A new and disposable electrochemical immunosensor was designed for detection of alpha-fetoprotein (AFP), as a model analyte, with sensitivity enhancement based on enzyme-catalyzed silver deposition onto irregular-shaped gold nanoparticles (ISGNPs). The assay was carried out with a sandwich-type immunoassay protocol by using ISGNP-labeled anti-AFP antibodies conjugated with alkaline phosphatase (ALP–Ab2) as detection antibodies. The enzymatically catalytic deposition of silver on the electrode could be measured by stripping analysis in KCl solution due to the Ag/AgCl solid-state voltammetric process. Several labeling protocols including spherical gold nanoparticle-labeled ALP–Ab2 and ISGNP-labeled ALP–Ab2 were investigated for determination of AFP, and improved analytical properties were achieved with the ISGNP labeling. With the ISGNP labeling method, the effects of incubation time and incubation temperature for antigen-antibody reaction, and deposition time of silver on the current responses of the electrochemical immunosensors were also monitored. Under optimal conditions, the electrochemical immunosensor exhibited a wide dynamic range from 0.01 ng mL−1 to 200 ng mL−1 with a detection limit of 5.0 pg mL−1 AFP. The immunosensor displayed a good stability and acceptable reproducibility and accuracy. No significant differences at the 95% confidence level were encountered in the analysis of 10 clinical serum samples between the developed immunoassay and the commercially available electrochemiluminescent method for determination of AFP. 相似文献
We describe an electrochemical immunosensor for the simultaneous determination of alpha-fetoprotein (AFP) and prostate specific antigen (PSA) via a modified glassy carbon electrode. Silica nanoparticles (200–300 nm i.d.) with good monodispersity and uniform shape were synthesized, and the following species were then consecutively immobilized on their surface: gold nanoparticles (AuNPs; 5–15 nm i.d.), secondary antibody (Ab2) and the redox-probes Azure A or ferrocenecarboxy acid (Fc). In parallel, two types of primary antibodies (Ab1) were co-immobilized on the surface of the dissolved reduced graphene oxide sheets (rGO) that were also decorated with AuNPs. In the presence of antigens (AFP or PSA), the Ab2/Si@AuNPs carrying Azure A and Fc are attached to the AuNP/rGO conjugate via a sandwich type immunoreaction. Differential pulse voltammetry (DPV) was employed to measure the resulting changes in the signal of Fc or Azure A. Two well-resolved oxidation peaks, one at −0.48 V (corresponding to Azure A) and other at + 0.12 V (corresponding to Fc; both vs. SCE) can be observed in the DPV curves. Under optimal conditions, AFP and PSA can be simultaneously determined in the range from 0.01 to 25 ng mL‾1 for AFP, and from 0.012 to 25 ng mL‾1 for PSA. The detection limits are 3.3 pg mL‾1 for AFP and 4.0 pg mL‾1 for PSA (at a signal-to-noise ratio of 3). The method was applied to (spiked) real sample analysis, and the recoveries are within 96.0 and 107.2 % for PSA, and within 100.9 and 105.8 % for AFP, indicating that this dual immunosensor matches the requirements of clinical analysis.
As IgM is the first isotype of antibody which appears in blood after initial exposure to a foreign antigen in the pattern
of primary response, detection, and quantification of this molecule in blood seems invaluable. To approach these goals, generation,
and characterization of a highly specific mAb (monoclonal antibody) against human IgM were investigated. Human IgM immunoglobulins
were used to immunize Balb/c mice. Spleen cells taken from the immunized animals were fused with SP2/O myeloma cells using
PEG (polyethylene glycol, MW 1450) as fusogen. The hybridomas were cultured in HAT containing medium and supernatants from
the growing hybrids were screened by enzyme-linked immunosorbent assay (ELISA) using plates coated with pure human IgM and
the positive wells were then cloned at limiting dilutions. The best clone designated as MAN-1, was injected intraperitoneally
to some Pristane-injected mice. Anti-IgM mAb was purified from the animals’ ascitic fluid by protein-G sepharose followed
by DEAE-cellulose ion exchange chromatography. MAN-1 interacted with human IgM with a very high specificity and affinity.
The purity of the sample was tested by SDS-PAGE and the affinity constant was measured
( K\texta = \text3.\text5 ×\text10\text9\textM\text - 1 ) \left( {{K_{\text{a}}} = {\text{3}}.{\text{5}} \times {\text{1}}{0^{\text{9}}}{{\text{M}}^{{\text{ - 1}}}}} \right) . Immunoblotting and competitive ELISA were done and the results showed that the harvested antibody recognizes a conformational
epitope on the μ chain of human IgM and there was no cross-reactivity with other subclasses of immunoglobulins. Furthermore,
isotyping test was done and the results showed the subclass of the obtained mAb which was IgG1κ. 相似文献
Abstract A sensitive enzyme immunoassay is described for the determination of the urea herbicide methabenzthiazuron. The assay is carried out with polyclonal antibodies, which were raised in rabbits by immunization with a methabenzthiazuron-BSA conjugate containing five methabenzthiazuron residues per molecule. The ELISA was optimized on microtiter plates with a peroxidase-methabenzthiazuron tracer. The middle of the test (50% B/B0) was found at 1.0 μg/l. The lower detection limit of methabenzthiazuron is c. 0.05 μg/l. Samples can be measured up to 10 μg/l methabenzthiazuron (upper detection limit). The assay does not require concentration or clean-up steps for drinking or ground water samples. Validation experiments showed a good accuracy and precision. Work with monoclonal antibodies is in progress. 相似文献
Herein is reported an immunochemical approach to determine hyodeoxycholic acid using hybridoma-secreting monoclonal antibodies. The hyodeoxycholic acid-specific antibody was produced by fusing splenocytes immunized with a hyodeoxycholic acid-bovine serum albumin conjugate with a hypoxanthine–aminopterin–thymidine-sensitive mouse myeloma cell line (SP2/0). The antibody was highly specific for hyodeoxycholic acid, with less than 0.05 percent cross-reactivity to over fifty structurally related compounds. The antihyodeoxycholic acid monoclonal antibody was then used to develop a rapid, specific, and sensitive indirect competitive enzyme-linked immunosorbent assay (icELISA) for the determination of hyodeoxycholic acid in pharmaceutical compounds. The linear dynamic range was from 0.48 to 62.5 nanograms per milliliter with an IC50 value of 8 nanograms per milliliter. The icELISA results correlated well with a conventional high-performance liquid chromatography method for the determination of hyodeoxycholic acid (R2 = 0.9982). This study shows that the icELISA method was successfully applied to the quantification of hyodeoxycholic acid in pharmaceutical products. 相似文献
Antiangiogenic monoclonal antibodies in combination with therapeutic radionuclides are potential targeted therapy agents in
cancer. In this study, bevacizumab was successively labeled with [166Ho]HoCl3 after conjugation with DOTA-NHS-ester with a radiochemical purity of higher than 95% (RTLC). The conjugates were purified
by molecular filtration, the average number of DOTA conjugated per mAb was calculated and total concentration was determined
by spectrophotometric method and the average chelate to antibody ratio (c/a) for the conjugate used in this study was 5.8:1
and protein integrity experiments (SDS-PAGE). The biodistribution studies in wild-type rats demonstrate a similar pattern
to the other radiolabeled anti-vascular endothelial growth factor A (VEGF-A) immunoconjugates. 166Ho-DOTA-bevacizumab is a potential compound for therapy/imaging of VEGF-A expression in oncology. 相似文献
Polyclonal antibody production in mammals is generally associated with multiple injections of antigens and adjuvant and repeated
blood sampling procedures. In the production of second polyclonal antibodies, a number of critical steps can be identified
that may influence the outcome of the animal experiment (immunological results and the pain and suffering of the animals).
The goal of this work was to evaluate critical steps in the production of these antibodies, and to optimize production protocols
that will ultimately result in effective antibody responses. This work was achieved through immunization of two healthy sheep
by purified Alpha feto protein (AFP) antigen. Also, the present study involved the preparation of AFP standards from human
cord blood. Furthermore, preparation of a radiolabeled AFP tracer of a high specific activity using 125I isotope by chloramine T method was undertaken. Moreover, this study provides that there was no observable difference between
the Scottish Antibody Production Unit (SAPU) and Sigma SAM IgG and the two second antibodies obtained from the local sheep
sera. 相似文献
Methods based on immunoassays have been developed for cancer biomarker alpha-fetoprotein (AFP), but most involve complicated or stripping procedures and are unfavourable for routine use. Herein we report the proof-of-concept of simple and point-of-care (POC) immunoassay for AFP in hepatocellular carcinoma on a portable personal glucometer (PGM) by using antibody-invertase cross-linkage nanoparticles as the signal-generation tags. Antibody-invertase cross-linkage nano tags were synthesized by using reverse micellar method with glutaraldehyde. The POC immunoassay was carried out on monoclonal anti-AFP capture antibody-coated microplate with a sandwich-type reaction mode. Introduction of invertase nano labels accompanying target AFP could hydrolyse sucrose into glucose and fructose. The produced glucose molecules could be determined on a portable personal glucometer. Relative to unimolecular invertase labelling, improved analytical features were acquired with antibody-invertase nano labelling. With the nano labels, PGM-based immunoassay exhibited good electrochemical responses for the detection of AFP, and allowed detection of AFP at a concentration as low as 5.4 pg mL−1. Moreover, a good repeatability and intermediate precision could be found down to 12.68 %. Good well-matched results were obtained for analysis of human serum specimens between POC immunoassay and commercial human AFP ELISA kit. 相似文献
A new technique that uses gold immunochromatographic strips enhances the detection sensitivity by inducing the clustering of additional gold nanoparticles (AuNPs) around the immunogold particles immobilized on nitrocellulose strips. The additional AuNPs provide an intense signal that can be detected by the naked eye. The AuNPs were synthesized and conjugated to monoclonal antibodies using self-assembly. Other antibodies were immobilized in a defined detection zone on the nitrocellulose membrane. The detection principle is based on a “sandwich” immunoreaction, where gold-labeled antibodies serve as signal vehicles. To improve the sensitivity of the strips, we use a mixture of 1% HAuCl4 and 10 mmol L−1 NH2OH·HCl to “enlarge” the gold nanoparticles. The detecting limits of Avian influenza virus (AIV) and Newcastle disease virus (NDV) are significantly increased. Compared with commercial test strips, this method is 100-fold more sensitive. This method is easy to perform and can be carried out on-site in test laboratories. 相似文献
In this study, magnetic multimodal nanoparticles with potential applications in magnetic- and nuclear-medicine imaging, magnetic resonance imaging, hyperthermia, and theranostic (therapeutic and diagnostic), applications were prepared by coating iron oxide nanoparticles with silica (core–shell), functionalizing with aminopropyltriethoxy silane and coupling with diethylenetriamine pentaacetic acid ligand (DTPA). Radiolabeling of core–shell–DTPA particles with 68Ga radiometal was carried out through chelation of 68Ga(III) ions by DTPA and was used for positron emission tomography. The biodistribution of the 68Ga-radiolabeled magnetic nanoparticles compared to free 68Ga(III) was checked in normal Balb/c mice up to 2 h. 相似文献
Competitive electrochemical enzyme-linked immunosorbent assays based on disposable screen-printed electrodes have been developed for quantitative determination of ochratoxin A (OTA). The assays were carried out using monoclonal antibodies in the direct and indirect format. OTA working range, I50 and detection limits were 0.05-2.5 and 0.1-7.5 μg L−1, 0.35 (±0.04) μg L−1 and 0.9 (±0.1) μg L−1, 60 and 100 μg L−1 in the direct and indirect assay format, respectively. The immunosensor in the direct format was selected for the determination of OTA in wheat. Samples were extracted with aqueous acetonitrile and the extract analyzed directly by the assay without clean-up. The I50 in real samples was 0.2 μg L−1 corresponding to 1.6 μg/kg in the wheat sample with a detection limit of 0.4 μg/kg (calculated as blank signal −3σ). Within- and between-assay variability were less than 5 and 10%, respectively. A good correlation (r = 0.9992) was found by comparative analysis of naturally contaminated wheat samples using this assay and an HPLC/immunoaffinity clean-up method based on the AOAC Official Method 2000.03 for the determination of OTA in barley. 相似文献
The preparation of [1-3H]cyprodime ( 2 ), which has a specific activity of 31.6 Ci/mmol was carried out by catalytic tritiodehalogenation of 1-bromocyprodime ( 3 ). Bromination of cyprodime was performed by using chloroperoxidase, KBr, and H2O2. 相似文献
In order to investigate relationships between human carcinogenesis and dietary carcinogens, one hybridoma cell line secreting a monoclonal antibody against 2-amino-3-methylimidazo(4,5-f)quinoline (IQ), a dietary carcinogen, was produced by fusing splenocytes from Balb/c mice immunized with IQ-Lysine(Lys)-Ascaris protein conjugate. The subclass of monoclonal anti-IQ antibody was determined by double immunodiffusion using culture medium and identified as IgG1. Monoclonal anti-IQ antibody was purified from ascites fluids of Balb/c mice with affinity chromatography on Protein A-Sepharose CL4B and analyzed concerning its cross-reactivity and sensitivity with RIA. Finally, we showed that our monoclonal antibody recognized IQ, 2-amino-3,4-dimethylimidazo(4,5-f)quinoline (MeIQ) and several beta-carbolines more intensely and that the sensitivity to IQ was 23 nmol in 50% displacement. 相似文献
A generic hapten (H1), 3-(4-Dimethoxyphosphorothioyloxy phenyl)propanoic acid, was synthesized to produce monoclonal antibodies (mAbs) for the determination of organophosphorus pesticides (OPs) in a class-specific manner. Six heterologous haptens were designed to study the effect of hapten heterology on immunoassay sensitivity. Several mice were immunized with this H1-BSA immunogen. Spleen cells of two immunized mice were fused with myeloma cells, and the resulting hybridomas were screened using H1-OVA. In a competitive indirect enzyme-linked immunosorbent assay (ELISA) format, three hybridoma cell lines (B4-C6, D12-B5, E5-H2) that produced mAbs with high selectivity and broad specificity were selected and expanded. The monoclonal antibody D12-B5 with higher titer was chosen for further study. In heterologous assay, the combination of D12-B5 and coating antigen H7-OVA constituted a particularly sensitive assay for competitive indirect ELISA and showed broad specificity for the determination of OPs, including parathion-methyl, chlorpyrifos-methyl, tolclofos-methyl, fenthion, malathion, fenitrothion. This combination resulted in the IC50 of 0.58-10.47 µg/ml. 相似文献
Bothrops jararacussu venom’s (Bj2015) batch was biomonitored quarterly for one year to assess phospholipase A2 (PLA2) activity, immunogenicity, neurotoxicity, and myotoxicity. In silico models were applied to evaluate losses using decay model and recoveries by predictive trend analysis. Mice were immunized with Bj2015. Antibodies were detected by double-immunodiffusion and total protein and albumin were measured. Neuromuscular blockade-induced by 40 μg mL?1 venom solution was carried out using mouse nerve phrenic-diaphragm preparation. Resulting muscles were submitted to light microscopy to evaluate the myotoxicity. PLA2 activity of 0.1 mg mL?1 Bj2015 was measured using 4-nitro-3-(octanoyloxy)benzoic acid as substrate. Over time, greater losses occurred in neurotoxicity than PLA2, but not in myotoxicity and immunogenicity. Concluding, the neurotoxicity decrease can be related to enzymatic losses, including PLA2. Depending on the purpose of use, the collected venom responds on a long time, avoiding unnecessary new collections, improving life quality of animals in captivity and increasing their longevity. 相似文献