Fl/rGO complex by competitive adsorption, inducing the fluorescence recovery which provides a quantitative readout for MB. Besides, this design was simply based on the competitive adsorption of rGO between dye and FD, and can be generally applied to other dyes for label-free detection. The fluorescence enhancement efficiency (FEE) is proportional to the dye concentration over the range of 7.60-420.00 ng mL(-1) MB and 7.28-400.25 ng mL(-1) SY, respectively. The linear regression equations were calculated as FEE(MB) = 0.0192c(MB)- 0.3103 for MB and FEE(SY) = 0.0142 c(SY)- 0.0427 for SY, with the detection limits of 1.03 and 1.15 ng mL(-1), respectively. The MB in waste water and SY in an orange-flavored sports drink sample were assayed with satisfactory results.     

Study of Immunoassay Methods for Recombinant Human Erythropoietin （rhEPO） Using Competitive ELISA

Two different immunoassay methods,competitive indirect enzyme-linked immunosorbent assay (CI-ELISA) and amplificative competitive indirect ELISA(ACI-ELISA) using biotin-avidin complex system were studied to detect rhEPO.The linear ranges were 50-20000ng/mL and 10-50000ng/mL for CI-ELISA and ACI-ELISA,respectively.The low detection limits of CI-ELISA and ACI-ELISA were 62.8ng/mL and 8.5ng/mL,respectively.     

Quantitation of Indian krait (Bungarus caeruleus) venom in human specimens of forensic origin by indirect competitive inhibition enzyme-linked immunosorbent assay

An indirect competitive inhibition enzyme-linked immunosorbent assay was reported to detect krait venom in human specimens of forensic origin. Polyclonal anti-krait venom antibodies were characterized by indirect antibody capture assay. The calibration plot was constructed based on linear regression analysis (y = 72.85 - 12.29x, r(2) = 0.98) with concentration ranges from 0.013 to 1000 ng/well of krait venom with a limit of detection of 0.2 ng/mL in the assay system. The IC50 (inhibitory concentration at 50% displacement) value of krait venom was observed to be 70 ng. Spiking studies indicated recoveries of 95-100% and 94-100% when various concentrations of krait venom were spiked to rat tissues (skin, liver, and kidneys) and pooled human serum, respectively. Polyclonal anti-krait venom antibodies showed no cross-reactivity with cobra and viper venom when tested in the assay system. The coefficient of variation of various concentrations of working range in intra-assay (n = 6) was <5%, whereas in interassay (n = 6) it was observed to be < or 7%. Further, the method was used to quantitate krait venom in human autopsy and biopsy specimens of forensic origin. Concentration of krait venom was found to be in the range of 4-172 ng/100 mg skin or skin scrapings and 64-378 ng/mL blood or serum. The methodology may find application in forensic laboratories to assess the cause of death in the cases of krait-bite victims.     

测定人体尿液中苄基丙二酸的免疫分析新方法研究

将苄基丙二酸(BA)与牛血清白蛋白通过活化酯法偶联制备人工免疫原,然后免疫新西兰大白兔制得抗苄基丙二酸多克隆抗体;使用该抗体建立了测定人体尿液中的苄基丙二酸的间接竞争酶联免疫吸附方法(ic-ELISA),方法线性范围为1.0×10-2～1.0×103ng/mL,最低检测浓度为0.01 ng/mL,板内差异为5.5%,板间差异在3.8%～7.8%的范围内;用于实际尿样中苄基丙二酸的测定,回收率为87.8%～115.1%。     

酶联免疫吸附分析法检测花生过敏原的研究

本文利用自己研制的花生过敏原免疫新西兰大耳兔,获得效价为200 000的抗花生过敏原特异性抗体,建立了花生过敏原蛋白的间接竞争酶联免疫(ELISA)检测方法.结果表明:花生抗原在0.01～100 ng/mL范围内具有较好的线性关系,其竞争标准曲线为v=-0.1212x+0.9285(r=0.9819),IC50为34....     

Preparation of Anti-Nortestosterone Antibodies and Development of an Indirect Heterologous Competitive Enzyme-Linked Immunosorbent Assay to Detect Nortestosterone Residues in Animal Urine

The steroid 19-Nortestosterone (NT) has been illegally used in horse racing, dog games to boost physical performance, and also in animal husbandry to accelerate weight gain. This paper reports an indirect heterologous competitive enzyme-linked immunosorbent assay (icELISA) using polyclonal antibody for rapid, sensitive analysis of NT in animal urines. After derivation, NT haptens were conjugated to bovine serum albumin (BSA) and ovalbumin (OVA) through 1-Ethyl-3-(3-dimethylaminopropy) carbodiimide (EDC) method and mixed-anhydride technology respectively. Two antisera raised in rabbits and two coating antigens synthesized were screened and optimized using homologous and heterologous ELISA formats. The most sensitive heterologous icELISA (antisera generated from NT-17-BSA, with NT-3-OVA as coating antigen) gave an IC₅₀ value of 27 ng/mL, with a dynamic range from 4 to 198 ng/mL, and the limit of detection (LOD) of 1.9 ng/mL. Except for cross-reactivity (CR) toward 17α-Nortestosterone (67%) and β-Boldenone (16.4%), no significant CR was observed for other chemical analogues tested. When applied to the authentic animal urines, the intra- and inter-assay precision values were below 8.4%, while the recoveries were in the range of 95–110%. The correlation coefficient between the established icELISA and LC-MS/MS method was greater than 0.98.     

Development of ELISA and immunochromatographic assay for ofloxacin

Two rapid,sensitive and reliable immunoassay methods,namely competitive indirect enzyme-linked immunosorbent assay(CI- ELISA)and colloidal gold-based immunochromatographic assay(CGIA),were developed to detect ofloxacin(OFL).The linear range of the CI-ELISAwas from 0.5 to 128 ng/mL with a limit of detection(LOD)of 0.35 ng/mL.Good recoveries were obtained in analyzing simulated swine urine samples.The CGIA could accurately estimate OFL at concentrations as low as 10 ng/mL in less than 10 min,and test results were read visually without any instrument.     

Liquid chromatographic analysis of vitamin B6 in soy-based infant formula

A liquid chromatographic (LC) method is described for determination of total vitamin B6 in soy-based infant formula. Total vitamin B6 is quantitated by using ion-pair LC after precolumn transformation of phosphorylated and free vitamers into pyridoxol. The limit of detection is 0.3 ng and the limit of quantitation is 1.0 ng on-column (injection volume = 100 microL). Linear response ranged from 39 to 616 ng/mL (r2 = 0.99986). Analysis of a soy-based infant formula control fortified at 6 different concentration levels gave recoveries that averaged 104%. Assay of SRM 1846 gave results within the certified range (8.6 +/- 0.086 mg/kg versus the certified value of 8.4 +/- 1.0 mg/kg). The method provides a rapid and specific assay for the analysis of total vitamin B6 in fortified soy-based infant formula.     

Characterization and application of quantum dot nanocrystal–monoclonal antibody conjugates for the determination of sulfamethazine in milk by fluoroimmunoassay

Quantum dot (Qdot) nanocrystals have been increasingly used as fluorescence labels in fluoroimmunoassays recently because of their excellent optical characteristics. In this paper, a new monoclonal antibody (MAb) against sulfamethazine (SMZ) was successfully produced and linked to Qdot nanocrystals by covalent coupling. The Qdot–MAb conjugates were characterized by SDS-PAGE and high-performance capillary electrophoresis (HPCE). An enzyme-linked immunosorbent assay (ELISA) method was utilized to evaluate the antigen–antibody binding affinity and then a novel direct competitive fluorescence-linked immunosorbent assay (cFLISA) for the detection of SMZ in milk by using Qdots as fluorescent labels was evaluated. The results showed that the 50% inhibition values (IC₅₀) of the cFLISA were 4.3 ng/mL in milk and 5.2 ng/mL in PBS, and the limits of detection (LODs) were 0.6 ng/mL in milk and 0.4 ng/mL in PBS, respectively. The recoveries of SMZ from spiked milk samples at levels of 10–100 ng/mL ranged from 94 to 106%, with coefficients of variation (CVs) of 2.1–9.2%. Figure Shematic diagram of the direct cFLISA procedure Jianzhong Shen and Fei Xu contributed equally to this work.     

Colloidal gold-based immunochromatographic assay for detection of diethylstilbestrol residues

One-step membrane-based competitive colloidal gold-based immunoassays in immunochromatographic formats for the rapid detection of diethylstilbestrol (DES) were developed. Nitro-cellulose membrane strip was separately coated with goat anti-rabbit IgG (control line) and DES hapten-ovalubumin conjugate (test line). Anti-DES polyclonal antibody labeled with colloidal gold particles was first incubated with DES. A positive reaction as a result of the remaining antibody-gold conjugate combining with antigen coated on the membrane was obvious by visual detection, with detection limits for immunochromatographic of 0.5 microg/kg for detecting DES standard solution, and the limit of detection was 5 microg/kg for detecting the DES spiked in swine pork and liver. The assay time for test was less than 5 min, suitable for rapid testing on-site.     

使用限进介质色谱柱的柱切换高效液相色谱法直接进样测定大鼠血浆中舒必利

提出一种直接进样测定大鼠血浆中舒必利浓度的高效液相色谱方法,使用限进介质色谱柱作为预柱在线去除血浆蛋白后,将舒必利通过柱切换转移到分析柱中进行分析。限进介质色谱柱为CAPCELLPAKMFSCX阳离子交换柱（20&#215;4．0mmi．d．,5μm）,分析柱为Kromasil C18柱（150&#215;4．6mm i．d．,5μm）,限进介质柱预分离时流动相为PH=6．88的50mmol／L磷酸盐缓冲液乙腈（100：5,V／V）,切换后分析流动相为PH=6．83的50mmol／L磷酸盐缓冲液-乙腈（100：10,V／V）。流速均为1mL／min,检测波长为240nm。该方法检出限为17ng／mL,定量限为50ng／mL。舒必利在50～1400ng／mL之间线性良好（r=0．9997）,高中低浓度的日内、日间相对标准偏差分别为1．5％～4．2％及2．0％～5．2％,方法回收率为98．8％～104．1％．     

Enzyme-linked immunosorbent assay for okadaic acid: investigation of analytical conditions and sample matrix on assay performance

Okadaic acid (OA) is a lipophilic marine biotoxin. In this study, OA was coupled with the carrier proteins keyhole limpet hemocyanin and bovine serum albumin as immunity and detection antigens by an active ester method. The polyclonal antibody against OA was prepared successfully, an indirect competitive ELISA (ciELISA) developed for the detection of OA in shellfish, and the reactive conditions of ciELISA optimized. The LOD (15% inhibition concentration) for the microwell plates was 1.28 +/- 0.38 ng/mL, corresponding to 12.8 +/- 3.8 ng/g. Two extraction methods were used to remove shellfish matrix interference with high recovery of spiked samples, and the methanol extraction of shellfish mussel was analyzed after dilution in phosphate-buffered saline. For validation of the optimized ciELISA, spiked and natural samples were analyzed by ciELISA, and HPLC with fluorescence detection. The correlation of linear regression equation was y = 1.0064x - 10.234, and the correlation coefficient was 0.9347. From the results of the comparative study, the established ciELISA assay using polyclonal antibody against OA could be used in preliminary screening of suspicious shellfish samples.     

Dual-signal immuno-competitive determination of brain natriuretic peptide based on magnetic nanozyme

Brain natriuretic peptide (BNP) has been a disease marker in the diagnosis of heart failure. In this study, gold nanoparticles modified with Hemin (H-AuNPs) as nanozymes were used to oxidize ABST and MB to amplified colorimetric and electrochemical redox signals respectively. BNP was combined with H-AuNPs (BNP-H-AuNPs) through electrostatic adsorption to construct competitive nanozyme probes. Target BNP in the sample compete with BNP-H-AuNPs to bind the antibody-modified magnetic nanoparticles (AntiBNP-MNPs). Due to the excellent catalytic performance of the nanozyme, BNP can be observed well by colorimetric and electrochemical assays. Electrochemical method ensured more accurate detection of BNP with a wide detection range (1–200 pg/mL) and a low detection of limit (0.03 pg/mL). Meanwhile, the results of the experiment can be easily observed with the naked eye by simple colorimetric method with a range from 5 ng/mL to 25 ng/mL and a limit of detection down to 80.3 pg/mL. Thus, based on the important role of H-AuNPs, this assay has exhibited potential value of detection the other small proteins through this competitive nanozyme method.     

Preparation of a Monoclonal Antibody against a Kallikrein-Like Enzyme from Agkistrodon halys pallas Venom and Its Application in a Pharmacokinetic Study

Snake venom contains bioactive materials for drug development, diagnosis, and treatment. After separating and purifying the kallikrein-like enzyme (AHP-Ka) from Agkistrodon halys pallas venom for the first time, a monoclonal antibody against AHP-Ka was prepared and characterized. An indirect sandwich enzyme-linked immunosorbent assay (ELISA) based on the monoclonal antibody was developed and validated for the pharmacokinetic analysis of AHP-Ka in rat plasma. The method was calibrated using rat plasma and 1:100 dilution of plasma was selected to prepare a calibration curve to validate the precision, accuracy, and stability of the ELISA method. A good linear relationship was obtained in a working range from 3.9 ng/mL to 62.5 ng/mL with a limit of detection of 2.94 ng/mL. Intra- and inter-batch precision were less than 10%. The average recovery ranged from 94.6% to 104.4% in rat plasma at the concentrations of 5 ng/mL, 15 ng/mL, and 45 ng/mL, respectively. The ELISA method was successfully used for the pharmacokinetic study of AHP-Ka in Sprague-Dawley rat plasma after intravenous administration. The work is expected to contribute to future preclinical development of AHP-Ka.