DEVELOPMENT AND EVALUATION OF A RAPID ENZYME-LINKED IMMUNOFILTRATION ASSAY (ELIFA) AND AN ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) FOR THE DETECTION OF STAPHYLOCOCCAL ENTEROTOXIN B

An enzyme-linked immunofiltration assay (ELIFA) and a microtitre plate enzyme-linked immunosorbent assay (ELISA) were developed and compared for their ability to detect staphylococcal enterotoxin B (SEB). The double antibody capture format was used for both assays. Factors which improved the sensitivity of the ELIFA system were (1) addition of casein and thimerosal to the antigen dilution buffer; (2) addition of polyethylene glycol (MW 6000) to the detection and conjugate antibody dilution buffers; and (3) washing with diethanolamine buffer prior to addition of the substrate/chromogen. The ELIFA system had a turnaround time of approximately 1 h and a detection limit of 1 ng/mL of purified SEB. The ELISA had a total turnaround time of 21 h, or 3 h using plates pre-coated overnight with the capture antibody. The detection limit of the ELISA for purified SEB was 0.05 ng/mL. The detection limit of SEB in cheese samples spiked with purified enterotoxin and subjected to a simple extraction procedure was 1 ng/mL and 0.1 ng/mL of extract, with the ELIFA and the ELISA, respectively.     

Protein A-based antibody immobilization onto polymeric microdevices for enhanced sensitivity of enzyme-linked immunosorbent assay

Highly efficient antibody immobilization is extremely crucial for the development of high-performance polymeric microdevices for enzyme-linked immunosorbent assay (ELISA). In this article, a site-selective tyrosinase (TR)-catalyzed protein A strategy for antibody immobilization was developed to enhance the sensitivity of ELISA in poly-(methyl methacrylate) (PMMA) microchannels for interferon-gamma (IFN-gamma) assay. To effectively immobilize the target antibodies, oxygen plasma was first used to activate the inert PMMA. This is followed by poly(ethyleneimine) (PEI) coating, an amine-containing functional polymer. For comparison, protein A was also immobilized through the commonly used amine-glutaraldehyde (GA) chemistry. Oxygen plasma treatment effectively increased the amount of PEI attachment and subsequent binding efficiency of the primary antibody. The antibody immobilized via TR-catalyzed protein A was able to provide much better specific antigen capture efficiency than GA chemistry due to the optimal spacing and orientation. Consequently, by using this new method, the detection signal and the signal-to-noise ratio of the ELISA immunoassay in microdevices were all significantly improved. In comparison to the standard assay carried out in the 96-well microtiter plate, the treated microchannels exhibited a broader detection range and a shorter detection time. And the detection limit was also decreased to 20 pg/mL, much lower than that obtained in other microdevices.     

Development of a sensitive ELISA for the quantification of human tumour necrosis factor-alpha using 4 polyclonal antibodies

Despite the availability of many assays to measure concentrations of tumour necrosis factor alpha (TNF-alpha) in body fluids, these assays often lack specificity or sensitivity and are often of questionable reliability, resulting in inconsistent results. Therefore, we have developed an ELISA that is sensitive, reliable and not susceptible to disturbances by interfering substances such as heterophilic antibodies. The assay involves a combination of four polyclonal antibodies. The antibodies, which capture the analyte, were raised in chicken and the trapping anti-analyte antibodies were raised in rabbit. The immobilization of capture antibodies was achieved via a coating antibody raised in a duck against chicken IgY and the recognition of trapping antibodies was achieved by a detection antibody raised in a goat against rabbit IgG and labelled with HRP. The analytical and functional sensitivities of the ELISA are 8 pg/mL and 13 pg/mL, respectively. The assay showed good precision and, in contrast to our in-house RIA, excellent parallelism in serial dilutions. The recovery of TNF-alpha spiked to plasma samples ranged from 97% to 119%. Comparison of the newly developed, sensitive ELISA with our in-house RIA showed that the median TNF-alpha value obtained by RIA (range: 0.095-10.0, median 0.578 ng/mL) was found to be 1.5-2 times higher than that obtained with the ELISA (range 0.008-5.84, median 0.213 ng/mL). Spearman correlation was 0.755 (p < 0.0001). In addition, analysis of the TNF-alpha concentrations in blood from healthy individuals and from patients suffering from tuberculosis, with RIA and ELISA, showed the same differences although TNF-alpha levels obtained with ELISA were lower. We feel that this ELISA is a major improvement compared to the currently available assays for TNF.     

Immunodetection and immunopurification of aflatoxins using a high affinity monoclonal antibody to aflatoxin B1

A monoclonal antibody was obtained from BALB/c mice immunized with aflatoxin Bl (AFB1) conjugated to bovine serum albumin. This IgG2a antibody, ASCI, with K light chain has a high specificity for AFB1. In an indirect enzyme-linked immunosorbent assay the antibody litre in ascites fluid was 1: 6000 for 50% binding to plates coated with aflatoxin-poly-L-lysine. The assay is sensitive to 2.5 pg aflatoxin/assay. ASCI cross-reacts with closely related aflatoxin metabolites such as AFB2, AFM1 and AFG1. However, ASCI displays negligible cross-reactivity with other related aflatoxin analogues such as AFM2, AFP1, AFQ1 and aflatoxicol. An immunoabsorbent was prepared by coupling ASCI antibody to Ultrogel AcA 22. This immunomatrix was used to purify aflatoxins at 0–1 ng/ml levels from contaminated body fluids such as bovine milk. The antibody affinity column was regenerated and re-used several times. Owing to its high specificity for AFB1 and AFM1, ASCI will be of value in immunodetection and immunopurification of these toxins in various foodstuffs.     

Microfluidic electrochemical immunoarray for ultrasensitive detection of two cancer biomarker proteins in serum

A microfluidic electrochemical immunoassay system for multiplexed detection of protein cancer biomarkers was fabricated using a molded polydimethylsiloxane channel and routine machined parts interfaced with a pump and sample injector. Using off-line capture of analytes by heavily-enzyme-labeled 1 μm superparamagnetic particle (MP)-antibody bioconjugates and capture antibodies attached to an 8-electrode measuring chip, simultaneous detection of cancer biomarker proteins prostate specific antigen (PSA) and interleukin-6 (IL-6) in serum was achieved at sub-pg mL?1 levels. MPs were conjugated with ～90,000 antibodies and ～200,000 horseradish peroxidase (HRP) labels to provide efficient off-line capture and high sensitivity. Measuring electrodes feature a layer of 5 nm glutathione-decorated gold nanoparticles to attach antibodies that capture MP-analyte bioconjugates. Detection limits of 0.23 pg mL?1 for PSA and 0.30 pg mL?1 for IL-6 were obtained in diluted serum mixtures. PSA and IL-6 biomarkers were measured in serum of prostate cancer patients in total assay time 1.15 h and sensor array results gave excellent correlation with standard enzyme-linked immunosorbent assays (ELISA). These microfluidic immunosensors employing nanostructured surfaces and off-line analyte capture with heavily labeled paramagnetic particles hold great promise for accurate, sensitive multiplexed detection of diagnostic cancer biomarkers.     

Sensitive detection of multiplex toxins using antibody microarray

Using a newly developed fluorescent nanoparticle (NP) that gives rise to a high-intensity and stable fluorescent light, a sensitive antibody (Ab) microarray assay system has been developed for specific detection of bioterrorism agents, as exemplified by ricin, cholera toxin (CT), and staphylococcal enterotoxin B (SEB). The Ab microarray uses a sandwich format that consists of capture Abs, analytes (toxins), biotinylated detection Abs, and avidin-conjugated NP. In all three cases, polyclonal Abs (pAbs) displayed superiority over monoclonal antibodies (mAbs) in capturing toxins on microarray slides even when the pAbs and mAbs had similar affinity as determined by enzyme-linked immunosorbent assay (ELISA). The detection system was successfully used to detect toxins spiked in milk, apple cider, and blood samples. We were able to detect ricin at 100 pg/ml in buffer and at 1 ng/ml in spiked apple cider or milk, whereas CT and SEB were detected at 10 pg/ml in buffer and 100 pg/ml in spiked apple cider or milk. High specificities were also demonstrated in the detection of mixed toxin samples with similar sensitivities. The matrix effect of blood samples on the detection of mixed toxins seems to be minimal when the toxin concentration is at or above 100 ng/ml. The current study highlights the significant role of pAb and NP in increasing selectivity and sensitivity of toxin detection in a microarray format.     

MUC1粘蛋白的分离纯化及其抗体的制备

经高速离心从正常人乳中获得人乳汁颗粒膜(HMFGM),产量约0．4g／L。经进一步破碎、脱脂及sepharose CL-4B柱纯化,获得含MUC1粘蛋白的组分,并经SDS—PAGE、Western—blot及ELISA鉴定后,免疫家兔制备多抗。结果表明,进一步凝胶过滤获得MUC1粘蛋白,行SDS—PAGE后经希夫试剂和考马斯亮蓝染色呈单一条带,表观相对分子质量大干205000。Western—blot及ELISA结果表明可与MUC1特异性抗体结合。制备获得的多抗经ELISA测定效价为1：64000～1：128000。表明建立了MUC1粘蛋白的纯化方法,获得的MUC1粘蛋白及其抗体可进一步用于MUC1检测及其功能的研究。     

Protein chip based miniaturized assay for the simultaneous quantitative monitoring of cancer biomarkers in tissue extracts

A multiplexed fluorescence immunoassay using a novel planar waveguide technology-based microarray system, ZeptoMARK (Zeptosens), was developed to detect simultaneously urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1), and vascular endothelial growth factor (VEGF) in extracts of breast cancer tissues. The three analytes assay was cross-validated with single-analyte ELISA/chemiluminescence immunosorbent assay tests, revealing good correlations and enhanced assay sensitivities (LODs) of 1 pg/mL for uPA, 33 pg/mL for PAI-1, and 1 pg/mL for VEGF. Values were well within the 80-120% limits for assay recovery and within the +/-20% limits for assay precision. The uPA, PAI-1, and VEGF results obtained from 50 breast cancer cytosols using the protein array system demonstrated that the microarray-based multiplexed assay is a sensitive and robust tool to be used for the simultaneous quantification of cancer markers in small breast cancer tissue samples (core biopsies). The miniaturized, multiplexed assay format has a potential to be used for the quantitative analysis of a larger set of validated markers with significance in disease management.     

Detection of circulating immune complexes by the enzyme-linked immunosorbent assay in sera from cattle infected with Fasciola hepatica

Polyethylene glycol-precipitated immune complexes (PIC) from the sera of 5 calves with Fasciola hepatica worm burdens ranging between 27 and 70 flukes were examined for parasite antigen content at 2, 4, 6, 8, 10, and 16 wk postinfection (PI) by the enzyme-linked immunosorbent assay (ELISA). Three assays were devised using an affinity-processed rabbit antibody to worm excretory/secretory (FhES) antigens. The PIC plate assay detected parasite antigen by adherence of anti-FhES antibody to PIC incubated overnight on ELISA plates, and tests were visualized using anti-rabbit peroxidase-linked antibody. The serum complex and PIC capture assay utilized the anti-FhES immunoglobulin as an antigen capture antibody linked to the solid phase. The attached complexes were then detected by the adhering bovine antibody, either soluble complexes in serum or as PIC. All assays showed circulating immune complex (CIC) values elevated at 6-8 wk PI, which generally coincided with increased host circulating antibody to FhES antigens. The greatest detection rate for all of the immune complex (IC) detection assays occurred with the PIC capture assay. It detected antigen in almost 90% of sera tested at 6 and 8 wk PI. Both the serum complex and PIC capture assay detected greatest amounts of CIC in those animals with the largest worm burdens, whereas the PIC plate assay showed no such trend. This study shows that F. hepatica antigen detection in CIC can be used to aid immunologic diagnosis of fascioliasis.     

Simultaneous detection of multiple chemical residues in milk using broad-specificity antibodies in a hybrid immunosorbent assay

In this study, a novel immunoassay using 2 types of sensors (QDs and an enzyme) were simultaneously used for detecting multiple structurally different molecules in milk. The method integrates the fluorescence-linked immunosorbent assay (FLISA) using QD605 and QD655 as probes and an enzyme-linked immunosorbent assay (ELISA) using horseradish peroxidase (HRP) labeled secondary antibody. The FLISA was produced by anti-sulfonamide and anti-quinolone broad-specificity monoclonal antibodies (MAbs) for simultaneously detecting 6 sulfonamides and 11 quinolones. Combined with the FLISA, an ELISA was utilized for detecting melamine from the same milk samples. The cross-reactivity of the MAbs was retained while binding the QDs by using avidin and a secondary antibody as bridges. Milk samples were detected using this hybrid immunoassay, with limits of detection (LOD) of the quinolones (0.18 ng mL(-1)), sulfonamides (0.17 ng mL(-1)) and melamine (7.5 ng mL(-1)), respectively. The results demonstrated that the detection limits of the integrated methods were better than required and simplified the sample pretreatment process. The developed immunoassay is suitable for high-throughput screening of low-molecular weight contaminants.     

改良DAS-Dot-ELISA检测西瓜细菌性果斑病菌

以硝酸纤维素膜为载体,对Dot-ELISA法的封闭条件、包被抗体浓度、点样量等反应条件进行优化,建立改良DAS-Dot-ELISA法快速检测西瓜细菌性果斑病菌。研究发现,以含乙二胺四乙酸二钠(EDTA)的脱脂奶粉液高温处理后用于封闭,可有效降低背景;轻微振荡可提高杂交效率,减少非特异性结合。改良DAS-Dot-ELISA可快速、经济的检测西瓜果斑病菌,灵敏度达1.9×105CFU/mL。在对两批次种子样品的检测中,改良DAS-Dot-ELISA法检测带菌率分别为8.0%和6.0%,与微孔板ELISA结果完全一致;对每粒种子的检测结果,改良DAS-Dot-ELISA法与微孔板ELISA吻合率平均达99.0%,显示较好的实用前景,同时为快速检测西瓜果斑病菌提供一种新途径。     

Direct competitive chemiluminescence immunoassays based on gold‐coated magnetic particles for detection of chloramphenicol

Direct competitive chemiluminescence immunoassays (CLIA) based on gold‐coated magnetic nanospheres (Au‐MNPs) were developed for rapid analysis of chloramphenicol (CAP). The Au‐MNPs were modified with carboxyl groups and amino groups by 11‐mercaptoundecanoic acid (MUA) and cysteamine respectively, and then were respectively conjugated with CAP base and CAP succinate via an activating reaction using 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N‐hydroxysuccinimide (NHS). NSP‐DMAE‐NHS, a new and effective luminescence reagent, was employed to label anti‐CAP antibody (mAb) as a tracer in direct CLIA for CAP detection using a ‘homemade’ luminescent measurement system that was set up with a photomultiplier tube (PMT) and a photon counting unit linked to a computer. The sensitivities and limits of detection (LODs) of the two methods were obtained and compared according to the inhibition curves. The 50% inhibition concentration (IC₅₀) values of the two methods were about 0.044 ng/mL and 0.072 ng/mL respectively and LODs were approximately 0.001 ng/mL and 0.006 ng/mL respectively. To our knowledge, they were much more sensitive than any traditional enzyme‐linked immunosorbent assay (ELISA) ever reported. Moreover, the new luminescence reagent NSP‐DMAE‐NHS is much more sensitive and stable than luminol and its derivatives, contributing to the sensitivity enhancement. Copyright © 2015 John Wiley & Sons, Ltd.     

Enhanced Sensitivity for Detection of HIV-1 p24 Antigen by a Novel Nuclease-Linked Fluorescence Oligonucleotide Assay

The relatively high detection limit of the Enzyme-linked immunosorbent assay (ELISA) prevents its application for detection of low concentrations of antigens. To increase the sensitivity for detection of HIV-1 p24 antigen, we developed a highly sensitive nuclease-linked fluorescence oligonucleotide assay (NLFOA). Two major improvements were incorporated in NLFOA to amplify antibody-antigen interaction signals and reduce the signal/noise ratio; a large number of nuclease molecules coupled to the gold nanoparticle/streptavidin complex and fluorescent signals generated from fluorescent-labeled oligonucleotides by the nuclease. The detection limit of p24 by NLFOA was 1 pg/mL, which was 10-fold more sensitive than the conventional ELISA (10 pg/mL). The specificity was 100% and the coefficient of variation (CV) was 7.8% at low p24 concentration (1.5 pg/mL) with various concentrations of spiked p24 in HIV-1 negative sera. Thus, NLFOA is highly sensitive, specific, reproducible and user-friendly. The more sensitive detection of low p24 concentrations in HIV-1-infected individuals by NLFOA could allow detection of HIV-1 infections that are missed by the conventional ELISA at the window period during acute infection to further reduce the risk for HIV-1 infection due to the undetected HIV-1 in the blood products. Moreover, NLFOA can be easily applied to more sensitive detection of other antigens.     

Simultaneous analysis of human plasma catecholamines by high-performance liquid chromatography with a reversed-phase triacontylsilyl silica column

The clinical importance of simultaneous analysis of 3,4-dihydroxyphenylglycol with other human plasma catecholamines has been investigated to better understand the sympathetic nervous system. However, previous reports have had analytical difficulties with both resolution and extraction. The current study uses a reversed-phase triacontylsilyl silica (C30) column under the mobile phase condition without ion-pair reagents to separate catecholamines and their metabolites, with above 91% recoveries for intra-assay, above 85% for inter-assay, and less than 10% (n=5) coefficient of variation. Lower detection limits (S/N=4) and quantification limits (S/N=6) were 40 and 100 pg/mL for norepinephrine, 3,4-dihydroxyphenylglycol, and 3,4-dihydroxyphenylalanine, 10 and 20 pg/mL for epinephrine, 10 and 40 pg/mL for dopamine. Linear ranges were from 40 to 5000 pg/mL for norepinephrine and 3,4-dihydroxyphenylalanine, from 100 to 5000 pg/mL for 3,4-dihydroxyphenylglycol, and from 10 to 2000 pg/mL for epinephrine and dopamine. The C30 column may prove clinically useful, as it provides a convenient and simultaneous method of evaluation of human plasma catecholamines.     

Detection of trace aflatoxin M1 in human urine using a commercial ELISA followed by HPLC

Aflatoxin is a liver carcinogen, and rapid, inexpensive methods to detect its urinary biomarkers are needed. We used a commercial enzyme-linked immuno-sorbent assay (ELISA) for aflatoxin M1 in urine (Helica Biosystems) to test 52 Haitian samples. Using this ELISA, we detected traces above the limit of detection (0.2?ng/ml urine) but below the limit of quantitation (0.4?ng/ml) in 14 samples. Liquid chromatography of all 52 Haitian urine samples revealed that only 11 had quantifiable AFM1 (mean: 29.5?pg/ml, standard error: 10.8, range: 2.94–96.5?pg/ml). The Helica ELISA may have detected forms of aflatoxin other than AFM1 in the Haitian samples, or matrix enhancement may have affected results at low AFM1 concentrations. This ELISA may serve as an initial, qualitative indicator of aflatoxin exposure for epidemiological purposes. But this method’s utility as a precise and specific indicator of AFM1 concentrations will require additional refinement and validation.     

A real-time immuno-PCR assay for routine ultrasensitive quantification of proteins

A fast and robust assay, based on the combination of the highly sensitive immuno-PCR (IPCR), employing standardized self-assembled DNA-protein conjugates as reagents, and the well-established, reliable, and fast real-time PCR detection by means of the TaqMan principle is introduced in this work. The use of anti-species immunoglobulin reagents allows one for easy adaptation of this assay to basically any existing ELISA application. The use of an internal competitor in the real-time IPCR (rtIPCR) further increases the sensitivity and significance of this assay; 0.1-0.01 amol (500-50 fg/mL) IgG from several species (mouse, rabbit, goat, and human) were detectable using direct, indirect, and sandwich model rtIPCR assays, thereby increasing the detection limit of the analogous ELISA tests about 100- to 1000-fold. The robustness of this method was demonstrated in two typical applications by detecting 40 pg/mL of the novel anti-cancer drug rViscumin in human plasma samples as well as 100 pg/mL of a research antibody in cell culture media. In both cases, a comparable ELISA was 1000-fold less sensitive.     

人CD137抗体促进NK细胞对乳腺癌细胞特异性杀伤作用的体外研究

目的探讨人自然杀伤(NK)细胞在CD137抗体作用下通过抗体依赖性细胞毒性作用(ADCC)介导对乳腺癌细胞的杀伤作用。方法NK细胞表型和细胞因子检测实验分组:阴性对照组(未用人CD137抗体处理的NK细胞)、CD137抗体处理组(10μg/mL人CD137抗体处理4 h的NK细胞);NK细胞毒性检测实验分组:根据体系中是否添加NK细胞、人CD137抗体和西妥昔单抗分为8组。流式细胞术检测两组NK细胞表面CD16分子的表达情况,酶联免疫吸附测定(ELISA)法检测两组NK细胞培养上清液中干扰素(IFN)-γ和肿瘤坏死因子(TNF)-α的浓度,乳酸脱氢酶(LDH)法检测8组反应体系中表皮生长因子受体(EGFR)高表达乳腺癌细胞系MDA-MB-231和EGFR低表达乳腺癌细胞系MDA-MB-453的杀伤比例,并采用t检验或析因分析进行统计学分析。结果与阴性对照组比较,CD137抗体处理组CD16+NK细胞比例(79.57﹪±0.92﹪比90.43﹪±0.67﹪)、细胞因子IFN-γ浓度[(388.90±7.02)pg/mL比(523.90±1.90)pg/mL]和TNF-α浓度[(20.59±4.09)pg/mL比(47.22±2.14)pg/mL]均升高,差异有统计学意义(P<0.05);MDA-MB-231细胞杀伤的三因素析因分析结果显示:NK细胞、人CD137抗体和西妥昔单抗3个因素分别对MDA-MB-231细胞的杀伤都有作用(F=5227.276、201.473、1792.242,P均<0.001),3个因素两两之间的交互作用对MDA-MB-231细胞的杀伤也都有作用(F=183.903、1517.187、33.483,P均<0.001),3个因素的二级交互作用差异有统计学意义(F=41.505,P<0.001)。结论人CD137抗体可增强NK细胞分泌细胞毒性因子IFN-γ和TNF-α的能力,同时可上调NK细胞表面CD16分子的表达,从而使得NK细胞可能通过西妥昔单抗介导的ADCC作用增强对表皮生长因子受体(EGFR)高表达乳腺癌细胞的杀伤作用。     

Comparison between inhibitory indirect ELISA and HPLC methods to quantify free and adducted aflatoxins in human urine

HPLC and Inhibitory Indirect ELISA (I.I. ELISA) methods for quantitation of aflatoxins (AF) in human urine were compared in terms of specificity, sensitivity, easiness and cost. I.I. ELISA was optimized in kind of antibody in use, type of plastic plate, adduct synthesis technique, peroxidase and antibody dilutions, etc. Both polyclonal (Cuban) and monoclonal (British) anti-AF antibodies were statistically studied and the process was standardized. HPLC and electrophoresis were performed while synthetizing AFB(1)-DNA and AFB(1)-Cl-Ovalbumin (AFB(1)-Cl-Ov) adducts. Costar polystyrene plate had the best adherence. Optimum coating dilution was 10 ng of AFB(1)-Cl-Ov per well. Dilutions of 1:1000 of monoclonal antibody from purified culture or 1:300 from monoclonal antibody from tissue culture and 1:1000 of peroxidase anti-mouse conjugate were the best. Optimum separation with HPLC was obtained isocratically with 60% MeOH and 40% distilled water mobile phase. ELISA had a sensitivity of 1 pg mL(-1) AFB(1) and HPLC sensitivity was 0.1 ng mL(-1) AFB(1) with fluorescence detector and 4.5 ng mL(-1) with UV detector. Monoclonal antibody gave more accurate results for determination of free and adducted AFB(1) in urine analysis.     

Electrochemical immunosensor array using a 96-well screen-printed microplate for aflatoxin B1 detection

A novel analytical immunosensor array, based on a microtiter plate coupled to a multichannel electrochemical detection (MED) system using the intermittent pulse amperometry (IPA) technique, is proposed for the detection of aflatoxin B1 (AFB1). In the present work, the electrochemical behaviour and electroanalytical performance of the thick-film carbon sensors (also designated as screen-printed electrodes) incorporated in the multichannel electrochemical plate were first evaluated. Then the 96-well screen-printed microplate was modified in accord with a competitive indirect enzyme-linked immunoassay (ELISA) format for aflatoxin B1 detection. The measurements were performed using both spectrophotometric and electrochemical procedures and the results of the calibration curves, detection limit (LOD), sensitivity and reproducibility of the respective assay systems were evaluated. The immunoassay was then applied for analysis of corn samples spiked with AFB1 before and after the extraction treatment, in order to study the extraction efficiency and the matrix effect, respectively. These studies have shown that using this system, AFB1 can be measured at a level of 30 pg/mL and with a working range between 0.05 and 2 ng/mL. Good recoveries (103+/-8%) were obtained, demonstrating the suitability of the proposed assay for accurate determination of the AFB1 concentration in corn samples. The specificity of the assay was assessed by studying the cross-reactivity of PAb relative to AFB1. The results indicated that the PAb could readily distinguish AFB1 from other aflatoxins, with the exception for AFG1.     

Antibody microarray analysis of inflammatory mediator release by human leukemia T-cells and human non small cell lung cancer cells.

Cytokines and chemokines are responsible for regulating inflammation and the immune response. Cytokine and chemokine release is typically measured by quantitative enzyme-linked immunosorbant assay (ELISA) or Western blot analysis. To expedite the analysis of samples for multiple cytokines/chemokines, we have developed slide-based Thermo Scientific ExcelArray Antibody Sandwich Microarrays. Each slide consists of 16 subarrays (wells), each printed with 12 specific antibodies in triplicate and positive and negative control elements. This 16-well format allows for the analysis of 10 test samples using a six-point standard curve. The array architecture is based on the "sandwich" ELISA, in which an analyte protein is sandwiched between an immobilized capture antibody and a biotinylated detection antibody, using streptavidin-linked Thermo Scientific DyLight 649 Dye for quantitation. The observed sensitivity of this assay was <10 pg/mL. In our experiments, the Jurkat cell line was used as a model for human T-cell leukemia, and the A549 cell line was used as a model for human non-small cell lung cancer. To evoke a cytokine/chemokine response, cells were stimulated with tumor necrosis factor alpha (TNFalpha), phorbol-12-myristate-13-acetate (PMA, TPA), and phytohemagglutinin (PHA). Cell supernatants derived from both untreated and stimulated cells were analyzed on four different arrays (Inflammation I, Inflammation II, Angiogenesis, and Chemotaxis), enabling the quantitation of 41 unique analytes. Stimulated cells showed an increase in the expression level of many of the test analytes, including IL-8, TNF-alpha, and MIP-1alpha, compared to the non-treated controls. Our experiments clearly demonstrate the utility of antibody microarray analysis of cell-culture supernatants for the profiling of cellular inflammatory mediator release.