酶联免疫分析结合生物素-亲合素放大体系测定血清中的雌三醇

通过简单的全合成获得了共价结合的雌三醇-生物素复合物(E3-Biotin),结合辣根过氧化物酶标记的亲合素(HRP-Avidin)作为免疫分析探针,建立了灵敏的酶联吸附免疫分析新方法以测定血清中的雌三醇.在稀释酶的缓冲溶液中加入小牛血清可有效地抑制非特异性吸附.实验结果表明,此方法具有很高的灵敏度和抗非特异性吸附干扰的能力,同时具有很好的稳定性和重现性.标准曲线线性范围为0.1～10ng/mL,检出限为0.06ng/mL.     

A secondary antibody format chemiluminescence immunoassay for the determination of estradiol in human serum

A competitive immunoassay for estradiol (E2) based on secondary antibody format was established. The donkey anti-rabbit IgG was used as the secondary antibody to coat micro-plates, and the horseradish peroxidase (HRP)-luminol-H₂O₂ chemiluminescent system with high sensitivity was chosen as the detection system. The addition of sodium trichloroacetate (CCl₃COONa) in the enzyme buffer as a replaceable packing material can realize directly analysis of E2 in human serum without extraction, which improved reproducibility and resolution of the assay. Additionally, the method showed specific recognition of estrogen, without cross-reaction for the major steroids (estrone (E1), estriol (E3), dihydrotestosterone (DHT), androstenedione, testosterone (T)) commonly found in human serum. The chemiluminescence immunoassay with secondary antibody can be applied to detect E2 with good precision at concentrations as low as 1.48 pg mL⁻¹. The proposed method has been successfully applied to the determination of E2 in 97 human sera and showed a good correlation compared with the commercially radioimmunoassay (RIA) kit with a correlative coefficient of 0.9881. This method has exhibited great potential in the fabrication of diagnostic kit and can be used in the clinical analysis of E2 in human serum.     

Enzyme immunoassay for the drug of anti-ulcer using avidin-biotin system.

We have developed a competitive enzyme immunoassay for a drug, which was a newly synthesized anti-ulcer agent, using an enzyme immunoassay. The polyclonal anti-drug antibody coupled to biotin, peroxidase labeled drug derivatives as a tracer, and a small column of Sepharose 4B covalently bound to avidin were used in the assay. This assay is simple and rapid, and the sensitivity and the measuring range can be controlled by the flow rate of the substrate solution. The correlation between serum drug concentrations (0.1-10 micrograms/ml) measured by gas chromatography and this assay was good (r = 0.991). This principle for the assay is very practical and applicable to the enzyme immunoassay for small and large molecules.     

Quantitative analysis of 17beta-estradiol in river water by fluorometric enzyme immunoassay using biotinylated estradiol.

A sensitive and simple immunoassay to determine 17beta-estradiol (E2) in fresh water was developed. The method is based on a solid-phase avidin-biotin binding assay and solid phase extraction. The binding event of E2 to the antibody is detected indirectly by the competitive reaction between E2 and biotinylated estradiol (BE) as a tracer for the limited binding sites of antibodies immobilized onto the wall of a microtiter plate. Namely, E2 concentrations are determined from the strong interaction between BE and avidin conjugated with horseradish peroxidase (avidin-HRP). In order to achieve a sensitive measurement for the binding of BE to the antibody immobilized on the microtiter plate substrate, QuantaBlu fluorogenic peroxidase substrate (QFPS) was employed. The detection limit and the linear range of E2 determination were 27 pM and 27 - 7480 pM, respectively. The relative standard deviations (RSD) for the E2 assay were between 0.3 and 12.0% (n = 3). The cross-reactivities of several other estrogens in this assay system were also investigated. No serious influences from any cross-reaction caused by other estrogens tested in this experiment were observed. The determination of E2 in water samples from eight rivers and a marsh in Hokkaido was performed by the immunoassay combined with solid phase extraction. It was found that the concentration of E2 was in the range between 0.06 and 67 pM.     

Improvement of an Enzyme Immunoassay for the Determination of Mercury(II)

Three systems were tested for the optimization of a heterogenous non-competitive enzyme immunoassay (EIA) for the determination of Hg (II). The sensitivity of the non optimized Hg-EIA with a detection limit of 2.1 μg/L Hg (II) was improved by an avidin-biotin-complex (ABC) amplification system to a 2-fold lower detection limit (1.1 μg/L Hg (II)). A conventional competitive ElA with the competition reaction between bound and free Hg (II) for antibody (ab) binding sites yielded a detection limit of 1.0 μg/L Hg (II). Further improvement of sensitivity could be achieved by a competitive displacement EIA. In this case ab molecules bound to immobilized haptens are displaced in the next step by free Hg (II). The detection limit of the displacement approach is 0.4 μg/L Hg (II).     

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.     

Determination of 3,5,6-trichloro-2-pyridinol (TCP) in water by a continuous competitive immunoassay system based on the streptavidin-biotin interaction

A competitive continuous immunoassay system for the determination of 3,5,6-trichloro-2-pyridinol (TCP), the major degradation product of the insecticide chlorpyrifos, in water is described. The immunoassay system is based on the transient retention of the specific LIB-MC2 monoclonal antibody anti-TCP as a biotinylated derivative using the streptavidin-biotin interaction. The permanent immobilization of streptavidin on controlled-pore-glass provides an adequate active support for the transient retention of the biotinylated monoclonal antibody anti-TCP. In a subsequent step, the immuno-competitive reaction between the biotinylated LIB-MC2 and the TCP/hapten-POD mixture takes place. This competitive assay relies on the determination of the biocatalytic action of peroxidase, retained in the active support, on a derivatization reaction which yields a fluorescent product. The method exhibits a determination range of 0.01-200 microg L(-1) of TCP (r2=0.9919, n=9) with a precision, expressed as RSD, lower than 4.2% and a sampling frequency of 3 h(-1). The approach has been applied to the determination of TCP in water with recoveries of 89.7-105.6%.     

Characterization of s-triazine antibodies and comparison of enzyme immunoassay and biotin-avidin enzyme immunoassay for the determination of s-triazine

Triazines have been used widely as herbicides and known to cause environmental contamination. For developing ELISA of s-triazines, six kinds of s-triazine derivatives (one from simazine, one from atrazine and four from cyanuric chloride) which contained a C3- or C6-carboxylic acid group bridge were prepared. These derivatives were conjugated to bovine serum albumin (BSA) for the use of immunogens and to KLH for the use of coating ligands on the microtiter plate wells. Polyclonal antibodies produced from rabbit or sheep using atrazine-BSA (1b-BSA) and simazine-BSA (1a-BSA) immunogens. These antibodies were characterized and biotinylated for the use of enzyme immunoassay (EIA). We evaluated EIA and biotin-streptavidin mediated EIA in terms of the sensitivities and specificities with these antibodies. The results in both assay systems showed that coating ligand synthesized from atrazine derivative was better than that from simazine derivative. Comparing binding ability between C3-carboxylic acid and C6-carboxylic acid spacers of N-alkyl group in s-triazine ring, C3-carboxylate-KLH (2c-KLH) derived from atrazine showed better sensitivity for the both assay systems. The detection limit was found to be 0.01 ppb in biotin-streptavidin mediated EIA (B-Av EIA). Comparing IC₉₀ values of EIA (0.5 ppb) and B-Av EIA (0.05 ppb), B-Av EIA was able to detect one order lower concentration range of atrazine than EIA.     

Enzyme immunoassay of thyrotropin releasing hormone (TRH).

A sensitive and specific double-antibody enzyme immunoassay (EIA) for a thyrotropin releasing hormone (TRH)-like immunoreactive substance has been developed. In order to synthesize TRH-labeled beta-D-galactosidase (beta-gal), a newly devised TRH derivative, pGlu-His-Pro-NH-(CH2)6-NH2 (TRH-Hex), was employed. TRH-Hex was linked to beta-gal by the N-(epsilon-maleimidocaproyloxy) succinimide coupling procedure. For competitive reactions, the TRH antibody was incubated with standard TRH and TRH-Hex-beta-gal (delayed addition). Free and antibody-bound enzyme hapten were separated by using an anti-rabbit immunoglobulin G coated immunoplate. Activity of the enzyme on the plate was fluorometrically determined. The present immunoassay allows detection of 0.8 to 100 pmol/well of TRH.     

Characteristics of a liposome immunoassay on a poly(methyl methacrylate) surface

Liposome immunoassay (LIA) is based on enzyme immunoassay (EIA) but the detection sensitivity could be significantly enhanced by using antibody-coupled immunoliposomes encapsulating HRP (horse radish peroxidase). Here, we applied LIA to non-porous poly(methyl methacrylate) (PMMA) and polystyrene (PS) surfaces to compare its detection sensitivity with that of EIA, using rabbit IgG (a ligand molecule) and anti-rabbit IgG antibody (a capture molecule) as the model system. LIA developed much stronger color signals than EIA, especially at a lower concentration range (< ca. 1 μg mL⁻¹). PMMA showed higher affinity toward rabbit IgG than the PS surface, and the anti-rabbit IgG antibody adsorbed on PMMA was more stable than that on PS. Furthermore, the effects of spot volume and antibody concentration on the signal density were analyzed. The signal density increased as the antibody concentration increased, but it was not significantly affected by the spot volume (2.5–20 μL). In conclusion, LIA on PMMA as a solid support is a very useful, highly sensitive microarray detection system. Sang Youn Hwang and Yoichi Kumada have same rights on this paper.     

New highly sensitive enzyme immunoassay for the determination of pravastatin in human plasma

New highly sensitive enzyme immunoassay (EIA) has been developed and validated for the determination of pravastatin (PRV) in human plasma samples. PRV was coupled to keyhole limpt hemocyanin (KLH) and bovine serum albumin (BSA) via its terminal carboxylic acid group by carbodiimide reagent. PRV-KLH conjugate was used as an immunogen for raising anti-PRV polyclonal antibody in rabbits. The generated anti-PRV antibody recognized PRV with high affinity and selectivity. PRV-BSA conjugate was immobilized onto microwell plates and used as a solid phase. The assay involved a competitive binding reaction between PRV, in plasma sample, and the immobilized PRV-BSA for the binding sites on a limited amount of the anti-PRV antibody. The anti-PRV antibody bound to the plate wells was quantified with horseradish peroxidase-labeled anti-immunoglobulin second anti-rabbit IgG antibody and 3,3′,5,5′-tetramethylbenzidine as a substrate for the peroxidase enzyme. The concentration of PRV in the sample was quantified by its ability to inhibit the binding of the anti-PRV antibody to the immobilized PRV-BSA and subsequently the color development in the assay wells. The conditions of the proposed EIA were investigated and the optimum conditions were employed in the determination of PRV in plasma samples. The assay limit of detection was 0.2 ng mL⁻¹ and the effective working range at relative standard deviation (RSD) of ≤5% was 0.5-20 ng mL⁻¹. The mean analytical recovery of PRV from spiked plasma was 100.9 ± 2.98%. The precision of the assay was satisfactory; RSD was 2.61-3.70 and 3.96-4.17% for intra- and inter-assay precision, respectively. The analytical procedure is convenient, and one can analyze ∼200 samples per working day, facilitating the processing of large-number batch of samples. The proposed EIA has a great value in the routine analysis of PRV in plasma samples for its therapeutic monitoring and pharmacokinetic studies.     

Development of a sensitive, rapid, biotin-streptavidin based chemiluminescent enzyme immunoassay for human thyroid stimulating hormone

A highly sensitive "two-site" chemiluminescent immunoassay specific for human thyroid stimulating hormone (TSH) was developed. The signal amplification was achieved via a biotin-streptavidin system (BSAS). The HRP-luminol-H(2)O(2) chemiluminescent system with high sensitivity was chosen as the detection system. Biotinylated anti-TSH monoclonal antibody (MAb) and HRP-labeled streptavidin were first synthesized. Then the signal amplification was achieved through the interaction between the biotinylated anti-TSH MAb and the HRP-streptavidin conjugate. The light intensity developed was in proportion to the TSH present in the samples. The assay showed little cross-reactivity with three other glycoprotein hormones (human chorionic gonadotropin (HCG), luteinizing hormone (LH), follicle stimulating hormone (FSH)) due to the high specificity of the antibody. The working range for human thyroid stimulating hormone was 0.1-40 mU L(-1). Both the intra-assay and inter-assay coefficients of variation were less than 10% for the BSAS based chemiluminescent enzyme immunoassay (CLEIA). The proposed assay had a sensitivity of 0.01 mU L(-1) which was 10-fold higher than the HRP-MAb conjugate based TSH immunoassay. Thus the higher sensitivity facilitated the clinical testing for thyroid states. The effects of several reaction parameters, such as incubation time, temperature, and reaction volume of the method, were also studied. This method has been successfully applied to the evaluation of TSH in human serum. Compared with the commercial enzyme chemiluminescent immunoassay, the correlation was satisfied.     

New fluorometric enzyme immunoassay for 17beta-estradiol by homogeneous reaction using biotinylated estradiol

A new fluorometric enzyme immunoassay for 17β-estradiol (E2) using biotinylated estradiol (BE) as a probe ligand, is described. In this method, E2 is detected indirectly by a solid-phase avidin-biotin binding assay, in which the biotin is immobilized on a microtiter plate (biotin-plate). After the competitive reaction between E2 and BE for the anti-E2 antibody in solution, the free E2 and BE are separated from the bound forms by means of ultrafiltration. The concentration of BE in the solution is determined from the reaction between the biotin immobilized on the plate and the free BE for the limited biotin binding sites of avidin conjugated with horseradish peroxidase (avidin-HRP), which is added to the solution. The enzymatic reaction of HRP was measured by a fluorometric analysis with the QuantaBlu™ Fluorogenic Peroxidase Substrate (QFPS) in order to detect of the avidin-biotin binding with a high degree of sensitivity. The detection limit and linear range for the determination of E2 were 0.12 nM and from 0.12 to 25 nM, respectively. The relative standard deviations (R.S.D.) for the E2 assay were between 2.2 and 9.1% (n = 3). The cross-reactivity for several other estrogens was also evaluated.     

Enzyme immunoassay screening procedure for the synthetic anabolic estrogens and androgens diethylstilbestrol, nortestosterone, methyltestosterone and trenbolone in bovine urine

Immunoassays are often used for the screening of anabolic residues in edible tissues and excreta (urine, faeces) from inspected animals. Radioimmunoassays have been used for ten years for the determination in biological samples of the main natural and synthetic anabolic estrogens and androgens. In order to simplify the sample preparation and analysis and to reduce the cost, competitive enzyme immunoassays (EIA) were developed for the main synthetic anabolics used illegally in livestock fattening. EIA are based on a competition between the analyte (hormone or metabolite) and the enzyme-labelled hormone for binding to specific antibodies immobilized in wells of a microtitration plate. Two enzymes were evaluated: horseradish peroxidase (HRP) and Bacillus licheniformis beta-lactamase (BLL) using hydrogen peroxide-o-phenylenediamine or benzylpenicillin-starch-iodine as substrates, respectively. The same derivative was used for chemical coupling of the hormone to enzyme (tracer preparation) and to bovine serum albumin to produce specific antibodies in rabbits. Hormone doses that inhibited 50% of the tracer (HRP-hormone) binding to antibody (ID50) were 18, 8, 6 and 11 pg per well for diethylstilbestrol, nortestosterone, methyltestosterone and trenbolone, respectively. These values were lower than those observed in RIA. The reproducibility and accuracy of EIA in urine analysis were similar to those of RIA. Very small amounts of urine were needed (2.5 microliters). This simple method may require less than 2 h. With the BLL-hormone tracer, the enzymatic activity remaining in the wells and hence the hormone content of the sample could be estimated with the naked eye using benzylpenicillin-starch-iodine as substrate.     

Rapid extraction and quantitative detection of the herbicide diuron in surface water by a hapten-functionalized carbon nanotubes based electrochemical analyzer

A solid phase extraction micro-cartridge containing a non-polar polystyrene absorbent matrix was coupled with an electrochemical immunoassay analyzer (EIA) and used for the ultra-sensitive detection of the phenyl urea herbicide diuron in real samples. The EIA was fabricated by using carboxylated carbon nanotubes (CNTs) functionalized with a hapten molecule (an amine functionalized diuron derivative). Screen printed electrodes (SPE) were modified with these haptenized CNTs and specific in-house generated anti diuron antibodies were used for bio-interface development. The immunodetection was realized in a competitive electrochemical immunoassay format using alkaline phosphatase labeled secondary anti-IgG antibody. The addition of 1-naphthyl phosphate substrate resulted in the production of an electrochemically active product, 1-naphthol, which was monitored by using differential pulse voltammetry (DPV). The assay exhibited excellent sensitivity and specificity having a dynamic response range of 0.01 pg mL(-1) to 10 μg mL(-1) for diuron with a limit of detection of around 0.1 pg mL(-1) (n = 3) in standard water samples. The micro-cartridge coupled hapten-CNTs modified SPE provided an effective and efficient electrochemical immunoassay for the real-time monitoring of pesticides samples with a very high degree of sensitivity.     

A highly sensitive enzyme immunoassay for evaluation of 2′-deoxycytidine plasma level as a prognostic marker for breast cancer chemotherapy

A highly sensitive competitive enzyme immunoassay (EIA) has been developed and validated for the determination of the plasma level of 2′-deoxycytidine (dCyd), the potential prognostic marker for breast cancer chemotherapy. This assay employed a monoclonal antibody that recognizes dCyd with a high specificity, and 5′-succinyl-dCyd (5′sdCyd) conjugate of bovine serum albumin (5′sdCyd-BSA) immobilized onto microplate wells as a solid phase. The assay involved a competitive binding reaction between dCyd, in plasma sample, and the immobilized 5′sdCyd-BSA for the binding sites of the anti-dCyd antibody. The bound antibody was quantified with horseradish peroxidase-labeled anti-immunoglobulin second antibody and 3,3′,5,5′-tetramethylbenzidine as a peroxidase substrate. The concentration of dCyd in the sample was quantified by its ability to inhibit the binding of the antibody to the immobilized 5′sdCyd-BSA and subsequently the color formation in the assay. The assay limit of detection was 8 nM and the effective working range at relative standard deviations (R.S.D.s) of ≤10% was 20-800 nM. No cross-reactivity from the structurally related nucleobases, nucleosides, and nucleotides was observed in the proposed assay. Mean analytical recovery of added dCyd was 98-100 ± 3.2-8.2%. The precision of the assay was satisfactory; R.S.D. was 3.4-4.2 and 4.3-8.9% for intra- and inter-assay precision, respectively. The proposed EIA was compared favorably with HPLC method in its ability to accurately measure dCyd spiked into plasma samples. The analytical procedure is convenient, and one can analyze 200 samples per working day, facilitating the processing of large-number batch of samples. The proposed EIA is expected to contribute in further evaluation of dCyd as a prognostic marker for breast cancer chemotherapy and elucidation of the role of dCyd in various biological and biochemical systems.     

Novel and Sensitive Noncompetitive Enzyme Immunoassay to Measure Peptides by Biotinylation

Abstract

A novel and sensitive noncompetitive enzyme immunoassay for angiotensin I as a peptide model is described. Angiotensin I in buffer containing bovine serum albumin or in plasma was biotinylated using sulfosuccinimidyl-6-(biotinamido)hexanoate. the biotinylated angiotensin I was trapped onto anti-angiotensin I IgG-coated polystyrene balls and, after washing to eliminate other biotinylated substances, was eluted with HCl. the biotinylated angiotensin I eluted was reacted with anti-angiotensin I Fab'-peroxidase conjugate and trapped onto streptavidin-coated polystyrene balls. Peroxidase activity bound to the polystyrene balls was assayed by fluorimetry. the detection limit of angiotensin I was 13 fg (10 amol)/tube and 6.5 ng/1 of plasma, which was 10 to 480-fold lower than that previously reported by competitive radioimmunoassay and competitive enzyme immunoassay. and other peptides could also be measured more sensitively by the present method than by competitive radioimmunoassay.     

Enzyme immunoassay of a substance P-like immunoreactive substance in human plasma and saliva.

A sensitive and specific double-antibody enzyme immunoassay (EIA) for a substance P (SP)-like immunoreactive substance (SP-IS) was developed. For competitive reactions, the SP-antibody was incubated with SP standard (or sample) and beta-D-galactosidase labeled Tyr8-SP (delayed addition). Free and antibody-bound enzyme hapten were separated by using an anti-rabbit immunoglobulin G coated immunoplate. Activity of the enzyme on the plate was fluorometrically determined. The present immunoassay allows detection of 0.4 to 10 fmol/well of SP. Using the present EIA, SP-ISs in human saliva and plasma were determined. The level of SP-IS in human saliva was about 7 pmol/l, which was almost three times higher than that in human plasma.     

Universal quantum dot-based sandwich-like immunoassay strategy for rapid and ultrasensitive detection of small molecules using portable and reusable optofluidic nano-biosensing platform

A universal sandwich-like immunoassay strategy based on quantum-dots immunoprobe (QD-labeled anti-mouse IgG antibody) was developed for rapid and ultrasensitive detection of small molecules. A portable and reusable optofluidic nano-biosensing platform was applied to investigate the sandwich-like immunoassay mechanism and format of small molecules, as well as the binding kinetics between QD immunoprobe and anti-small molecule antibody. A two-step immunoassay method that involves pre-incubation mixture of different concentration of small molecule and anti-small molecule antibody, and subsequent introduction of QD immunoprobe into the optofluidic cell was conducted for small molecule determination. Compared with the one-step immunoassay method, the two-step immunoassay method can obtain higher fluorescence signal and higher sensitivity index, thus improving the nano-biosensing performance. Based on the proposed strategy, two mode targets, namely, microcystin-LR (MC-LR) and Bisphenol A (BPA) were tested with high sensitivity, rapidity, and ease of use. A higher concentration of small molecules in the sample led to less anti-small molecule antibody bound with antigen-carrier protein conjugate immobilized onto the sensor surface, and less QD immunoprobes bound with anti-small molecule antibody. This phenomenon lowered the fluorescence signal detected by nano-biosensing platform. Under optimal operating conditions, MC-LR and BPA exhibited a limit of detection of 0.003 and 0.04 μg/L, respectively. The LODs were better than those of the indirect competitive immunoassay method for small molecules via Cy5.5-labeled anti-small molecule antibody. The proposed QD-based sandwich-like immunoassay strategy was evaluated in spiked water samples, and showed good recovery, precision and accuracy without complicated sample pretreatments. All these results demonstrate that the new detection strategy could be readily applied to the other trace small molecules in real water samples.