Preparation of Anti-Cadmium-EDTA Complex Polyclonal Antibody and Its Application for Determination of Cadmium in Aqueous Solution

Abstract

A polyclonal antibody that can recognize cadmium-ethylenediaminetetraacetic acid (CD-EDTA) complex was prepared via the injection of New Zealand white rabbits with Cd-1-(4-isothiocyanatobenzyl) ethylenediamine-N,N,N′,N′-tetraacetic acid–BSA (Cd-ITCBE-BSA). The polyclonal antibody displayed high levels of affinity for Cd-1-(4-isothiocyanatobenzyl) ethylenediamine-N,N,N′,N′-tetraacetic acid-OVA (Cd-ITCBE-OVA) with favorable titer of 1.28 × 10⁶. A simple, reliable, and economical indirect competitive immunoassay based on this polyclonal antibody was developed and validated for detection of cadmium. Assay optimization was performed with respect to chelator concentration, ionic strength, blocking solution, pH, and reaction time. The detection limit of the assay was 0.21 µg L^?1, and the effective linear range was from 10^?1 to 10³µg L^?1. The coefficient of variation (CV) of intra- and interassay were 1.0–8.2% and 2.3–6.9%, respectively. Results yielded low cross-reactivity of the assay to other tested metals such as Pb²⁺, Ni²⁺, Mg²⁺, Ca²⁺, Cu²⁺, Mn²⁺, Zn²⁺, Co²⁺, Cr³⁺, and Fe³⁺, except Hg²⁺, which showed a cross-reactivity of 7.4%. Spike recoveries of ultrapure water, tap water, and samples of the Yangtze River were 85.5–116.3%. These results show that this assay is suitable for quantitative detection of cadmium at trace levels in water samples.     

Construction of Three Single‐Chain Antibody Fragment Variable Fusion Structures for Sensitive Detection of Nucleocapsid Protein of Hantaan Virus

Abstract

Three single‐chain fragment variable (scFv) fusion structures were constructed for use in rapid and sensitive detection of nucleocapsid protein (NP) of Hantaan virus. The detection of NPs on glass chips was signalized by enzyme labeling or fluorescence dye Cy3, or Cy5 cluster nanoparticles. The sensitivity of the methods with different signal systems was evaluated and compared. The detection limits of scFv‐alkaline phosphatase fusion, fluorescence labeling (scFv‐Cy3), and nanoparticles labeling (scFv‐SBP‐streptavidin‐nanoparticle) were 0.1 µg/mL, 1 ng/mL, and 0.1 ng/mL NP, respectively, which were all lower than that in a conventional enzyme‐linked immunosorbent assay (ELISA) (1 µg/mL). Twenty Hantaan virus isolates were detected using the proposed methods.     

A Simultaneous Enzyme-Linked Immunoassay for Anti-Thyroglobulin Autoantibody in Human Serum

Abstract

A “simultaneous” enzyme-linked immunoassay for the measurement of anti-thyroglobulin autoantibody in human serum was studied, in which human thyroglobulin-coated silicone rubber rod, ß-D-galactosidase- or horseradish peroxidase-conjugated human thyroglobulin and serum sample were mixed at the same time. In order to obtain a suitable range of the measurable antibody, an appropriate amount of human thyroglobulin-enzyme conjugate was carefully selected. A successful simultaneous assay for antithyroglobulin was obtained using thyroglobulin-ß-D-galactosidase conjugate, in which the minimum amount of detectable antibody was approximately 10 ng/ml using 5 μl of serum. This sensitivity was 20-fold higher than that in two-step sandwich enzyme-linked immunoassay reported previously.     

Rabbit Monoclonal Antibody-Based Lateral Flow Immunoassay Platform for Sensitive Quantitation of Four Sulfonamide Residues in Milk and Swine Urine

Based on the available rabbit monoclonal antibody (RabMAb), a rapid and sensitive lateral flow immunoassay (LFA) platform has been developed for quantitative detection of four sulfonamide residues(SRs) of sulfadiazine (SD), sulfathiazole (STZ), sulfapyridine (SP), and sulfamethoxazole (SMX).Within the designed LFA competitive format assay, which was based on antigen-antibody properties, the hapten conjugate N¹-[4-(carboxymethyl)-2-thiazolyl] sulfanilamide linked to protein ovalbumin (TS-OVA) and goat anti-rabbit antibody were sprayed as capture and control reagents, respectively, and then the antibody was conjugated to colloidal gold particles as the detection reagent. With quantitative assessment aided by a colorimetric strip reader, the sensitivities of the established LFA method for SD, STZ, SP, and SMX were 0.91 ng mL^?1, 0.10ng mL^?1,0.12ng mL^?1, and 2.13ng mL^?1, and the half-maximum inhibition concentrations (IC₅₀) were 5.19 ng mL^?1, 1.25 ng mL^?1, 0.66 ng mL^?1, and 24.14 ng mL^?1, respectively. The recoveries at three spiked levels (5, 20, 50 ng mL^?1for SD, STZ, and SP; 20, 50, 100 ng mL^?1 for SMX) were in the range of 78.02–135.10% and 76.40–137.16% for milk and swine urine, respectively. More importantly, the detection performance of the established platform was consistent with that of in-parallel LC-MS/MS analysis. In conclusion, the proposed LFA platform has showed the potential for fast, sensitive and relatively accurate quantification of four sulfonamide residues in practical uses.     

Development of a Time-Resolved Fluoroimmunoassay for the Rapid Detection of Methyl-3-Quinoxaline-2-Carboxylic Acid in Porcine Tissues

A time-resolved fluoroimmunoassay for the specific determination of methyl-3-quinoxaline-2-carboxylic acid in animal tissues, a marker residue of olaquindox, was developed. The IC₅₀ of the assay was found to be 1.46 ± 0.19 ng/mL of methyl-3-quinoxaline-2-carboxylic acid in phosphate-buffered saline samples and the detection limit was 0.16 ± 0.03 ng/mL. For porcine liver and muscle samples spiked with 5, 10, and 15 ng/g, the recovery ranges were 95.7–112.3% and 98.5–116.2% and the coefficients of variation were 9.3–11.5% and 8.9–14.2%, respectively. The time-resolved fluoroimmunoassay results correlated well with high performance liquid chromatography results (correlation coefficients of 0.991 for liver and 0.988 for muscle). This study suggests that this method is simple, fast, and sensitive for the high-throughput determination of methyl-3-quinoxaline-2-carboxylic acid in animal tissues.     

The Synthesis of N-Morphine Hapten and Production of Monoclonal Antibody

The drug overdose and addiction is a serious international problem. Therefore developing a rapid, accurate, convenient and cheap method for detecting morphine in urea is useful and necessary, especially for investigation of epidemiology, identification of medical jurisprudence and determination of drug addict etc. There are many compounds that are similar to morphine in chemical structure and tend to have cross-reaction with the anti-morphine antibody. In order to reduce the cross reaction a…     

可溶性人源含硒抗体酶GPx的研究

对噬菌体展示人单链抗体库进行筛选,得到与半抗原S-二硝基苯取代的谷胱甘肽二丁酯特异结合的单链抗体3B10。用计算机模拟分析了单链抗体的空间结构,发现抗原结合的CDR3区位于抗体的表面,推测其可能进一步参加硒化反。利用突变引物,在大肠杆菌中表达了可溶性抗体蛋白,并用化学方法将催化必需基团硒代半胱氨酸（Sec）组装到3B10抗原结合部位,获得了具有谷胱甘肽过氧化酶活力的人源抗体酶。动力学研究结果表明,抗体酶和天然酶一样,符合乒乓反应机制。     

嗜硫色谱纯化抗体技术的研究进展

嗜硫色谱在高盐环境下对抗体及其他某些蛋白质产生特异性吸附,再在低盐条件下洗脱,可获得高纯度及高回收率的蛋白质产品,是一种新型蛋白质纯化技术。该文对嗜硫色谱及其在抗体纯化中的应用做了综述。     

4-羟基苯乙烯基吡啶为HRP底物的酶荧光免疫传感体系测定布氏杆菌抗体

本文合成了一种新型辣根过氧化物酶（HRP）荧光底物—4-羟基苯乙基吡啶（pHSP）,并首次将它运用于酶联荧光免疫传感体系。对pHSP化学性质的研究证实,pHSP在空气中较稳定,对HRP、H2O2的荧光响应性能优于传统HRP荧光底物如对羟苯乙酸、Amplex Red和佳味醇等。pHSP本身只有极弱的荧光,在HRP催化下可被 H2O2氧化成二聚体产物,该二聚体在300 nm的激发光下能发射波长为437 nm的强荧光,并且反应体系的荧光增加与HRP量在一定浓度范围内成线形相关。根据此原理,建立了兔布氏杆菌抗体的酶联荧光传感分析新方法。运用制备的传感装置测定兔布氏杆菌抗体的线形范围为110-5 1.6 10-3 g/L,抗体检出限为110-5 g/L,相对标准偏差为4.1%（n=11）。 pHSP的二聚体产物水溶性很低,利用设计的装置较好地解决了传统测定溶液体系方法灵敏度打折的问题。     

新型含磷抗原的合成

肽键的水解在生命化学中起着重要的作用, 利用人工合成的抗原诱导具备天然肽酶活性的抗体酶具有深远意义, 因而众多的工作集中于催化酰胺键和肽键水解的抗体酶的研究. 这些工作通常是以Paulling的过渡态理论为指导, 以磷为中心原子设计合成类似肽键或酰胺键水解过渡态结构的过渡态类似物. 然而按照这种方法得到的抗体酶很少具有肽酶活性. 金声等[1]曾以CPA的肽底物马脲酰苯丙氨酸为模型, 以四面体过渡态类似物为基础, 用硫代替磷, 设计合成了新型半抗原[2]. 制备了相应的具有活性的抗体酶, 然而这样的结果尚不能说明以硫原子代替磷原子的方法的优越性, 因此有必要以磷原子为中心原子合成出一个类似抗原, 将其诱导出的抗体相互比较, 才能得到有意义的结论. 本文报道了含磷抗原的合成, 合成路线如下: