High specificity detection of Pb2+ ions by p-SCN-Bz-DTPA immunogen and p-NH2-Bn-DTPA coating antigen

Only one bifunctional metal-chelator was used to prepare immunogen and coating antigen in all of the previous researches. However, the antibody-specific recognition to the spacer arm of the bifunctional metal- chelator might lower the specificity of heavy metal ions immunoassay. Two different bifunctional metal-chelators were adopted to prepare the immunogen and coating antigen respectively in our study to avoid this problem. The conjugates of keyhole limpet hemocyanin （KLH） and p-SCN-Bz-DTPA-Pb were used as immunogen, whereas the conjugates of bovine sentrn albumin （BSA） and p- NH2-Bn-DTPA-Pb were used as coating antigen. Poly- clonal antibodies specific to DTPA-Pb chelates were obtained from rabbits. Indirect competitive enzyme-linked immunosorbent assay （ELISA） was adopted to detect Pb^2＋ ion solutions prepared by Pb^2＋ standard solution and ultrapure water. In the mixing microplate, DTPA and Pb2＋ ions formed chelates and combined with specific anti- bodies. After incubation, the DTPA-Pb and the antibodies complex were added into the wells of the reaction microplate. The reaction microplate was coated by the conjugates ofBSA andp-NH2-DTPA-Pb, which competed for the specific antibodies. The result signals presented a good sigmoid curve when the Pb^2＋ concentration ranges from 0.01 to 100mg·L^-1 The IC50 of the indirect competitive ELISA is 0.23±0.04mg·L^-1 Pb2＋ ion. The cross-reaction with Cd^2＋, Cu^2＋, Fe^2＋, Mn^2＋, Zn^2＋, and other divalent ions were less than 5%.     

Production of monoclonal antibody against doxycycline for immunoassay of seven tetracyclines in bovine muscle and milk

The objective of this study was to produce a generic monoclonal antibody for multi-determination of the residues of tetracycline drugs in bovine muscle and milk. Two new immunogens of doxycycline were prepared that were used to produce the monoclonal antibodies. Results showed the obtained antibodies simultaneously recognized seven tetracycline drugs (doxycycline, tetracycline, chlortetracycline, oxytetracycline, minocycline, methacycline, demeclocycline). The obtained antibodies and three coating antigens were arranged into six combinations to optimize the reagents combination. After comparison of the performances of these combinations, a heterologous indirect competitive ELISA was then used to determine the seven tetracyclines in bovine muscle and milk. The crossreactivities to the seven analytes were in the range of 47%–102% and the limits of detection were in the range of 1.5–6.9 ng/mL depending on the compound. The recoveries of the seven drugs from fortified blank samples were in the range of 75.3%–106.8% with coefficients of variation lower than 10.9%. Therefore, this method could be used as a multi-analytes screen tool for routine monitoring of the residues of these tetracycline drugs in bovine muscle and milk.     

Prenatal diagnosis of cystic fibrosis using a monoclonal antibody specific for intestinal alkaline phosphatase

A monoclonal antibody (AAP-1), specific for the intestinal isoenzyme of alkaline phosphatase (ALP), has been used to develop an immunoassay for amniotic fluid samples. Values in the immunoassay correlated closely with those obtained by direct determination of phenylalanine-inhibitable ALP. A panel of 124 control second-trimester amniotic fluids and 21 fluids with a 1 in 4 risk of a cystic fibrosis fetus were examined in the immunoassay. Eight of 10 affected cases had values below an arbitrary cut-off of one third median, while all the non-affected cases were above this level. Almost identical results were obtained by enzymatic determination of phenylalanine-inhibitable ALP. However, in both systems the false positive rate (control fluids with values below one third median), was unacceptably high. It is pointed out that at present the most effective system for the prenatal diagnosis of cystic fibrosis is achieved by measuring the ratio of intestinal to total ALP in amniotic fluid supernatants. This is probably best effected by enzymatic assay in the presence of phenylalanine and homoarginine inhibition.     

重金属污染物的免疫学检测技术研究进展

重金属的免疫学检测是一种新型的重金属检测方法，与传统检测方法相比，具有省时、省力、费用低廉、便于携带、易于操作等优点，能用于重金属污染物的现场快速检测和常规检测，这对于重金属污染地区的补救和恢复工作具有很大的意义，因而发展和普及应用潜力很大。国外学者通过选择或合成双功能鳌合剂鳌合重金属离子并与载体蛋白偶联制备出完全抗原，进一步制备出金属特异性单抗。目前应用免疫学检测方法检测环境中的重金属离子还处于实验室的试验阶段，初步实验结果表明KinExA免疫检测法具有用作重金属免疫检测传感器的能力，并且越来越多的重金属检测模型被开发出来。金属特异性抗体一抗原的结合属性的初步研究表明，影响抗体对抗原识别的主要因素有：金属离子的半径、电子和形态上的并协性；鳌合剂的结构；金属离子一鳌合剂复合物三维结构和价态结构；抗体中的某些氨基酸残基能与抗原中金属离子直接配位以及与抗原中的鳌合剂部分发生相互作用（疏水作用、氢键作用等）。     

Fetal renal vein thrombosis,hydrops fetalis,and maternal lupus anticoagulant. A case report

Hydrops fetalis with fetal renal vein thrombosis in a mother with antiphospholipid antibody syndrome detected post-partum suggests an underlying pathogenetic association that may provide new strategies for treatment of a lethal disorder.     

重组BALB/C鼠冷诱导RNA结合蛋白CIRP多克隆抗体的制备与鉴定

冷诱导RNA结合蛋白(CIRP)是第一种在哺乳动物细胞中被发现的冷休克蛋白,伴随着细胞冷应激过程过量表达.为进一步研究冷诱导RNA结合蛋白CIRP的生物学功能,构建重组BALB/C鼠CIRP的原核表达载体,重组BALB/C鼠CIRP蛋白经NI-NTA亲和树脂纯化后免疫獭兔制备抗CIRP多克隆抗体,以间接ELISA和Western blot检测抗体效价和抗原特异性.本实验成功构建了CIRP的原核表达载体,在E coli XL-1-blue得到了高效表达,利用原核表达包涵体蛋白免疫獭兔获得了高效价的CIRP多克隆抗体,该抗体能识别肝脏和睾丸中天然的CIRP蛋白.图5参14     

Production of anti-amoxicillin ScFv antibody and simulation studying its molecular recognition mechanism for penicillins

The molecular recognition mechanism of an antibody for its hapten is very interesting. The objective of this research was to study the intermolecular interactions of an anti-amoxicillin antibody with penicillin drugs. The single chain variable fragment (ScFv) antibody was generated from a hybridoma cell strain excreting the monoclonal antibody for amoxicillin. The recombinant ScFv antibody showed similar recognition ability for penicillins to its parental monoclonal antibody: simultaneous recognizing 11 penicillins with cross-reactivities of 18–107%. The three-dimensional structure of the ScFv antibody was simulated by using homology modeling, and its intermolecular interactions with 11 penicillins were studied by using molecular docking. Results showed that three CDRs are involved in antibody recognition; CDR L3 Arg 100, CDR H3 Tyr226, and CDR H3 Arg 228 were the key contact amino acid residues; hydrogen bonding was the main antibody–drug intermolecular force; and the core structure of penicillin drugs was the main antibody binding position. These results could explain the recognition mechanism of anti-amoxicillin antibody for amoxicillin and its analogs. This is the first study reporting the production of ScFv antibody for penicillins and stimulation studying its recognition mechanism.     

Production of the broad specific monoclonal antibody against sarafloxacin for rapid immunoscreening of 12 fluoroquinolones in meat

The objective of this study was to produce a generic antibody for immunoassay of fluoroquinolone drugs in meat. Two novel haptens of sarafloxacin were synthesized that were used to produce the monoclonal antibodies. The obtained monoclonal antibodies simultaneously recognized 12 fluoroquinolone drugs (sarafloxacin, diflocaxin, marbofloxacin, ofloxacin, ciprofloxacin, enrofloxacin, norfloxacin, pefloxacin, lomefloxacin, amifloxacin, enofloxacin and danofloxacin). After evaluation of different coating antigen/antibody combinations, a heterologous competitive indirect enzyme linked immunosorbent assay (ELISA) was developed to determine the 12 drugs. The crossreactivities to these analytes were in the range of 18%–113% and the limits of detection were in the range of 0.8–6.5 ng/mL depending on the compound. Eight fluoroquinolones licensed as veterinary drugs in China were fortified into blank chicken for ELISA analysis. The recoveries were in the range of 67.6%–94.6% with coefficients of variation lower than 12.4%. Therefore, this method could be used as a screen tool for routine monitoring of the residues of these fluoroquinolone drugs in animal derived foods.     

Colloidal gold probe-based immunochromatographic assay for the rapid detection of lead ions in water samples

One-step immunochromatographic assay (ICA) has been developed using colloidal gold-labeled monoclonal antibody probe for the rapid detection of lead ions in water samples. The ICA was based on the theory of competitive reactivity, and the results can be easily judged based on the presence or absence of a red colored test line with visual detection. Under optimal conditions, this method shows high detecting sensitivity with a LOD (limit of detection) of 50 ng/ml. Stability test indicates that the immunochromatographic strips are stable for 8 weeks at room temperature. During practical application, nanometer TiO₂ is used to enrich the lead ions in water samples. The ICA is successfully applied in the measurement of lead ion concentrations in local water samples, and the results are highly consistent with that of ICP-MS. Detecting lead ions with ICA can be done within 4 min and is very useful for the rapid onsite testing.     

Preparation of hapten-specific monoclonal antibody for cadmium and its ELISA application to aqueous samples

High-affinity and specific monoclonal antibodies against cadmium-ethylene diamine tetraacetic acid (EDTA) complex have been produced using the hybridoma technique. A hapten was synthesized and characterized by Fourier Transform Infrared Spectroscopy (FT-IR) and UV-Vis. Competitive enzyme-linked immunosorbent assay (ELISA) for quantitative detection of cadmium in aqueous sample was developed. The monoclonal antibody with high level of binding affinity for Cd-IEDTA-BSA and high specificity for soluble Cd-EDTA complex showed less than 0.99% cross-reactivity with other 11 metals. The limit of detection was 0.10 μg·L^-1, and the effective linear range was 10^-1–10³ μg·L^-1. The intra- and inter-assay coefficient variations were 1.5%–6.3% and 3.2%–7.4%, respectively. The spike recovery in different water samples were between 98.5% and 110.3%. The detection limit of this assay was well below the allowable concentration of cadmium (3 μg·L^-1), and the working range was wider than that of other methods which showed the range of 2.19–86.38 and 0–10³ μg·L^-1. The competitive ELISA established in this paper was sensitive and accurate in the screening of cadmium in aqueous samples. The results will lay a solid foundation for construction of an immunoassay kit for cadmium.