Quantitative assessment of factors affecting the sensitivity of a competitive immunomicroarray for pesticide detection

Analytical protein microarrays offering highly parallel analysis can become an invaluable tool for a wide range of immunodiagnostic applications. Here we describe factors that influence the sensitivity of a competitive immunomicroarray that quantifies small molecules; in this case, the pesticides dichlobenil metabolite 2,6-dichlorobenzamide (BAM) and atrazine. Free pesticide concentrations in solution are quantified by the competitive binding of fluorescence-conjugated monoclonal antibodies to either surface-immobilized pesticide hapten-protein conjugates or pesticides in solution. We investigated the influence of antibody labeling techniques, microarray substrates, and spotting and incubation buffers. The results showed that microarrays immobilized on EasySpot or in-house fabricated agarose substrates printed with Genetix Amine Spotting Solution resulted in optimum results when the arrays were incubated with the sample/antibodies diluted in a Tris buffer supplemented with 0.05% each bovine serum albumin (BSA) and Tween 20. Furthermore, the application of directly labeled primary antibodies allowed for better sensitivity compared to secondary polyclonal antibody quantification.     

Chemiluminescent Enzyme Immunoassay for Measuring Leptin

Leptin is one of the representative adipocyte-derived protein hormones. Measuring the serum leptin concentration gives an important index for preventing and treating diabetes mellitus and other diseases. We constructed in this study a chemiluminescent enzyme immunoassay (CLEIA) for measuring leptin by using the anti-leptin polyclonal antibody and alkaline phosphatase (ALP). The method applies the IgG-conjugated ferrite particle to capture leptin in a sample and the ALP-conjugated Fab fragment to detect the captured leptin. We tested Block ace, CE510, and bovine serum albumin (BSA) for their abilities to block non-specific binding of ALP-conjugated anti-leptin Fab to the ferrite particle and found BSA to be the most effective. The measurable range with this ELISA for leptin was 0.1–1.0 pg/mL of leptin and the detection limit (blank+2SD) was 0.1 pg/mL of leptin. These results demonstrate sufficient sensitivity with our system to measure the serum leptin concentration and its clinical usefulness. The results also suggest that a sensitive enzyme immunoassay can be constructed by using only one polyclonal antibody.     

Chemiluminescent enzyme immunoassay for measuring leptin

Leptin is one of the representative adipocyte-derived protein hormones. Measuring the serum leptin concentration gives an important index for preventing and treating diabetes mellitus and other diseases. We constructed in this study a chemiluminescent enzyme immunoassay (CLEIA) for measuring leptin by using the anti-leptin polyclonal antibody and alkaline phosphatase (ALP). The method applies the IgG-conjugated ferrite particle to capture leptin in a sample and the ALP-conjugated Fab fragment to detect the captured leptin. We tested Block ace, CE510, and bovine serum albumin (BSA) for their abilities to block non-specific binding of ALP-conjugated anti-leptin Fab to the ferrite particle and found BSA to be the most effective. The measurable range with this ELISA for leptin was 0.1-1.0 pg/mL of leptin and the detection limit (blank+2SD) was 0.1 pg/mL of leptin. These results demonstrate sufficient sensitivity with our system to measure the serum leptin concentration and its clinical usefulness. The results also suggest that a sensitive enzyme immunoassay can be constructed by using only one polyclonal antibody.     

玉米赤霉烯酮单克隆抗体的制备及间接竞争ELISA检测方法的建立

玉米赤霉烯酮具有较强的生物毒性,检测谷物中的玉米赤霉烯酮在食品和饲料安全中具有重要的作用.将玉米赤霉烯酮与牛血清白蛋白的偶联物免疫BALB/c小鼠制备单克隆抗体,并建立基于单克隆抗体的酶联免疫法作为检测玉米赤霉烯酮的方法.结果共筛选到4株抗玉米赤霉烯酮单克隆抗体,3株抗体亚类为IgG1,1株为IgG2b.选择其中的一株杂交瘤细胞2C9制备小鼠腹水,纯化后测定了抗体效价为1/40 000.以此单抗建立的间接竞争ELISA方法,其半数抑制率(IC50)为1.90 ng/mL,检测限(IC10)为0.051 ng/mL,检测区间(IC20-IC80)为0.115-13.900 ng/mL;且对玉米赤霉烯酮有很好的特异性.回收率检测在样品含1.46-93.80 μg/kg时回收率为96.5％-113.0％.本实验建立的检测方法可用于多种谷物及饲料样本中玉米赤霉烯酮的检测.     

Detection of Bacillus anthracis spores and a model protein using PEMC sensors in a flow cell at 1 mL/min

Piezoelectric-excited millimeter-sized cantilever (PEMC) sensors of 4mm(2) sensing area were immobilized with antibody specific to Bacillus anthracis (anti-BA) spores or bovine serum albumin (anti-BSA). Detection of pathogen (Bacillus anthracis (BA) at 300 spores/mL) and BSA (1 mg/mL) were investigated under both stagnant and flow conditions. Two flow cell designs were evaluated by characterizing flow-induced resonant frequency shifts. One of the flow cells labeled SFC-2 (hold-up volume of 0.3 mL), showed small fluctuations (+/-20 Hz) around a common resonant frequency response of 217 Hz in the flow rate range of 1-17 mL/min. The total resonant frequency change obtained for the binding of 300 spores/mL in 1h was 90+/-5 Hz (n=2), and 162+/-10 Hz (n=2) under stagnant and flow conditions, respectively. Binding of antibodies, anti-BA and anti-BSA, were more rapid under flow than under stagnant conditions. The sensor was repeatedly exposed to BSA with an intermediate release step. The first and second responses to BSA were nearly identical. The total resonant frequency response to BSA was 388+/-10 (n=2) Hz under flow conditions. Kinetic analysis is carried out to quantify the effect of flow rate on antibody immobilization and the two types of detection experiments.     

Fluorescein and tetramethyl rhodamine as haptens in enzyme immunohistochemistry

Summary Fluorescein (Fl) and tetramethyl rhodamine (Rh) were evaluated as possible candidates for a double hapten sandwich system in enzyme immunohistology. Monoclonal antibodies were raised against Fl and Rh. Their fine-specificity was tested with a competition-like assay. A pair of Mab's was selected for immunohistology in which they functioned as a bridge between Fl/Rh conjugated antibodies and Fl/Rh labeled peroxidase and alkaline phosphatase, respectively. The binding of fluorescein labeled antibodies could be successfully demonstrated in histological slides. A large variability in the efficacy of staining was observed in the case of rhodamine labeled antibodies. The phenomenon is explained by assuming that tetramethyl rhodamine isothiocyanate reacts preferentially with lysine residues near to, or embedded in, hydrophobic regions in a protein. This condition may reduce the accessibility of the Rh moiety for anti-Rh antibodies.Abbreviations AEC 3-amino-9-ethylcarbazole - AP Alkaline phosphatase - BSA Bovine serum albumin - CGG Chicken gamma globulin - c-IF Cytoplasmic immunofluorescence - DAB 3,3-Diamino benzidine HCl - ELISA Enzyme linked immunosorbent assay - Fl Fluorescein - F/P ratio Number of fluorochrome molecules per molecule of protein - Ig Immunoglobulin - Mab Monoclonal antibody - OPD Ortho-phenylenediamine-dihydrochloride - PBS Phosphate buffered saline - PBS-T PBS with 0.2% Tween-20 - PO Peroxidase - RAM/PO Peroxidase labeled rabbit antiserum directed against mouse immunoglobulins - Rh Tetramethyl rhodamine - RT Room temperature In honour of Prof. P. van Duijn     

Comparison of different enzyme immunoassays for measuring indole-3-acetic acid in vegetative citrus tissues

An enzyme-linked immunosorbent assay (ELISA) using polyclonal antibodies, which were raised against indole-3-acetic acid (IAA) conjugated to bovine serum albumin (BSA) via the indolic nitrogen (IAA-N₁-BSA), has been developed. The sensitivity and specificity of these antibodies were compared to those of polyclonal and monoclonal antibodies raised against IAA conjugated to BSA via C₁ of the carboxyl group (IAA-C₁-BSA). The sensitivity of the assays improved in the following order: monoclonal antibodies > antibodies to IAA-C₁-BSA > antibodies to IAA-C₁-BSA. Antibodies against IAA-C₁-BSA had less cross-reactivity to indoles structurally related to IAA, excluding indole-3-pyruvic acid. A rapid and effective method for purification of IAA in citrus tissues before analysis by ELISA is described. Values of IAA in citrus ( Citrus sinensis [L.] Osbeck cv. Shamouti orange) shoot tips obtained with all three antibodies were similar. However, in leaf tissues which contain lower amounts of IAA compared to shoot tips, monoclonal antibodies gave higher values of IAA than polyclonal antibodies. Estimation of free IAA levels in purified extracts of citrus shoot tips, very young leaves, and mature leaves was ca 380, 248, and 74 ng (g fresh weight)⁻¹ respectively.     

Extremely sensitive and selective antibodies against the explosive 2,4,6-trinitrotoluene by rational design of a structurally optimized hapten

Antibodies are a promising tool for the fast and selective trace detection of explosives. Unfortunately, the production of high-quality antibodies is not trivial and often expensive. Therefore, excellent antibodies are a rare and limiting resource in fields such as biosensing, environmental analysis, diagnostics, cancer therapy, and proteomics. Here, we report the synthesis, bioconjugation, and application of the structurally optimized hapten 6-(2,4,6-trinitro)-phenylhexanoic acid to improve the selectivity and sensitivity of antibodies for the detection of one of the most important explosives, trinitrotoluene. With a conjugate of bovine serum albumin and a highly purified N-hydroxy-succinimide (NHS)-activated hapten, two rabbits were immunized to obtain polyclonal antibodies. The immunization process was monitored by enzyme-linked immunosorbent assay to gain information about the progress of antibody titer and affinity. Finally, the polyclonal antibodies reached an affinity constant of (5.1 ± 0.6) × 10(9) l/mol (rabbit R1) and (2.3 ± 0.2) × 10(9) l/mol (rabbit R2). The respective assays show a minimum test midpoint (IC(50) value) of 0.1 ± 0.01 μg/l (R1) and 0.2 ± 0.02 μg/l (R2) and a working range of 0.005 to 150 μg/l (R1) and 0.007 to 200 μg/l (R2), which corresponds to more than four orders of magnitude for both. This is quite remarkable for a competitive immunoassay, which is often believed to have a narrow dynamic range. The limit of detection was calculated to 0.6 ng/l (R1) and 1.5 ng/l (R2), which is up to 100 times improvement in relation to the assay of Zeck et al. (1999) on the basis of a monoclonal antibody. The excellent selectivity of the polyclonal antibodies was comprehensively examined by determining the cross-reactivity to common explosives and other nitroaromatics including nitro musk components. The widely held belief that polyclonal antibodies generally display higher cross-reactivities than monoclonals could be disproved.     

Monoclonal antibodies for use with 125iodine-labeled radioligands in progesterone radioimmunoassay

The interactions of heterologous and homologous 125I-iodinated radioligand with polyclonal and monoclonal antibodies directed against 11-hydroxyprogesterone hemisuccinate conjugated to bovine serum albumin were compared. Our data show that, with a polyclonal antibody, the use of the same bridge in the tracer and the antigen results in a low sensitivity while a heterologous tracer can decrease markedly the titer of antibody but increases the sensitivity of the radioimmunoassay. Due to the high specificity of monoclonal antibodies, the interaction with heterologous and homologous iodinated tracers is extremely variable from one antibody to another but the present results demonstrate that radioimmunoassays of very high sensitivity can be obtained with homologous and heterogeneous tracers. However, an appropriate tracer has to be selected to meet the requirement of such an assay.     

单克隆抗体与多克隆抗体配对ELISA方法比较

以人绒毛膜促性腺激素(HCG)为抗原,制备出对HCG的多克隆抗体和特异性单克隆抗体,并进行抗体纯化和特性分析,利用辣根过氧化物酶(HRP)分别对其进行了标记.采用双抗夹心ELISA试验,探讨了多克隆抗体与单克隆抗体配对的若干事项.结果表明,利用单克隆抗体和酶标多克隆抗体配对,并用含动物血清的稀释液稀释酶标抗体,可实现对检测原的高特异性和高灵敏度检测.     

Bioluminescent immunoassay for alpha-fetoprotein

A bioluminescent immunoassay of alpha-fetoprotein is described. It uses monoclonal antibodies labeled with glucose-6-phosphate dehydrogenase and polyclonal antibodies coimmobilized on Sepharose with bioluminescent enzymes from marine bacteria. The bioluminescent reaction which occurs in the immunosorbent is proportional to the amount of alpha-fetoprotein in the assay. The protocol is simple and rapid, and no separation step is required to remove the excess labeled antibodies. The assay can be performed directly on 25 microliters serum and it is as sensitive as other immunometric assays.     

Detection of staphylococcal enterotoxins by enzyme-linked immunosorbent assays and radioimmunoassays: comparison of monoclonal and polyclonal antibody systems.

Murine monoclonal antibodies reactive with at least one of the serological types of staphylococcal enterotoxin were examined for use in assay systems for the detection of enterotoxin at the level of 1.0 ng of enterotoxin per ml. An antibody sandwich enzyme-linked immunosorbent assay was devised for each toxin type by identifying an effective antibody pair. One antibody (the coating antibody) was coated onto a polystyrene plate and removed the enterotoxin from the test solution; the second antibody (the probing antibody) was conjugated to horseradish peroxidase and detected the captured toxin. Enterotoxins A and E could be detected in the same system by the use of cross-reacting monoclonal antibodies. All subtypes of enterotoxin C could be detected in one assay system. Two effective systems were described for each of types B and D. Each of these systems, when compared with the homologous enterotoxin-specific polyclonal rabbit antibody systems, was found to compare favorably. The monoclonal enzyme-linked immunosorbent assay systems for the detection of enterotoxins A and C2 were examined for a variety of food extracts; no abnormal interference could be detected from these extracts. The monoclonal antibody systems were also compared with the homologous enterotoxin-specific polyclonal serum for the detection of enterotoxin by the competitive radioimmunoassay (RIA). Single monoclonal antibodies generally did not perform as well in the RIA as did the homologous toxin-specific polyclonal serum. However, pools of monoclonal antibodies were prepared that approached the sensitivity and precision of the polyclonal system for the detection of each toxin by the RIA.     

Immunolocalization of abscisic acid by monoclonal antibodies in Lavandula stoechas L. leaves

A monoclonal antibody was used to localize abscisic acid in Lavandula stoechas L. plants treated with 1 M abscisic acid. ABA localization was performed using EDC as the only fixative, which preserves antigenicity and improves the specificity of monoclonal antibodies. A relationship was observed between the effect of abscisic acid on chloroplast ultrastructure and ABA accumulation. Gold particles accumulated on swollen chloroplasts. Our findings provide cytochemical evidence that the chloroplast is a trapping compartment, and yield valuable information on the relation between chloroplast ultrastructure and ABA accumulation.Abbreviations ABA abscisic acid - BSA bovine serum albumin - EDC 1-(3-dimethyl-aminopropyl)-3 ethyl carbodiimide - IgG immunoglobulin G - mAB monoclonal antibodies - pAB polyclonal antibodies     

Monoclonal antibody against a lactose epitope of glycosphingolipids binds to melanoma tumour cells

Mice were immunized with a neoglycoprotein consisting of a chemically modified carbohydrate moiety (reductively aminated 3-sialyllactose) linked to human serum albumin. By this procedure an antibody response to the normally non-immunogenic carbohydrate structure was obtained. Hybridomas were established, and monoclonal antibodies were selected in ELISA based on their binding to the saccharide hapten, or to a lactosylceramide-mimicking neoglycolipid, lactose-bis-sulfone. One of the selected antibodies, 2H4, was of particular interest, since it also bound to glycolipids present on melanoma cells. FACS analysis of a panel of 14 melanoma cell lines showed that the 2H4 antibody bound to the majority of these. In frozen, non-fixed sections or paraffin sections of biopsies the monoclonal antibody 2H4 stained melanoma cells, but not tumour infiltrating lymphocytes or normal skin. Detailed immunochemical analysis of 2H4, using thin layer chromatography revealed that it recognized an internal lactose epitope in several glycosphingolipids.Abbreviations BSA bovine serum albumin - FACS fluorescence activated cell sorter - ELISA enzyme-linked immunosorbent assay - HSA human serum albumin - LacCer lactosylceramide - MAb monoclonal antibody - PBS phosphate buffered saline     

Detection of staphylococcal enterotoxins by enzyme-linked immunosorbent assays and radioimmunoassays: comparison of monoclonal and polyclonal antibody systems

Murine monoclonal antibodies reactive with at least one of the serological types of staphylococcal enterotoxin were examined for use in assay systems for the detection of enterotoxin at the level of 1.0 ng of enterotoxin per ml. An antibody sandwich enzyme-linked immunosorbent assay was devised for each toxin type by identifying an effective antibody pair. One antibody (the coating antibody) was coated onto a polystyrene plate and removed the enterotoxin from the test solution; the second antibody (the probing antibody) was conjugated to horseradish peroxidase and detected the captured toxin. Enterotoxins A and E could be detected in the same system by the use of cross-reacting monoclonal antibodies. All subtypes of enterotoxin C could be detected in one assay system. Two effective systems were described for each of types B and D. Each of these systems, when compared with the homologous enterotoxin-specific polyclonal rabbit antibody systems, was found to compare favorably. The monoclonal enzyme-linked immunosorbent assay systems for the detection of enterotoxins A and C2 were examined for a variety of food extracts; no abnormal interference could be detected from these extracts. The monoclonal antibody systems were also compared with the homologous enterotoxin-specific polyclonal serum for the detection of enterotoxin by the competitive radioimmunoassay (RIA). Single monoclonal antibodies generally did not perform as well in the RIA as did the homologous toxin-specific polyclonal serum. However, pools of monoclonal antibodies were prepared that approached the sensitivity and precision of the polyclonal system for the detection of each toxin by the RIA.     

金霉素单克隆抗体的制备及检测方法的建立

采用羰基二咪唑法,将半抗原金霉素(AM)分别与牛血清白蛋白(BSA)和卵清蛋白(OVA)偶联制备金霉素免疫抗原AM-BSA和检测抗原AM-OVA,通过紫外光谱扫描检测偶联产物。采用细胞杂交瘤技术,制备抗金霉素单克隆抗体杂交瘤细胞株,建立了金霉素竞争ELISA检测方法,其灵敏度达到50ng/ml,且呈现良好的线性关系(r=0.9812),并且与其他抗生素无交叉反应。     

Immunochemical characterization of polyclonal and monoclonal Streptococcus group A antibodies by chemically defined glycoconjugates and synthetic oligosaccharides.

Synthetic oligosaccharides of increasing complexity that represent different epitopes of the Streptococcus Group A cell-wall polysaccharide were used as haptens and glycoconjugates of bovine serum albumin (BSA) and horse hemoglobin (HHb) to characterize polyclonal and monoclonal antibodies. Rabbits were immunized with the BSA glycoconjugates of a linear trisaccharide, branched trisaccharide, and branched pentasaccharide. The binding specificities of the polyclonal antisera were determined by a series of inhibition ELISA studies in which disaccharide through pentasaccharide haptens were used as inhibitors of antibody-glycoconjugate binding. Monoclonal antibodies derived from mice immunized with a killed bacterial vaccine were selected for their binding to native polysaccharide antigen coupled to BSA and the BSA glycoconjugates of the di- and linear tri-saccharides. Polyclonal antibodies were moderately specific for the oligosaccharide epitope of the immunizing glycoconjugate and only those antibodies raised to the branched pentasaccharide antigen showed cross-reaction with the bacterial antigen. The behaviour of selected monoclonal antibodies parallels the binding profile of polyclonal antibodies in that the two highest-titre antibodies were directed toward an epitope displayed by the branched pentasaccharide.     

Immunogenicity of a brominated protein and successive establishment of a monoclonal antibody to dihalogenated tyrosine

During inhalation of allergens and experimental sepsis, formation of brominated tyrosine has been reported. In this study, we first examined the immunogenicity of brominated protein prepared by treatment of N-bromosuccinimide (NBS). The immunized serum obtained reacted with brominated bovine serum albumin (BSA). The NBS dose-dependent formation of immunoreactivity, which was estimated by enzyme-linked immunosorbent assay, was observed, and the increase coincided with 3,5-dibromotyrosine (DiBrY) formation in the modified BSA, which was chemically determined by liquid chromatography/quadrupole tandem mass spectrometry (LC/MS/MS). Second, by use of immunized mice, monoclonal antibodies to the brominated one were prepared. The two established novel monoclonal antibodies obtained from the immunized mice reacted with DiBrY, 3,5-dichlorotyrosine (DiClY), and 3,5-diiodotyrosine (DiIY). Moreover, 3,5-dihalo-4-hydroxybenzoic acids (3,5-dichloro-4-hydroxybenzoic acid and 3,5-dibromo-4-hydroxybenzoic acid) were recognized by these antibodies. These results suggest that dihalogenated tyrosines (DiBrY, DiClY, and DiIY) are the epitopes. Lastly, we used the antibody in an immunohistochemical study. Lipopolysaccharide (LPS) was intraperitoneally administered to mice, and livers were removed. Positive staining of LPS-treated mouse liver tissues by both the anti-dihalotyrosine antibody and anti-myeloperoxidase antibody was estimated, suggesting that inflammatory tissue damage induces the formation of dihalotyrosine in vivo.     

Preparation of anti-2,4-dichlorophenol and 2,4-dichlorophenoxyacetic acid monoclonal antibodies

The ratios of hapten and bovine serum albumin (BSA) in an antigen conjugate were determined by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Hybridomas secreting monoclonal antibodies against 2,4-dichlorophenoxyacetic acid (2,4-D) were produced by fusing 2,4-D-BSA conjugate-immunized splenocytes with a HAT-sensitive mouse myeloma cell line, P3-X63-Ag8-653. A substantial cross-reaction was observed for 2,4-dichlorophenol (2,4-DP) when compared with that observed for 2,4-D. The full measurement range for this assay is 0.2–3 μg ml⁻¹ for 2,4-DP. On the other hand, the range for 2,4-D is between 1 and 20 μg ml⁻¹. This revised version was published online in July 2006 with corrections to the Cover Date.     

定量检测猕猴血清中重组人源化抗狂犬病毒单克隆抗体间接ELISA法的建立

目的：建立定量检测血清中重组人源化抗狂犬病毒单克隆抗体（HuMabs）NM57的间接ELISA法,为药代动力学研究提供一种简单快速的方法。方法：采用狂犬病毒糖蛋白包被酶标板、HRP标记的IgG-Fc段为标记抗体,建立定量检测HuMabsNM57的间接ELISA法,并对其特异性、灵敏度、精密度及准确度进行检测。结果：间接ELISA法检测HuMabsNM57的灵敏度为5ng／mL,组内及组间精密度分别为2．6％-6．0％、8．5％-11．3％。结论：建立了灵敏度高、特异性强的检测HuMabs NM57的间接ELISA法,精密度及准确度均符合药代动力学要求,可用于猕猴及人血清中HuMabsNM57的检测。