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.     

Production of monoclonal antibodies against fullerene C60 and development of a fullerene enzyme immunoassay

The aim of the present study was to produce monoclonal anti-fullerene C(60) antibodies and to develop the enzyme immunoassay for the detection in the first use of free fullerene C(60) both in solutions and in multicomponent biological probes. The immunization of mice with the conjugate of fullerene C(60) carboxylic derivative with thyroglobulin synthesized by carbodiimide activation led to the production of eight clones of anti-fullerene antibodies. The specificity of the antibody-fullerene binding was confirmed. Indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed for the determination of water-soluble protein-conjugated fullerene, the fullerene aminocaproic acid, fullerenol and for pristine fullerene in solution. To solubilize extremely hydrophobic free fullerene C(60) a specially selected water-organic mixture compatible with immunoassay was proposed. The detection limit of free fullerene C(60) in solution was 2 μg L(-1). Fullerene C(60) was also detected by ELISA in organ homogenates of rats intraperitoneally or intragastrically administered with fullerene. To reduce the influence of biomatrices on the assay results a technique was developed for the biological sample pretreatment by the extraction of C(60) from bioprobe by toluene followed by the evaporation of toluene and dissolution of the fullerene-containing extract in the selected water-organic media. The ELISA procedure in the first use allowed the detection of fullerene C(60) in different tissues.     

Cu-DNAzyme facilitates highly sensitive immunoassay

A simple Cu-DNAzyme system is used for signal transduction of a CuO nanoparticle-labeled immunoassay, which makes the immunoassay fast, simple, cost-effective, and sensitive, thus promising for biomedical applications and point-of-care testing.     

Cu-DNAzyme facilitates highly sensitive immunoassay

Fast and sensitive antigen detection is important in biomedical research and development. Despite, being invented 48 years ago, the enzyme-linked immunosorbent assay (ELISA) remains one of the most successful and widely employed bioanalytical techniques in research and clinical diagnostics due to its reliability and simplistic design. Recently, nanotechnology has offered efficient signal reporting. In spite of some improvements in these systems, there are typically increased material costs involved and the need for expensive equipment or complicated chemical processes, thus negating any possible benefits over ELISA. Herein, we communicate a simple Cu-DNAzyme system for signal transduction of a CuO nanoparticle-labeled immunoassay. The reported immunoassay amplifies signal generation similar to traditional ELISA and is fast, simple, cost-effective, and sensitive, holding promise for biomedical applications and point-of-care testing.     

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.     

Preparation of antibodies and development of a sensitive immunoassay with fluorescence detection for triazine herbicides

Specific polyclonal antibodies against s-triazine herbicides were obtained by preparing immunogens coupling home-synthesized haptens derivatives of simazine (6-chloro-N-ethyl-N′-ethyl-1,3,5-triazine-2,4-diamine) to lysine groups of hemocyanin from keyhole limpets and bovine serum albumin carrier proteins. Three highly sensitive rabbit antisera were obtained and evaluated with a battery of six enzyme tracers derived from triazine structures in an optimized ELISA format. The antiserum As8 and the HRP-2f tracer, which yield the best assay sensitivity for simazine (detection limit 0.11 ± 0.02 μg L⁻¹, IC₅₀ 0.88 ± 0.04 μg L⁻¹), were applied to the development of a sensitive flow-through immunoassay for the analysis of this herbicide. The automated assay was based on a direct competitive immunosorbent assay and fluorescence detection. The optimized method presents an IC₅₀ value of 0.35 ± 0.04 μg L⁻¹ with a detection limit of 1.3 ± 0.9 ng L⁻¹ and a dynamic range from 0.010 to 7.5 μg L⁻¹ simazine. The generic nature of the antiserum was shown by good relative cross-reactivities with other triazines such as atrazine (420%) or propazine (130%) and a lower response to terbutylazine (6.4%) and desethyl-atrazine (2.2%). No cross-reactivity was obtained for nonrelated pesticides such as 2,4-dichlorophenoxyacetic acid or linuron and the assay could be applied as a screening method for triazine herbicides. The total analysis time was 30 min per determination and the immunosensor could be reused for more than 150 cycles without significant loss of activity. The immunosensor has been successfully applied to the direct analysis of simazine in surface water samples at the nanogram per liter level. The results obtained by comparative analysis of the immunosensor with a chromatographic procedure for triazines showed a close correspondence.     

Development of a highly sensitive inhibition immunoassay for microcystin-LR

The development of a rapid one-step antigen-immobilized inhibition ELISA for microcystin-LR is described. For microplate coating a microcystin-biotin conjugate was synthesized. Using the commercially available monoclonal antibody MC10E7 in our newly established assay, IC₅₀ values of 0.045 μg l⁻¹ have been achieved. The detection limit for microcystin-LR was 4 ng l⁻¹. Considering the guidelines proposed by the world health organization (WHO) for microcystin-LR in drinking water (1 μg l⁻¹) the sensitivity of our test is more than sufficient. The period of assay processing could successfully be shortened to about 3 h without any loss in sensitivity. The suitability of the newly developed assay was evaluated with microcystin-LR spiked environmental water samples. Recovery rates for microcystin-LR between 60 and 165% were obtained in the linear range of the test format. The antigen-immobilized test format provides a highly reproducible, easy, and fast to perform detection system for microcystin allowing an internal retrospective quality control of the assay.     

Development of highly sensitive chemiluminescence enzyme immunoassay based on the anti-recombinant HC subunit of botulinum neurotoxin type A monoclonal antibodies

Botulinum neurotoxins (BoNTs) are the most poisonous substances ever known. The early detection of these toxins could bear more time for appropriate medical intervention. The standard method for detecting BoNTs is the mouse bioassay, which is time consuming (up to 4 days) and requires a large number of laboratory animals. The immunologic detection methods could detect the toxins within a day, but most of these methods are less sensitive compared with the mouse bioassay due to the lack of high-affinity antibodies. Recently, the recombinant H_C subunit of botulinum neurotoxin type A (rAH_C) was expressed as an effective vaccine against botulism, indicating that the rAH_C could be an effective immunogen that raises the monoclonal antibody (mAb) for detecting BoNT/A. After immunized BALB/c mice with rAH_C, 56 mAbs were generated. Two of these mAbs were selected to establish a highly sensitive sandwich chemiluminescence enzyme immunoassay (CLEIA), in which FMMU-BTA-49 and FMMU-BTA-22 were used as capture antibody and detection antibody, respectively. The calculated limit of detection (LOD) based on molecular weight of rAH_C and BoNT/A reached 0.45 pg mL⁻¹. This CLEIA can be used in the detection of BoNT/A in matrices such as milk and beef extract. This method has 20–40 fold lower LOD than that of the mouse bioassay and takes only 3 h to complete the detection, indicating that it can be used as a valuable method to detect and quantify BoNT/A.     

A highly sensitive fluorescent immunoassay based on avidin-labeled nanocrystals

Nanocrystals of the fluorogenic precursor fluorescein diacetate (FDA) were applied as labels in order to improve on the assay sensitivity achieved in our previous studies. Each FDA nanocrystal can be converted into ∼2.6×10⁶ fluorescein molecules, which is useful for improving immunoassay sensitivity and limits of detection. NeutrAvidin was simply adsorbed onto the surface of the FDA nanocrystals, which were coated with distearoylglycerophosphoethanolamine (DSPE) modified with amino(poly(ethylene glycol))(PEG(2000)-Amine) as an interface for coupling biomolecules. This can be applied to detect different kinds of analytes that are captured by corresponding biotinylated biomolecules in different bioanalytical applications. The applicability of the NeutrAvidin-labeled nanocrystals was demonstrated in an immunoassay using the labeled avidin–biotin technique. Biotinylated antibody and FDA-labeled avidin were applied to the assay sequentially. The performance was compared with the traditional sandwich-type assay for mouse immunoglobulin G detection. Following the immunoreaction, the nanocrystals were released by hydrolysis and dissolution instigated by adding a large volume of organic solvent/sodium hydroxide mixture. The limit of detection was lower (by a factor of 2.5–21) and the sensitivity was (3.5–30-fold) higher than immunoassays using commercial labeling systems (FITC and peroxidase). This study shows that using fluorescent nanocrystals in combination with the avidin–biotin technique can enhance assay sensitivity and provide a lower limit of detection without requiring long incubation times as in enzyme-based labels.     

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.     

A rapid and sensitive 384-microtiter wells format chemiluminescent enzyme immunoassay for clenbuterol

A fast and sensitive chemiluminescent (CL) enzyme immunoassay for clenbuterol (CLB) analysis in bovine urine has been developed. Clenbuterol (CLB) specific polyclonal antibodies were raised in rabbit using a CLB azo derivative conjugated with ovalbumin. Horseradish peroxidase (HRP) was used as label and conjugated with the same derivative. In the developed competitive method, antibodies were immobilized on 384-wells black polystyrene microtiter plates; the sample volume was 20 mul and HRP-labeled CLB activity was immediately measured, using different CL substrates, after 10 min incubation time. Emitted light was recorded using a sensitive back-illuminated, cooled CCD camera or a conventional, photomultiplier-based micrtotiter plate reader. The developed method fulfills all the requirements of precision (CV below 10%) and accuracy (mean recovery from 96 to 110%) with a detection limit of 0.08 ppb in urine matrix. The use of 384-wells microtiter plate allows a 5-fold reduction in reagent quantity and the CL detection improves the detectability of the HRP-labeled tracer, thus reducing analysis time. The developed method is therefore suitable for high-throughput screening of CLB in urine samples, with reduced costs as compared with conventional colorimetric enzyme immunoassays, thanks to the possibility to optimize the system in non-equilibrium immunological conditions and with a very fast chemiluminescence detection of the HRP-label activity.     

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.     

A highly sensitive caffeine immunoassay based on a monoclonal antibody

A new immunoassay has been developed based on a commercially available anti-caffeine monoclonal antibody and a de novo synthesized tracer, using horseradish peroxidase and UV–visible detection. Caffeine, which is frequently found in surface waters, can be quantified with a relative error lower than 20% for concentrations above 0.025 μg L^?1 (limit of quantitation, direct analysis). The limit of detection is 0.001 μg L^?1 and can be reduced by solid-phase extraction (SPE). Moreover, with minor adaptations, the assay can be used to quantify caffeine in several beverages, shampoo, and caffeine tablets. The results obtained by ELISA correlate well with those from liquid chromatography–tandem mass spectrometry (LC–MS–MS) for the tested matrices. Several surface waters from Berlin were analysed and all tested positive for caffeine, with concentrations higher than 0.030 μg L^?1. In one run 66 samples can be analysed within 2 h.

Stabilization of phospholipid polymer surface with three-dimensional nanometer-scaled structure for highly sensitive immunoassay

A phospholipid polymer platform and an antibody as a bioaffinity ligand were used to construct a biointerface for a highly sensitive immunoassay. The platform had a nanometer-scaled particle deposition surface and it was constructed with poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-p-nitrophenyloxycarbonyl poly(ethylene glycol) methacrylate (MEONP)] (PMBN) by an electrospray deposition (ESD) method. The PMBN surface could immobilize specific antibodies through covalent chemical bonding by the reaction between MEONP units and amino groups in the antibody. In addition, the PMBN could prevent nonspecific protein adsorption from an analyte. However, the nanometer-scaled structure of the PMBN lost its shape after immersion in an aqueous medium. To stabilize the nanometer-scaled structure in an aqueous medium, the PMBN was cross-linked with 1,4-butylenediamine and then heat-treated. These treatments effectively improved the stability of the nanometer-scaled structure, that is, the structure had a high porosity even after immersing in an aqueous medium. The stabilization affected the specific signal in the enzyme-linked immunosorbent assay (ELISA), that is, the specific signal in ELISA was enhanced.     

A highly sensitive europium nanoparticle-based lateral flow immunoassay for detection of chloramphenicol residue

A europium nanoparticle-based lateral flow immunoassay for highly sensitive detection of chloramphenicol residue was developed. The detection result could be either qualitatively resolved with naked eye or quantitatively analyzed with the assistance of a digital camera. In the qualitative mode, the limit of detection (LOD) was found to be 0.25 ng/mL. In the quantitative mode, the half-maximal inhibition concentration (IC50) was determined to be 0.45 ng/mL and the LOD can reach an ultralow level of 0.03 ng/mL, which is ~100 times lower than that of the conventional colloidal gold-based lateral flow immunoassay. Potential application of the established method was demonstrated by analyzing representative cow milk samples.

Development of a highly sensitive and specific immunoassay for enrofloxacin based on heterologous coating haptens

In the paper, an enzyme-linked immunosorbent immunoassay (ELISA) for detection of enrofloxacin was described using one new derivative of enrofloxacin as coating hapten, resulting in surprisingly high sensitivity and specificity. Incorporation of aminobutyric acid (AA) in the new derivative of enrofloxacin had decreased the IC₅₀ of the ELISA for enrofloxacin from 1.3 μg L⁻¹ to as low as 0.07 μg L⁻¹. The assay showed neglect cross-reactivity for other fluoroquinolones but ofloxacin (8.23%), marbofloxacin (8.97%) and pefloxacin (7.29%). Analysis of enrofloxacin fortified chicken muscle showed average recoveries from 81 to 115%. The high sensitivity and specificity of the assay makes it a suitable screening method for the determination of low levels of enrofloxacin in chicken muscle without clean-up step.     

免疫毛细管电泳-激光诱导荧光分析DNA加合物的方法学研究

DNA加合物是一类重要的生物标志物,可应用于人体致癌物暴露监测、癌症风险评价和人群易感性研究。DNA加合物作为生物标志物的应用需要安全、灵敏、快速的先进分析技术。我们利用免疫毛细管电泳-激光诱导荧光分析,发展了高灵敏的DNA加合物分析方法和技术。本文主要介绍了相关的仪器研制及方法学研究。方法学研究涉及DNA加合物荧光探针的合成和表征、抗体与DNA加合物的相互作用及其结合计量学、抗原-抗体复合物的稳定化和DNA驱动电泳聚焦技术。