Simultaneous electrochemical immunoassay using CdS/DNA and PbS/DNA nanochains as labels

An electrochemical method for the simultaneous detection of two different tumor markers, carcinoembryonic antigen (CEA) and α-fetoprotein (AFP), in one-pot, using CdS/DNA and PbS/DNA nanochains as labels was developed. Herein, magnetic beads (MBs) as bimolecule immobilizing carriers, were used for co-immobilization of primary anti-CEA and anti-AFP antibodies. The distinguishable signal labels were synthesized by in situ growth of CdS and PbS nanoparticles on DNA chains, respectively, which were further employed to label the corresponding secondary antibodies. A sandwich-type immunoassay format was formed by the biorecognition of the antigens and corresponding antibodies. The assay was based on the peak currents of Cd(2+) and Pb(2+) dissolved from CdS and PbS nanoparticles by HNO(3) using square wave stripping voltammetry. Experimental results show that the multiplexed electrochemical immunoassay has enabled the simultaneous monitoring of CEA and AFP in a single run with wide working ranges of 0.1-100ngmL(-1) for CEA and 0.5-200ngmL(-1) for AFP. The detection limits reach to 3.3pgmL(-1) for CEA and 7.8pgmL(-1) for AFP.     

Streptavidin-aequorin fusion protein for bioluminescent immunoassay

The fusion protein of streptavidin to aequorin (STA-AQ) was highly purified from inclusion bodies in Escherichia coli cells and applied to a bioluminescent sandwich immunoassay. α-Fetoprotein (AFP), which is a serological marker of liver cancer, was used as a model analyte to test STA-AQ in an immunoassay. The measurable range of AFP by the sandwich immunoassay, using the complex of STA-AQ and the biotinylated anti-AFP antibody, was 0.02-200 ng/mL with an average coefficient of variation of 4.9%. The detection sensitivity with the complex of STA-AQ and the biotinylated anti-AFP antibody was similar to that with the complex of biotinylated aequorin, streptavidin and the biotinylated anti-AFP antibody. STA-AQ would be a useful reporter protein for immunoassays.     

Streptavidin-Aequorin Fusion Protein for Bioluminescent Immunoassay

The fusion protein of streptavidin to aequorin (STA-AQ) was highly purified from inclusion bodies in Escherichia coli cells and applied to a bioluminescent sandwich immunoassay. α-Fetoprotein (AFP), which is a serological marker of liver cancer, was used as a model analyte to test STA-AQ in an immunoassay. The measurable range of AFP by the sandwich immunoassay, using the complex of STA-AQ and the biotinylated anti-AFP antibody, was 0.02–200 ng/mL with an average coefficient of variation of 4.9%. The detection sensitivity with the complex of STA-AQ and the biotinylated anti-AFP antibody was similar to that with the complex of biotinylated aequorin, streptavidin and the biotinylated anti-AFP antibody. STA-AQ would be a useful reporter protein for immunoassays.     

Development of a multipurpose time-resolved fluorescence immunoassay for rat insulin

We present a time-resolved fluorescence immunoassay (TR-FIA) for the measurement of rat insulin in cell extracts and culture media. This assay is based on the binding of two monoclonal antibodies to different parts of the insulin molecule in a 96-well microtiter plate. For the detection, europium-labeled streptavidin that interacts with the second biotinylated antibody is used. Samples of 25 μl could be analyzed in less than 2 days with a measuring range between 5 and 1250 pg (0.2-50 μg/L or 34.4-8600 pM). The inter- and intraassay percentage coefficients of variation were less than 8.3 and 5.1, respectively. Recoveries of 0.48 to 40 μg/L rat insulin, added to culture medium, ranged between 94 and 107%. Results were significantly correlated with those of an in-house radioimmunoassay (RIA) for rodent insulin (P < 0.0001, r² = 0.99). The TR-FIA method had a similar detection limit (0.16 μg/L), but its working range was at least 5-fold larger. Additional advantages include the lower cost, the applicability to measurements in tissue and serum, and the quantification of insulin from other species.     

Detection of carcinoembryonic antigen using single-domain or full-size antibodies stained with quantum dot conjugates

Compact single-domain antibodies (sdAbs) are nearly 13 times smaller than full-size monoclonal antibodies (mAbs) and have a number of advantages for biotechnological applications, such as small size, high specificity, solubility, stability, and great refolding capacity. Carcinoembryonic antigen (CEA) is a tumor-associated glycoprotein expressed in a variety of cancers. Detection of CEA on the tumor cell surface may be carried out using anti-CEA antibodies and conventional fluorescent dyes. Semiconductor quantum dots (QDs) are brighter and more photostable than organic dyes; they provide the possibility for labeling of different recognition molecules with QDs of different colors but excitable with the same wavelength of excitation. In this study, the abilities for specific detection of CEA expressed by tumor cells with anti-CEA sdAbs biotinylated in vitro and in vivo, as well as with anti-CEA mAbs biotinylated in vitro, were compared using flow cytometry and the conjugates of streptavidin with QDs (SA-QDs). The results demonstrated that either in vitro or in vivo biotinylated anti-CEA sdAbs are more sensitive for cell staining compared to biotinylated anti-CEA mAbs. The data also show that simultaneous use of biotinylated sdAbs with highly fluorescent SA-QDs can considerably improve the sensitivity of detection of CEA on tumor cell surfaces.     

A new homogeneous enzyme immunoassay. Its application to measurement of alpha-fetoprotein

A rapid and sensitive homogeneous enzyme immunoassay (homogeneous EIA) was developed for determination of serum proteins such as alpha-fetoprotein (AFP). There are two assay systems, one is a competitive system including horseradish peroxidase (HRP)-labeled antigen, antibody and substrate, and the other is a non-competitive system including HRP-labeled antibody and substrate. When the aggregate was formed through the binding of HRP-labeled AFP and anti-AFP antibody or through the binding of HRP-labeled anti-AFP antibody and AFP, HRP of the aggregates, as compared with HRP of free conjugates, exhibited marked activity in the presence of 35 mM H2O2. The extent of stimulation of HRP activity depended on the amount of AFP. This new assay method is very simple and sensitive, and can be used for the determination of any kind of protein, hormone, or drug.     

甲胎蛋白在体外对人肝癌细胞生长的影响

本文用MTT比色法观察了甲胎蛋白(AFP)在体外对人肝癌细胞生长的影响。结果表明,AFP能促进SMMC-7721人肝癌细胞的生长。当AFP与AFP抗体合用时,AFP抗体能减弱AFP对SMMC-7721细胞生长的促进作用;AFP抗体单用对此种细胞的生长亦有抑制作用。另一方面,在相同的实验条件下,AFP和AFP抗体对HL-60人白血病细胞的生长无明显影响;提示AFP的促生长作用具有一定的肿瘤细胞特异性,并非一种蛋白质对培养细胞的非特异性营养作用。此外,AFP亦能促进MCF-7人乳腺癌细胞的生长,AFP抗体对此种细胞的生长有抑制作用。由于MCF-7细胞存在功能性AFP受体,也能合成和分泌AFP。这就提示,人肝癌细胞中的AFP很可能与其受体特异性结合,产生促生长效应。确切机制尚待进一步阐明。     

Non-competitive immunoassay for alpha-fetoprotein using micellar electrokinetic capillary chromatography and laser-induced fluorescence detection

A non-competitive immunoassay based on micellar electrokinetic capillary chromatography (MECC) with laser-induced fluorescence (LIF) detection has been developed for the determination of alpha-fetoprotein (AFP). The anti-AFP antibody was labeled with fluorescein isothiocyanate (FITC) and the product was used as a fluorescent tracer, then AFP was mixed with the labeled antibody. After incubation, the immune AFP-antibody complex was separated from labeled free antibody by MECC. The parameters affecting separation such as the concentration of sodium dodecyl sulfate (SDS), the buffer pH and separation voltage were investigated and the following conditions were selected: 20 mM tetraborate containing 100 mM SDS at pH 9.50, and 20 kV separation voltage. The detection limit of this assay was 0.1 ng/ml with a linear range spanning two orders of magnitude. This method was applied to determine AFP in human serum.     

基于上转磷光颗粒的AFP免疫层析定量检测

目的：建立了上转磷光免疫层析快速定量检测AFP的方法,用于原发性肝癌的诊断。方法：利用上转磷光标记的双抗夹心免疫层析技术检测血清中AFP的含量。将AFP单克隆抗体分别标记上转磷光颗粒并包被硝酸纤维膜,制成免疫层析检测试纸。评价该试纸的灵敏度、特异性和精密度。并通过临床50例血清样本,探讨上转磷光技术与化学发光检测方法的一致性。结果：该方法能在20min内完成,检测灵敏度达5ng/ml。浓度范围从5-1000ng/ml作标准曲线,呈现较好的线性。通过三组平行实验探讨其精密度,变异系数都在10%以内。应用于其它肿瘤标志物如CEA、CA199、AFU和NSE的检测,无交叉反应。与化学发光相比,决定系数R2=0.9692,一致性较好。结论：该方法简便、快速、廉价,可以实现定量,尤其适合基层门诊和体检现场使用。     

Comparison of CEA distribution in lesions and tumors of salivary glands as determined with monoclonal and polyclonal antibodies

Immunohistochemical localization of carcinoembryonic antigen (CEA) with conventional antibody to CEA (anti-CEA), nonspecific crossreacting antigen (NCA)-absorbed polyclonal antibody to CEA (NCAa-CEA), and monoclonal antibody to CEA (Mono-CEA) have been compared in obstructive lesions and salivary gland tumors. Normal salivary glands gave strong staining of the luminal borders of acinar cells with anti-CEA, whereas no staining occurred with Mono-CEA. Obstructive lesions showed occasionally marked staining with anti-CEA in some acinar cells, but there was no reaction with Mono-CEA. Of 69 pleomorphic adenomas examined, 34 were positively stained with anti-CEA, 18 with NCAa-CEA and 8 with Mono-CEA along the luminal borders of the tumor cells. The frequency of positive staining of material within tubular lumina was similar with all three immunoreagents. Neoplastic cells were positive with Mono-CEA in only three cases, while eight cases were positive with NCAa-CEA and 11 cases with anti-CEA. In salivary gland tumors true CEA is be found mainly at the border of tumor cells, but the frequency of positive reactions is low.     

Expression and characterization of a recombinant Fab fragment derived from an anti-human alpha-fetoprotein monoclonal antibody

Alpha-fetoprotein (AFP) is a well-known molecular marker indicating the development of cancer as well as fetal abnormalities such as open neural tube defects. Accordingly the measurement of serum AFP is important for the diagnosis of hepatocellular carcinoma (HCC) and other abnormalities. Monoclonal antibodies (McAb) to AFP were produced to develop an immunoassay kit, and to study the possibility of an antibody (Ab) therapy. The immunoglobulin genes were cloned from hybridoma cells, and expressed in E. coli as an Fab soluble into a culture medium. The Fab of anti-AFP McAb exhibited binding to AFP and similar affinity compared to the original IgG. This recombinant antibody can be studied further for in vivo imaging and immunotherapeutics.     

A highly sensitive method for the demonstration of carcinoembryonic antigen in normal and neoplastic colonic tissue

Summary A highly sensitive method for the immuno-histochemical localisation of carcinoembryonic antigen (CEA) is described. This method is based on the binding of a peroxidase-antiperoxidase complex (PAP) to anti-CEA antibodies by means of an anti-gamma-globulin which reacts with both the anti-CEA and the antiperoxidase antibodies. Using the technique described here, CEA was localised in conventionally processed normal and cancerous colonic tissue. In normal as well as in neoplastic tissues, a CEA-specific staining of cell membranes and cytoplasm was demonstrated. In frozen sections of normal colonic tissue CEA was found even at high dilutions of the first antibody; this indicates the high sensitivity of the method. The applicability of the method to conventionally processed and thereby well preserved tissue specimens opens the possibility to identify CEA by light microscopy even at very low concentrations.     

Development of a new sensitive and specific time-resolved fluoroimmunoassay (TR-FIA) of chlormadinone acetate in the serum of treated menopausal women

We describe the development of a serum chlormadinone acetate (CMA) time-resolved fluoroimmunoassay (TR-FIA). We prepared haptens (3-CMO-chlormadinone acetate and 6-chloropregna-4,6-dien-17,20-diol-3-one-20-hemisuccinate), biotinylated tracers (3(biotinylaminopropylamido) 3-CMO-chlormadinone acetate and 3-(6-chloropregna-4,6-dien-17,20-diol-3-one-20-hemisuccinylamino)1-biotinylaminopropane), and immunogens necessary for eliciting two antibodies (anti-chlormadinone acetate 3-CMO/BSA and anti-chlormadinone 20-hemisuccinate/BSA). The specificity of the assay was rigorously studied to eliminate possible interference by polar metabolites of CMA, particularly 17 alpha-acetoxy-6-chloro-3beta-hydroxypregna-4,6-diene-20-one (3beta-hydroxy metabolite), employing an easy-to-use ethylene glycol chromatographic step prior to immunoassay, so as to separate the polar metabolites, in particular the 3beta-hydroxy-CMA metabolite, from the intact CMA. The choice of the anti-CMA antibody was guided by the high assay sensitivity obtained with the anti-CMA 3-CMO/BSA antibody. The detection limit was 51pg/ml. Interassay reproducibility CVs were between 2.6 and 4.5%. This TR-FIA thus appeared to be a sensitive, specific, precise, and consequently well-suited method for measurement of serum CMA during a pharmacokinetic study in women.     

Effect of alpha-fetoprotein on the growth of human hepatoma cells in vitro]

The stimulatory activity of human alpha-fetoprotein (AFP) on the growth of mouse hepatoma-22 cells had been reported in our previous paper. The present work aimed at further investigation of the effect of AFP on human hepatoma cell growth by MTT colorimetric assay. The results showed that AFP could stimulate the growth of SMMC-7721 human hepatoma cells in vitro. The present results also showed that the stimulatory effect of AFP on the growth of SMMC-7721 cells was decreased by the anti-serum of AFP. The anti-AFP antibody alone could suppress the growth of SMMC-7721 cells. On the other hand, AFP and anti-AFP antibody had no effect on the growth of HL-60 human leukemia cells, indicating that the tumor cell growth stimulating effect of AFP was not simply due to non-specific addition of exogenous protein and this effect of AFP showed strict tumor cell specificity. In addition, MCF-7 human breast cancer cell growth was also promoted by AFP and inhibited by anti-AFP antibody. Because AFP cell-surface receptors have been detected in MCF-7 breast cancer cells, and AFP could also be produced and secreted by MCF-7 cells, the possibility may be considered: AFP may bind with its receptors on tumor cell membrane for the purposes of growth stimulation.     

A new specific and sensitive time resolved-fluoroimmunoassay of 11-deoxycortisol in serum

A biotinylated 11-deoxycortisol tracer was synthesized from 11-deoxycortisol-3-carboxymethyloxime and the conjugate obtained by acylation of biotinylaminopropylammonium trifluoroacetate. This biotinylated tracer was used to develop an 11-deoxycortisol time-resolved-fluoroimmunoassay (TR-FIA). The tracer was quantified after adding streptavidine-Europium. A TR-FIA sensitive standard curve, with displacement of 20, 50, and 80% of tracer was obtained with 12.4, 70.7, and 512.8 pg of 11-deoxycortisol, respectively. After extraction followed by Celite chromatography, purified serum samples were simultaneously assayed by RIA and TR-FIA. The results obtained by the two methods were practically identical, however, this new specific, non-isotopic 11-deoxycortisol assay has the advantage of being more sensitive than RIA, thus well-suited to accurate measurement in endocrinological studies, particularly when serum 11-deoxycortisol levels in patients are just above the highest normal values. Moreover, this non-isotopic assay is cheaper than RIA.     

Amperometric immunosensor based on toluidine blue/nano-Au through electrostatic interaction for determination of carcinoembryonic antigen

A new current amplified immunosensor for the determination of carcinoembryonic antigen (CEA) was demonstrated in this work. The electrode architecture was fabricated by positively charged toluidine blue (TB) coated on negatively charged poly-sulfanilic acid (PSAA) modified glassy carbon electrode (GCE) surface through electrostatic interactions to form a TB/PSAA film, which provided an interface containing amine groups to assemble gold nanoparticles (nano-Au) for immobilization of carcinoembryonic antibody (anti-CEA) and horseradish peroxidase (HRP) instead of bovine serum albumin (BSA) to block sites against non-specific binding. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were employed to characterize the electrochemical properties of the modified processes. The CVs reduction current of the immunosensor charged linearly in two concentration ranges of CEA from 0.5 to 5.0 and 5.0 to 120.0 ng/ml in presence of 0.3mM H2O2 in analyte solution, and the detection limit was 0.2 ng/ml at three times background noise. The proposed method is economical, efficient and potentially attractive for clinical immunoassays.     

Sensitive sandwich electrochemical immunosensor for alpha fetoprotein based on prussian blue modified hydroxyapatite

A sandwich electrochemical immunosensor for the sensitive determination of alpha fetoprotein (AFP) has been fabricated. Prussian blue modified hydroxyapatite (PB@HAP) was firstly prepared and used as electrochemical label due to the wonderful conductivity and good biocompatibility of HAP. The results proved that the immunosensor fabricated using the label based on PB@HAP loaded with horse radish peroxidase (HRP) and secondary anti-AFP antibody (Ab(2)) (PB@HAP-HRP-Ab(2)) had high sensitivity, and the sensitivity of the label PB@HAP-HRP-Ab(2) was much higher than labels of PB@HAP-Ab(2), PB-HRP-Ab(2) and HAP-HRP-Ab(2). The mixture of graphene sheet (GS) and thionine (TH) was not only used to immobilize anti-AFP antibody (Ab(1)) but also took part in the signal amplification. The amperometric signal increased linearly with AFP concentration in the range of 0.02-8 ng/mL with a low detection limit of 9 pg/mL. The immunosensor had the advantages of high sensitivity, good selectivity and good stability, and was applied to the analysis of AFP in serum sample with satisfactory results. Due to the low-cost and easy synthesis of PB@HAP, the screen-printed electrodes could be used instead of the bare glass carbon electrode in order to achieve mass production. In addition, it had potential application in the detection of other tumor markers.     

Selective cytotoxic effects of immunotoxin--monoclonal anti-AFP-abrin-A chain conjugate on several human hepatoma cell lines.

A highly specific monoclonal antibody (anti-AFP) against alpha-fetoprotein (AFP) was linked to N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) to form conjugates which were purified with a protein A-sepharose CL-4B affinity column. The conjugate, PDP-Anti-AFP was then covalently coupled to the toxic abrin-A chain to synthesize immunotoxins. The immunotoxin, anti-AFP-abrin-A conjugate, which was also purified with a protein A-sepharose CL-4B affinity column, had a molecular weight of 180,000 and had 80% antigen-binding activity that of anti-AFP activity and 92% toxicity of abrin-A chain. The immunotoxin showed selective cytotoxicities toward the AFP secreting human hepatoma cell lines, such HepG2 and Hep3B, but not toward AFP non-secreting human hepatoma cell line, PLC/PRF/5.     

甲种胎儿球蛋白(AFP)定性定量酶联免疫试盒剂的研制

甲胎蛋白是一种癌胚相关蛋白,它在胎儿血清中含量每毫升数毫克之多,在正常人血清中低于25μg/ml,在原发性肝细胞癌者血清中甲胎蛋白的阳性率和滴度都很高,所以血清中甲胎蛋白的检测是诊断肝癌的特异指标。本方法采用双抗体夹心法原理,可定性及定量检测血清和血浆标本中甲胎蛋白含量,并配制AFP系列标准,为原发性肝癌的早期诊断及普查具有重要的临床意义。     

Monoclonal antibodies against human alpha-fetoprotein more reactive to cell-surface alpha-fetoprotein than to free alpha-fetoprotein.

This paper describes the finding of monoclonal antibody (MoAb) more reactive to cell-surface alpha-fetoprotein (AFP) than to free AFP by using a simple in vitro system. Twelve mouse MoAbs, ten IgG1, one IgG2a and one IgG2b, against human AFP from hepatocellular carcinoma were obtained by the cell fusion technique. Each hybridoma supernatant was assayed by enzyme-linked immunosorbent assay (ELISA) to solid-phase AFP. The assay results showed that two MoAbs, 67D and 80G, were most reactive to AFP. 80G had a higher affinity constant than 67D, while the both reactions were similarly difficult to inhibit by free AFP in ELISA. 67D and 80G reacted with AFP on the surface of ethanol-fixed cells from the human hepatoma cell line HuH-7 and this reaction was also difficult to inhibit by free AFP in Cell ELISA. Furthermore, Western blot analysis showed that 67D and 80G were more reactive to membrane-bound AFP than other antibodies. These findings first suggest that there could be anti-AFP MoAbs preferably binding to cell-surface AFP rather than to serum AFP.