人血清中cTnI的化学发光酶免疫分析研究——实验条件的优化

采用高效化学发光试剂3-(2'-螺旋金刚烷)-4-甲氧基-4-(3"-羟基)苯-1,2-二氧杂环丁烷磷酸(AMPPD)作为检测底物, 并将传统的ELISA两步双抗夹心法改为一步法, 得到了高灵敏测定人血清中心肌肌钙蛋白I(cTnI)的化学发光酶免疫分析优化条件. 采用单因素变化法和方阵滴定法得到的最佳实验条件为: 捕获抗体包被浓度为10.0 μg/mL, 以pH=7.0的PBS作为免疫反应缓冲底液, 以含质量分数为1.0%的BSA pH=9.6的碳酸盐溶液缓冲液, 于4 ℃封闭过夜, 生物素-检测抗体(Biotin-IgG2)以及碱性磷酸酶-亲和素(ALP-Avidin)结合物均采用1:2000稀释度, 免疫反应条件为37 ℃, 孵育时间60 min, 以去离子水作为洗涤剂, 以1:100稀释的AMPPD作为发光反应底物, 发光反应时间10 min(37 ℃). 检出限为0.02 ng/mL, 比现行ELISA法灵敏度提高一个数量级; 测定周期约75 min, 比两步法ELISA快得多; 线性范围(0.04~36.20 ng/mL)比ELISA法扩宽了两个数量级; 加标回收率97.5%~102.8%, 对标准样品的测定结果与用ELISA法的测定结果吻合; 重复性好, 3个样品批内变异系数均小于8.5%(n=12).     

MCM-41 mesoporous material modified carbon paste electrode for the determination of cardiac troponin I by anodic stripping voltammetry

An anodic stripping voltammetric method for the determination of cardiac troponin I (cTnI) at a MCM-41 mesoporous material modified carbon paste electrode (MCM-MCPE) was investigated. The test was based on the dual monoclonal antibody “sandwich” principle using colloidal gold as a labeled substrate. Four main steps were carried out to obtain the analytical signal, i.e. electrode preparation, immunoreaction, silver enhancement, and anodic stripping voltammetric detection. The anodic stripping peak current increased linearly with the concentration of cTnI over the range of 0.8-5.0 ng/ml. A detection limit of 0.5 ng/ml was obtained. The established method was applied to detect cTnI in acute myocardial infarction (AMI) samples using routine enzyme-linked immunoadsorbent assay (ELISA) for comparison analysis, and good results were obtained.     

心衰标志物可视化联合检测方法研究

基于夹心免疫原理和表面免疫吸附模型,采用Image Pro Plus软件对硅片表面磁颗粒分布图像进行定量化处理,建立了硅片表面心衰标志物可视化联合检测方法。研究表明,该方法从抗体包被到获得检测结果不超过1 ｈ,操作简单,在单张硅片上可实现心衰标志物NT-proBNP和cTnI的联合检测。NT-proBNP和cTnI的最低检出限分别为21.8 pg/mL和0.034 ng/mL,检测范围分别为21.8~40000 pg/mL和0.034~50 ng/mL。83例不同程度心血管疾病患者的血清样本的检测结果与商品化免疫分析仪检测结果一致,相关系数r>0.98。因此,该快速免疫检测方法可以满足临床检测心衰标志物的需要。     

IgG anti-gliadin determination with an immunological microfluidic system applied to the automated diagnostic of the celiac disease

In the present article, a novel microfluidic immunosensor coupled with electrochemical detection for anti-gliadin IgG antibody quantification is proposed. This device represents an important tool for a fast, simple, sensitive, and automated diagnostic for celiac disease, which is carried out through detection of anti-gliadin IgG antibodies present in human serum samples. Celiac disease (CD) is an autoimmune disease generated by gluten protein fractions called prolamins. This pathology affects about one in 250 people around the world, produces intestinal inflammation, villous atrophy, and crypt hyperplasia, which causes a range of symptoms including altered bowel habits, malnutrition and weight loss. Our immunosensor consists of a Plexiglas device coupled to a gold electrode, with a central channel containing 3-aminopropyl-modified controlled pore glass (AP-CPG). The quantification of anti-gliadin IgG antibodies was carried out using a heterogeneous, non-competitive enzyme-linked immunosorbent assay (ELISA) in which IgG antibodies bound to gliadin protein, immobilized on AP-CPG, were determined by alkaline phosphatase (AP) enzyme-labeled second antibodies specific to human IgG. The p-aminophenyl phosphate (p-APP) was converted to p-aminophenol (p-AP) by AP, and the electroactive product was quantified on a gold electrode at 0.250 V. The calculated detection limits for electrochemical detection and the ELISA procedure were 0.52 and 2.72 UR mL⁻¹, respectively, and the within- and between-assay coefficients of variation were below 5.8%. The optimized procedure was applied to the determination of anti-gliadin IgG antibodies in human serum samples.     

Recombinant OmpL1 Protein as a Diagnostic Antigen for the Detection of Canine Leptospirosis

Microscopic agglutination test (MAT) is the standard method for the diagnosis of leptospirosis, which is laborious and the interpretation of the results is subjective. The present work describes the use of recombinant-based IgG ELISA for the serodiagnosis of leptospirosis. We used recombinant outer membrane protein OmpL1 as an antigen for conducting IgG enzyme-linked immunosorbent assay (ELISA). A total of 475 canine serum samples were subjected to IgG ELISA; 294 sera were positive to ELISA, while 283 were positive to MAT. All samples that were positive to MAT were positive to ELISA also, however, few samples which were negative to MAT were positive to ELISA, which suggested that recombinant-based IgG ELISA showed 100 % sensitivity when compared to MAT. Thus, this present study showed that recombinant OmpL1-based IgG ELISA appears to be a better alternative to MAT for the diagnosis of leptospirosis and rOmpL1 protein could be used as a potential diagnostic antigen in different assay formats for leptospirosis.     

Enzyme-linked immunosorbent assays based on peroxidase labels and enzyme-amplified lanthanide luminescence detection

The enzyme-amplified lanthanide luminescence (EALL) detection is developed and applied for the determination of peroxidase as marker in enzyme-linked immunosorbent assays (ELISA). The detection scheme is based on the peroxidase catalysed dimerization of 4-hydroxyphenylpropionic acid (pHPPA) and the subsequent formation of a ternary complex with Tb(III)EDTA. Quantum yields and fluorescence lifetimes of the luminescent species are presented to give an estimate of the potential of this procedure. Two different ELISA were performed with the EALL detection scheme. For the first, a model ELISA for the determination of goat anti-rabbit IgG, a limit of determination of 3 micrograms dm-3 (2 fmol) of the antibody could be achieved. As second model assay, a commercial ELISA kit was successfully validated for the new detection scheme. Photometric and EALL detection were in good agreement for the determination of human anti-gliadin IgA in serum.     

A Highly-Sensitive Colorimetric Assay Method for Antibody Array Based on an Tyramide Signal Amplification System

In this work, a highly-sensitive and cost-effective detection approach based on the integration of tyramide signal amplification with a silver enhancement method (SEM-TSA) has been developed successfully. To demonstrate the feasibility of this approach, human IgG as a model target protein was employed and its concentration was assayed based on colorimetric detection. The analytical parameters including the concentrations of detection antibody, streptavidin-horseradish peroxidase, biotinyl tyramide, and streptavidin-nanogold were systematically optimized. The quantitative analysis was performed and a dynamic range was obtained from 0.18 ng/mL to 39.1 ng/mL, while no detectable images could be observed when the silver enhancement method (SEM) without TSA was used. The detection limits were 0.18 ng/mL and 21 ng/mL for SEM-TSA and SEM, respectively. The results showed that sensitivity of the presented colorimetric assay significantly increased by two-orders of magnitude. In addition, this method has been successfully applied in analyzing normal human serum samples. The results suggested the colorimetric detection method based on TSA-SEM has promising potential applications in biomedical analysis and clinical diagnosis.     

Novel enzyme-linked immunosorbent assay for determination of fluvastatin in plasma at picogram level

For the first time, an enzyme-linked immunosorbent assay (ELISA) has been developed and validated for the determination of fluvastatin (FLV) in plasma samples at picogram level. The assay employed a polyclonal antibody that specifically recognizes FLV with high affinity, and FLV conjugate of bovine serum albumin (FLV-BSA) immobilized onto microplate wells as a solid-phase. The assay involved a competitive binding reaction between FLV, in plasma sample, and the immobilized FLV-BSA for the binding sites on a limited amount of the anti-FLV antibody. The bound anti-FLV antibody was quantified with horseradish peroxidase-labeled second anti-rabbit IgG antibody (HRP-IgG) and 3,3′,5,5′-tetramethylbenzidine (TMB) as a substrate for the peroxidase enzyme. The concentration of FLV in the sample was quantified by its ability to inhibit the binding of the anti-FLV antibody to the immobilized FLV-BSA and subsequently the color intensity in the assay wells. The conditions for the proposed ELISA were investigated and the optimum conditions were employed in the determination of FLV in plasma samples. The assay limit of detection was 10 pg mL⁻¹ and the effective working range at relative standard deviations (RSD) of ≤5% was 20-1000 pg mL⁻¹. Analytical recovery of FLV from spiked plasma was 97.1-102.7 ± 2.85-6.25%. The precision of the assay was satisfactory; RSD was 2.46-5.37 and 3.19-6.64% for the 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 ELISA has a great value in routine analysis of FLV for its therapeutic monitoring and pharmacokinetic studies.     

Preparation of Anti—Cardiac Troponin I Monoclonal Antibodies and Their Characterization with Surface Plasmon Resonance Biosensor

IntroductionIn fast and slow skeletal and cardiac muscles,troponin I,the inhibitory protein of the troponin-tropomyosin complex,exists in three isotype formsencoded by three separated genes.The amino acidsequences of the two skeletal and one cardiac Tn Iforms( s Tn I and c Tn I,respectively) exhibit40 %dissimilarity[1] .Moreover,human cardiac Tn I has31 additional residues on the N - terminal end,which do not exist in skeletal forms,thus it pro-vides a high potential for obtaining cardiac-…     

Solid substrate room temperature phosphorescence immunoassay based on an antibody labeled with tetramethylrhodamine B isothiocyanate

Solid substrate room temperature phosphorescence immunoassay (SS-RTP-IA) based on an antibody labeled with tetramethylrhodamine B isothiocyanate (TRITC) was described. The SS-RTP properties of rabbit anti-goat antibody labeled with TRITC (RAGAb-TRITC) and its immune complex with goat-immunoglobulin G (G-IgG) were studied on a polyamide membrane (PM). The results showed that RAGAb-TRITC can react specifically with G-IgG on a PM, and retain the excellent SS-RTP property of TRITC, λ_ex^max/λ_em^max=558 nm/700 nm. The dependence of the phosphorescence intensity on the amount of antigen G-IgG (in a sample volume of 0.4 μl) was linear. Compared with enzyme-linked immunosorbent assay (ELISA), this assay showed a lower detection limit (0.37 pg per spot), a better linear relationship and a wider dynamic range (0.62–30 pg). The method was applied directly to determination of G-IgG in goat serum and the results agreed well with those obtained by ELISA. Therefore, this study shows the high sensitivity in SS-RTP and specificity of the immunological reaction. Moreover, it is a simple but powerful procedure.     

Differential pulse voltammetric enzyme-linked immunoassay for the determination of Helicobacter pylori specific immunoglobulin G (IgG) antibody

A differential pulse voltammetric enzyme-linked immunoassay for the determination of helicobacter pylori (H. pylori) specific IgG antibody in human serum has been developed. The method is based on coupling the oxidation reaction of 3,3',5,5'-tetramethylbenzidine (TMB)-H(2)O(2) that is catalysed by horseradish peroxidase-IgG(HRP-IgG) conjugate with the electro-reduction of the enzymatic product to measure the activity of HRP-IgG. The latter reaction exhibits a sensitive differential pulse voltammetric response at 0.1 V (versus Ag AgCl ) in pH 4.0 acetate buffer solution. So, the H. pylori specific IgG antibody could be detected. The detection limit of present method for H. pylori specific IgG antibody was 1.0 units ml(-1), which was about seven times lower than that obtained by traditional spectrophotometric ELISA procedure.     

A new strategy for electrochemical immunoassay based on enzymatic silver deposition on agarose beads

A novel, sensitive electrochemical immunoassay in a homogeneously dispersed medium is described herein based on the unique features of agarose beads and the special amplified properties of biometallization. The immunochemical recognition event between human immunoglobulin G (IgG) and goat anti-human IgG antibody is chosen as the model system to demonstrate the proposed immunoassay approach. Avidin-agarose beads rapidly react with the biotinylated goat anti-human IgG antibody to form agarose beads-goat anti-human IgG conjugate (agarose bead-Ab). Agarose bead-Ab, alkaline phosphatase conjugated goat anti-human IgG antibody (ALP-Ab) and the human IgG analyte are mixed to form sandwich-type immunocomplex followed by the addition of the enzymatic silver deposition solution to deposit silver onto the surface of proteins and agarose beads. The silver deposited are dissolved and quantified by anodic stripping voltammetry. The influence of relevant experimental variables was examined and optimized. The logarithm of the anodic stripping peak current depended linearly on the logarithm of the concentration of human IgG in the range from 1 to 1000 ng/ml. A detection limit as low as 0.5 ng/ml human IgG was attained by 3σ-rule. The R.S.D. of the approach is 9.65% for eight times determination of 10 ng/ml human IgG under same conditions. Optical microscope and TEM graphs were also utilized to characterize agarose beads and silver nanoparticles formed.     

Detection of antisperm antibody in human serum using a piezoelectric immunosensor based on mixed self-assembled monolayers

A novel piezoelectric immunosensor based on mixed self-assembled monolayers (mixed SAMs) formed by short-chain amine- and carboxyl-terminated thiols has been developed to immobilize antigens onto gold electrodes for detecting antisperm antibody (AsAb) in human serum samples. The properties and the enhanced performance of the affinity biosensor interface based on mixed SAMs are investigated. Most importantly, analytical results of several human serum samples using the developed technique are in satisfactory agreement with those given by the enzyme-linked immunosorbent assay (ELISA) method in the concentration ranging from 32.3 to 300.0 mU/ml. It means the procedure proposed in this paper is likely to have a great potential in research and may play an important clinical role in a few years later.     

Determination of 19-nortestosterone residues in aquaculture tissues by enzyme-linked immunosorbent assay and comparison with liquid chromatography and tandem mass spectrometry

19-Nortestosterone (17β-NT) was oximated by carboxymethoxylamine and then coupled with bovine serum albumin (BSA) in a mixed-anhydride reaction in order to produce an antibody. The conjugate rate of 17β-NT and BSA was estimated to be 24 by ultraviolet spectrophotometry. Polyclonal antibody of 17β-NT was acquired from the animal immunized with the conjugate. Through an indirect enzyme-linked immunosorbent assay (ELISA), which demonstrated that the synthesis of immunogen was successful, the titre of antiserum was found to be 6.4?×?10⁵. Based on the purified antibody, a competitive indirect ELISA was developed. ELISA revealed that the limit of detection (LOD) was 0.07?ng?g^?1, the recovery (in edible tissues) was 71–89%, and the working range was 0.05–31.25?ng?g^?1. The preliminary evaluation of assay performance through specificity, sensitivity, precision, and accuracy revealed that this ELISA method could be used in the practical detection of 17β-NT in tissue samples. Moreover, this method was compared with high-performance liquid chromatography tandem mass spectrometry, for which the transition for quantification of 17β-NT was 275.4/109.1.     

Homogeneous fluorescence-based immunoassay via inner filter effect of gold nanoparticles on fluorescence of CdTe quantum dots

Homogeneous immunoassays are becoming more and more attractive for modern medical diagnosis because they are superior to heterogeneous immunoassays in sample and reagent consumption, analysis time, portability and disposability. Herein, a universal platform for homogeneous immunoassay, using human immunoglobulin (IgG) as a model analyte, has been developed. This assay relies upon the inner filter effect (IFE) of gold nanoparticles (AuNPs) on CdTe QDs fluorescence. The immunoreaction of antigen and antibody can induce the aggregation of antibody-functionalized AuNPs, and after aggregation the IFE of AuNPs on CdTe QDs fluorescence is greatly enhanced, resulting in a decrease of fluorescence intensity in the system. Based on this phenomenon, a wide dynamic range of 1-100 pg mL(-1) for determination of IgG can be obtained. The proposed method shows a detection limit of 0.3 pg mL(-1) for human IgG, which is much lower than the corresponding absorbance-based approach and compares favorably with other reported fluorescent methods. This immunoassay method is simple, rapid, cheap, and sensitive. The proposed method has been successfully applied to measuring IgG in serum samples, and the obtained results agreed well with those of the enzyme-linked immunosorbent assay (ELISA).     

Enzyme-amplified protein microarray and a fluidic renewable surface fluorescence immunoassay for botulinum neurotoxin detection using high-affinity recombinant antibodies

Two immunoassay platforms were developed for either the sensitive or rapid detection of botulinum neurotoxin A (BoNT/A), using high-affinity recombinant monoclonal antibodies against the receptor binding domain of the heavy chain of BoNT/A. These antibodies also bind the same epitopes of the receptor binding domain present on a nontoxic recombinant heavy chain fragment used for assay development and testing in the current study. An enzyme-linked immunosorbent assay (ELISA) microarray using tyramide amplification for localized labeling was developed for the specific and sensitive detection of BoNT. This assay has the sensitivity to detect BoNT in buffer and blood plasma samples down to 14 fM (1.4 pg mL⁻¹). Three capture antibodies and one antibody combination were compared in the development of this assay. Using a selected pair from the same set of recombinant monoclonal antibodies, a renewable surface microcolumn sensor was developed for the rapid detection of BoNT/A in an automated fluidic system. The ELISA microarray assay, because of its sensitivity, offers a screening test with detection limits comparable to the mouse bioassay, with results available in hours instead of days. The renewable surface assay is less sensitive but much faster, providing results in less than 10 min.     

High-performance liquid affinity chromatography for the purification of immunoglobulin A from human serum using jacalin

A high-performance liquid affinity chromatographic method for the purification of serum immunoglobulin A (IgA) using a jacalin column is described. The automated procedure takes about 2 with minimal manipulation. The yields of the isolated IgA and of its IgG and IgM contamination were studied by enzyme-linked immunosorbent assay (ELISA) of 30 sera. Purity was assured by immunoelectrophoresis. The ratio of IgA1 to total IgA was unchanged after purification, as verified by ELISA. The results showed that greater than 90% IgA could be recovered with less than 0.5% total IgG and greater than 2.0% total IgM remaining in the fractions containing purified IgA.     

Trace analysis of microcystins in water using enzyme-linked immunosorbent assay

Routine monitoring of microcystin in natural waters is difficult because the concentration of the toxin is usually lower than the detection limits. As a more sensitive detection method for microcystin, we developed a competitive enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibodies. New monoclonal antibodies against the microcystin leucine-arginine variant (MCLR), a cyclic peptide toxin of the freshwater cyanobacterium Microcystis aeruginosa, were prepared from cloned hybridoma cell lines. We used keyhole limpet hemocyanin (KLH)-conjugated MCLR as an immunogen for the production of mouse monoclonal antibody. The immunization, cell fusion, and screening of hybridoma cells producing anti-MCLR antibody were conducted. In the ELISA test, a microtiter plate coated with MCLR-bovine serum albumin conjugate was incubated with standard microcystin samples. The amount of antibody bound was determined by the reaction of peroxidase-labeled anti-mouse IgG with its substrate, 3,3′,5,5′-tetramethyl benzidine (TMB). Since the ELISA test was highly sensitive, the newly developed ELISA can be suitable for the trace analysis of cyanobacterial hepatotoxins, microcystins in water. The linear responses of monoclonal antibodies with different concentrations of microcystin LR were established between 30 and 1600 pg/mL.     

Antibody-biotemplated HgS nanoparticles: extremely sensitive labels for atomic fluorescence spectrometric immunoassay

In this work, antibody goat anti-human IgG as a scaffold was employed for the synthesis and biofunctionalization of HgS nanoparticles (NPs) via a facile one-pot process. After a complete sandwich-type immunoreaction among primary antibody, human IgG and secondary antibody labeled with HgS NPs, a large number of mercury ions released from captured HgS NPs dissolution were quantitatively detected by chemical vapor generation atomic fluorescence spectrometry (CVG-AFS). Taking advantage of the signal amplification property of HgS NPs and the high sensitivity of CVG-AFS, the assay detected human IgG with a limit of detection (S/N = 3) of 0.6 ng mL(-1) (4.0 fmol mL(-1) or 0.4 fmol) and the response was linear over a dynamic range from 1.0 to 5.0 × 10(4) ng mL(-1) with a correlation coefficient of 0.996. A relative standard deviation (RSD) of 1.0 × 10(2) ng mL(-1) human IgG was 1.5% for within-batch (intra-assay) and 4.5% for between-batch (inter-assay). Other proteins, such as goat anti-rabbit IgG, goat anti-human IgG, rabbit anti-human IgG, carcinoembryonic (CEA), α-fetoprotein (AFP), human serum albumin (HSA) and bovine serum albumin (BSA) did not significantly interfere with the assay for human IgG. The analytical result of HgS NPs with AFS-based immunoassay technology for the quantification of human IgG in human serum from patients is in good agreement with the result obtained by conventional immunoturbidimetric method. The consequence shows that the novel immunosensor possessed satisfactory precision, extremely high sensitivity, high selectivity and could be applied for the quantification analysis of real samples.     

Homogeneous immunoassay based on aggregation of antibody-functionalized gold nanoparticles coupled with light scattering detection

A universal platform of homogeneous noncompetitive immunoassay, using human immunoglobulin (IgG) as a model analyte, has been developed. The assay is based on aggregation of antibody-functionalized gold nanoparticles directed by the immunoreaction coupled with light scattering detection with a common spectrofluorimeter. In phosphate buffer (pH 7.0) solution, the light scattering intensity of the gold nanoparticles functionalized with goat-anti-human IgG can be greatly enhanced by addition of the human IgG. Based on this phenomenon, a wide dynamic range of 0.05-10 microg ml(-1) for determination of human IgG can be obtained, and the detection limit can reach 10 ng ml(-1). The proposed immunoassay can be accomplished in a homogeneous solution with one-step operation within 10 min and has been successfully applied to the determination of human IgG in serum samples, in which the results are well consistent with those of the enzyme-linked immunosorbent assay (ELISA), indicating its high selectivity and practicality. Therefore, the gold nanoparticle-based light scattering method can be used as a model to establish the general methods for protein assay in the fields of molecular biology and clinical diagnostics.