[Preparation of an anti-cotinine monoclonal antibody and its application in immunological detection].

OBJECTIVE: To prepare monoclonal antibody against cotinine (COT) and to establish immunoassay for detecting COT in human urinary samples. METHODS: BALB/c mice were immunized with synthesized cotinine-bovine serum albumin (COT-BSA) to screen monoclonal antibody with technique of cell fusion. The monoclonal antibody was used for the indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and colloidal gold immunochromatographic strip assay for the detection of COT in human urine. RESULTS: The monoclonal antibody against COT was identified by ic-ELISA with a 50%inhibitive concentration (IC50) value of 21 ng/mL; and it was also identified by colloidal gold immunochromatographic strip assay with a cut-off value of 100 ng/mL. For ic-ELISA, the range of detection was 0-100 ng/mL with a minimal limit of 0.1 ng/mL; the recovery of assay was 99.41%-117.98%, and the intra-assay and inter-assay coefficient variations were not higher than 15.31%and 15.07%, respectively. For colloidal gold immunochromatographic strip assay, the accuracy of stability and repeatability both were 100%. CONCLUSIONS: The ic-ELISA and colloidal gold immunochromatographic strip assay using the prepared monoclonal antibody against COT have been proved to be reliable for the rapid detection of COT in human urines, which may be used for monitoring of environmental tobacco smoke.

Investigation of diethylstilbestrol residue level in human urine samples by a specific monoclonal antibody.

Diethylstilbestrol (DES) is used as a kind of animal feed additive and affects people's health through the food chain. The purpose of this study is to detect the residue level of DES in 576 human urine samples directly. DES-BSA was used to immunize Balb/c mice. The monoclonal antibody was produced by hybridoma that was screened through cell fusion techniques. Finally, we developed the indirect competitive ELISA method to analyze 576 human urine samples from Zhejiang Province, China. The IC50 of this method was 3.33 ng/mL. The LOD and LOQ were 0.16 and 0.54 ng/mL. Linear range of the standard curve was from LOD to 12.50 ng/mL. There was no cross-reactivity with two kinds of estrogens and two structural analogs with DES. Five hundred seventy-six urine samples were analyzed by the indirect competitive ELISA method, and the detection rate was 98.78%. The mean concentration and geometric mean were 4.70 and 3.50 ng/mL. The indirect competitive ELISA method based on monoclonal antibody was sensitive and reliable for the detection of DES in human urine samples. The results warned us to pay more attention to human health and food safety.

An ultra-sensitive monoclonal antibody-based enzyme-linked immunosobent assay for dibutyl phthalate in human urinary.

Dibutyl phthalate (DBP) has been extensively used as a plasticizer in many daily products, which is highly toxic to human, notably affecting the reproductive and developmental function. As the previous method is expensive, time-consuming, low sensitivity and just focused on the environment. Present study was aimed to establish an ultra-sensitive and simple method based on good quality monoclonal antibody, applying to evaluate excretion level of DBP in urine samples of Chinese population directly. A monoclonal antibody was generated and characterized after fusion of myeloma cells with spleen cells isolated from BALB/c mouse. The mouse was previously immunized using a specially designed amino derivative of DBP conjugated with bovine serum albumin (BSA) as immunogen. Cross-reactivity values of the monoclonal antibody against DBP, di-isobutyl phthalate (DIBP) were observed 100% and 1.25%, while for dimethyl phthalate (DMP), butyl benzyl phthalate (BBP) and didecyl phthalate (DDP) the values were <0.06%. The standard curve was constructed at 0-50 ng mL(-1) and good linearity (R(2)=0.994) was achieved. The observed IC50 (7.34 ng mL(-1)) and LOD (0.06 ng mL(-1)) values was improved 1000-fold to polyclonal antibody and 5-fold to other monoclonal antibodies. A total 1246 urine samples were analyzed and the detection frequency of DBP was observed 72.87% by ic-ELISA. The 95th percentile and mean concentration of DBP were 12.07 and 3.00 ng mL(-1). Acceptable recovery rates of DBP were 97.8-114.3% and coefficients variation 5.93-11.09%. The concentrations of DBP in females were found significantly higher (p<0.05) than males. Similarly, the DBP in middle aged and low educated individuals was found higher (p<0.001) than the others. Considering the adverse health effects, DBP internal exposure in the Chinese population should be reduced. The ic-ELISA method has been proved as a cost effective, specific, and highly sensitive screening tool to detect DBP in urinary samples.

Development of analytical method for ultrasensitive detection of salbutamol utilizing DNA labeled-immunoprobe.

Salbutamol (SAL) is known as quick-acting ß-2 receptor agonist and its use in humans for pulmonary diseases and/or in animal feed is limited because of associated potential hazardous effects on health. Several techniques are available for the detection of SAL, but are expensive and time consuming. Here for the first time, a novel pre-assembled DNA immunoprobe-based immuno-PCR assay was developed to investigate the levels of SAL in human urine samples and compared the proposed immuno-PCR assay for the detection of SAL with that of direct competitive ELISA. Under the optimized conditions, the assay showed a linear range over seven orders of magnitude, whereas the limit of detection of SAL in buffer was found to be 21 fg/mL. Current immuno-PCR assay exhibited a 300-fold better detection limit for SAL as compared to one achieved with direct competitive ELISA using the same antibody. In conclusion, it has been found that the developed method is highly sensitive and simple method that can significantly enhance the detection sensitivity for small molecules. Moreover it can be modified and used for detecting other small molecules and/or chemicals that exist in the environment and are responsible for the environmental pollution.

Development and comparison of two competitive ELISAs for estimation of cotinine in human exposed to environmental tobacco smoke.

We simultaneously set up two competitive (direct and indirect) enzyme-linked immunosorbent assays (ELISAs) based on the same antibody for estimation of cotinine (COT) in pregnant women especially and population generally exposed to environmental tobacco smoke. The results show that the limits of detection (LODs) for direct competitive ELISA and indirect competitive ELISA were 0.04 µgL(-1) and 0.1 µgL(-1), respectively. Direct competitive ELISA was found to be more sensitive than indirect competitive ELISA. Thereafter, we applied our direct competitive ELISA for the detection of COT from urinary samples taken from 450 volunteers from the Zhejiang Province of China. COT was detected in 100% of participants with concentration ranging from LOD to 5358.0 µgL(-1). The GM and 95th percentile concentration of COT in pregnant women were 6.3 µgL(-1) and 57.2 µgL(-1), respectively. Males had statistically higher COT concentrations than females (P < 0.0001), active smokers had statistically higher COT concentrations than non-smokers (P < 0.0001), whereas, non-pregnant women were found to have higher COT concentration than pregnant women. We conclude that our developed direct competitive ELISA is useful for detecting the COT in urinary concentration of human. The human urinary data obtained in this study indicated that common people generally and pregnant women especially were highly exposed to COT. Further studies are needed to focus on the sources of exposure, potential health effects and risk assessment of exposure to COT.

Estimation of urinary concentration of aflatoxin M1 in Chinese pregnant women.

Aflatoxin M1 (AFM1 ) is a main cause of hepatocarcenogenoma in Chinese population. Measurement of aflatoxin exposure in human may help in providing clear evidence for the exposure of specific environmental pollutants in certain population. "One child policy" in China offered parents more careful to choose safe food during pregnancy, but no reports published on the efficacy of their endeavor. In present study, we aimed to assess the exposure of AFM1 in Chinese pregnant women. The urine samples were collected from 600 volunteers from Zhejiang province, China and the urinary concentration of AFM1 was measured using ELISA kit. AFM1 was detected in 84% of the pregnant women. The geometric mean and 95th percentile concentration of AFM1 in pregnant women were 50.3 ng/L and 633.5 ng/L, respectively. Our results point out that pregnant women especially are at the high risk of exposure to AFM1 . Our results also indicate that although "one child policy" offered parents to pay more attention for the selection of safe food, but detection of AFM1 in urine of pregnant women indicate that more foods containing AFM1 still need to be detected. Highest exposure of AFM1 in pregnant women indicates that awareness campaigns must be started especially in the rural areas of China regarding the possible hazardous effects of AFM1 exposure in pregnant women.

[Determination of several environmental contaminants in human body].

OBJECTIVE: To detect common environmental pollutants in human body. METHODS: Urine samples were collected from 80 healthy subjects. Chromatography mass spectrometry (GC-MS), HPLC and ELISA were applied to detect several common environmental pollutants in urine samples. RESULTS: DBP and methylbenzene were present in 75.3% and 41.2% of urine samples. The methanal and AFM1 were found in most of urine samples (approximately 91≊97%). By contrast, PCBs, CPZ, 4, 5-DCC were found in less than 5 samples, but there was no TMT detected. CONCLUSION: Some of the environmental pollutants including carcinogens are detected in urine samples in this study.

[Preparation of salbutamol polyclonal antibodies and development of indirect competitive enzyme-linked immunoassay].

OBJECTIVE: To prepare the antibodies against salbutamol (SAL) with high sensitivity and to develop an indirect competitive enzyme-linked immunoassay (ic-ELISA) for fast detection of SAL. METHODS: The New Zealand white rabbits were immunized with SAL in a small dose and long period mode. The method of ic-ELISA was optimized and adopted for the detection of a series of SAL samples, then the standard curve of SAL was established. The precision and the recoveries of the method were determined. RESULTS: The antibodies with high sensitivity towards SAL were prepared with a IC50 of 12.21 ng/ml. The ic-ELISA method for SAL measurement was established, the recoveries of measurement was between 95%-105% and the CV was <3%. CONCLUSION: The antibodies against salbutamol have been prepared and an indirect competitive enzyme-linked immunoassay for fast and specific detection of SAL has been developed.

[Synthesis of artificial diethylstilbestrol antigen for preparation of polyclonal antibodies].

OBJECTIVE: To synthesize artificial diethylstilbestrol (DES) antigen and to prepare DES polyclonal antibody with high titer and sensitivity. METHODS: The derivative of DES (DES-HS) was synthesized from diethylstilbestrol, ethyl bromoacetate,bovine serum albumin (BSA) and chicken ovalbumin (OVA) with the nucleophilic substitution reaction; the compound was identified by electrospray ionization mass spectrometry(ESI-MS). The DES-HS and the carrier proteins (BSA, OVA) were cross-linked to prepare the artificial antigen; the UV absorption spectrophotometry and high performance liquid chromatography (HPLC) were used to identify the prepared artificial antigen. The rabbits were immunized with the DES artificial antigen to prepare the DES polyclonal antibodies. RESULTS: The DES-HS was synthesized. The DES artificial antigen was prepared successfully with a coupling rate of 22:1. The DES polyclonal antibodies with a titer of 1:25 600 and IC50 of 10.81 ng/ml were prepared with DES artificial antigen. CONCLUSION: A set of methods to synthesize DES artificial antigen and to prepare the DES polyclonal antibodies has been developed successfully.