Development Of Immunological Rapid Detecting Technology For Doxycycline Residues

Doxycycline (DOX) is an important member of the tetracycline family of antibiotics, which are often used to treat infectious diseases in animals. They are also used as feed additives to prevent diseases and promote the growth of animals. Notably, the antibiotic residues may remain in animal tissues for a while after withdrawal and present in the commercial meats. The presence of tetracycline residues in animal tissues can lead to an increase in the spreading of drug-resistant bacteria in human community, which is a serious health concern in humans.Many government authorities have made great efforts to control this problem by establishing programs for antibiotic determination in foods. New policies have been established for the control of maximum amount of antibiotics in supplied meats. For example, the maximum residue limitations (MRL) for DOX are set at 100μg/kg in muscle,300μg/kg in liver and 600μg/kg in kidney, respectively, in China and Europe. Therefore, it is necessary to study a rapid method to monitor their residues in animal tissues.Traditionally, the residues of DOX are measured by analytical methods, such as liquid chromatography-mass spectrometry (LC-MS), high performance thin layer chromatography (HPTLC), capillary electrophoresis, thin-layer chromatography (TLC), continuous-flow chemiluminometric, flow-injection, high performance liquid chromatography (HPLC), spectrofluorimetric analysis, time-resolved spectroscopy.Methods for the detection of DOX in animal tissues were mainly instrumental methods, which were based on high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). Although sensitive and accurate, most of the chromatographic methods are expensive, time-consuming, and unsuitable for ananysis of many samples. Enzyme immunoassay is enough sensitive, specific, rapid and simple to be a good and economical alternative to instrumental methods for screening DOX residues. At present, Immunological analytical products for detection of tetracyclines (TCs) residues in animal food have been developed, but immunological analytical technique for detecting DOX was uncompleted. So, developing an immunological analytical product for detecting DOX residue could be significant.This study aimed at immunological rapid determination of DOX residues in animal food, including the immunogenicity of DOX, synthesis of artificial antigen, preparation and special properties of DOX rabbit polyclonal antibody (Anti-DOX pAb), DOX monoclonal antibody (Anti-DOX mAb) and indirect competition ELISA mathed (ic-ELISA), the preparation and quality of immunological gold-labeled strip by HPLC. The results of this study were as follows:1. Immunogens and coating antigens synthesis and verificationThe immunogen doxycycline-para-aminobenzoic acid-bovine serum albumin (DOX-PABA-BSA) and the coating antigen doxycycline-para-aminobenzoic acid-ovalbumin (DOX-PABA-OVA) were synthesized by the diazonium coupling reaction and mixed anhydride methods. DOX-BSA and DOX-OVA were synthesized by the Hoffman degradation reaction and diazonium coupling methods. DOX-PABA-BSA and DOX-BSA were synthesized successfully and its conjugation ratio of DOX to BSA was about 12.3:1 and 5.8:1 by identification with infrared, ultraviolet and SDS-PAGE. These conjugates were identified with ultraviolet spectrum. Their spectrums were far different from carrier protein and hapten. The results showed that the conjugates were prepared successfully. Conjugates DOX-BSA and DOX-PABA-BSA were selected as immunogens. Balb/c mice were vaccinated with based and some intensified immunization. The high-titers and good sensitive, specific DOX antiserum of Balb/c mice immunized with DOX-PABA-BSA (or DOX-BSA) had been produced by identification with indirect ELISA.2. Production of Anti-DOX pAb and Anti-DOX mAb and evaluation of their performancePolyclonal antibodies were produced by immunization of New Zealand rabbits using various immunogen doses and time-scales with two conjugates (DOX-BSA and DOX-PABA-BSA), and Anti-DOX pAb had been produced. The results were pAbⅡof 2# rabit inhibiting effection was better, and the Anti-DOX pAb had the high titers of 1: 3.2×105 by indirect ELISA, a good sensitivity with 50% inhibitive concentration (IC50) of 10.65μg/L and low levels of cross-reactivity to TC (2.34%), CTC (1.46%), and OTC (5.07%) were observed.Mice were immunized with DOX-PABA-BSA or DOX-BSA to prepare mAb. The lymphocytes from Balb/c mouse spleen were fused with NSO myeloma cells. Three hybridoma lines of 2.3/3A6,2.1/2F8 and 2.1/4H10 that secrete Anti-DOX mAb were screened by indirect ELISA. The hybridoma cell lines of 2.3/3A6 secrete high affinity supernatant antibodies and ascites antibodies, which were screened by indirect ELISA. The titers of supernatant antibodies and ascites antibodies were 1:1280 and 1:32000. The immunoglobulin isotypes of the Anti-DOX mAb were identified with the Immunoglobulin Isotype Kit as IgGl. The chromosome number of the hybridomas was 90-101, the average number was 96.5. The IC50 of ascites antibodies to DOX was 36.19μg/L, low levels of cross-reactivity (CR) to TCs (CR<0.3%).3. Development and Evaluation of ic-ELISA methodUsing the antibodies produced by DOX-PABA-BSA, an ic-ELISA for determination of DOX was developed with Anti-DOX pAb. The standard curve regression equation of the ic-ELISA was typical sigmoid curve fitted to the four parameters logistic equation with the linear determination of 2.5 to 80μg/L. The values of IC50 were 10.65,16.46 and 16.99μg/L in PBS, swine muscle and liver samples, respectively. These results indicated that the matrix had minor interference on the detection of DOX by the ic-ELISA using the sample preparation procedure described. The limits of detection were about 2.65μg/kg of DOX in muscles and 2.96μg/kg in livers. The ic-ELISA was able to detect DOX with a dynamic range of 1.79-80μg/L. The recovery values ranged from 88.24%to 89.19%for muscle samples and 84.61%to 85.54%for liver samples. The coefficients of intra-assay variations (CVs) were 2.60%-13.17%, and the inter-assay CVs were 8.61%-11.38%. Therefore, the reproducibility of ic-ELISA for detection of DOX appeared to be better than that reported with another method. The specificity of ic-ELISA was determined using a set of similar antibiotics for potential cross-recognition by Anti-DOX pAb. Low levels of cross-reactivity to TC (2.34%), CTC (1.46%), and OTC (5.07%) were observed. Therefore, the ic-ELISA was able to detect DOX in swine muscles and livers.4. Preparation and performance evaluation of immunochromatographic Test stripsBasis on principle of gold immunochromatography assay (GICA), a rapid test strip of DOX (Test-strip) was developed with Anti-DOX mAb of 2.3/3A6. Test-strip was developed and applied to the screening for DOX. The calibration curve of DOXpAb-Strip was typical sigmoid curve fitted to the four parameters logistic and the determination limits were 11μg/L by BioDot-TSR3000 and 20μg/L by eye. Test-strip had no cross reaction to other antibiotics by eye-measurement.5. Comparison of ELISA and the Test strip with HPLC methodsTo further evaluate the validity of the quantitative response of ELISA method and the strip, groups of Duroc castrated pigs weighing 15±3 kg were housed individually in our animal facility for one week.10 pigs were randomly divided into control and test groups. The control group (n=2) was mock treated with another drug, intramuscularly. The test group (n=8) was treated intramuscularly with DOX at a dose of 2.5 mg/kg bodyweight (BW) daily for 4 consecutive days. In this study, the animals were mercifully slaughtered at days 0,3,6,10 and 16 after the withdrawal of the DOX treats. Their muscles and livers were collected and used for DOX analysis by ELISA method, lateral flow test strip and HPLC.After preparation, tissue samples were analyzed by established ELISA and high performance liquid chromatography (HPLC) method. The results showed that the ELISA method had a good correlation with HPLC for detecting muscle (r=0.9980) and liver (r=0.9991) samples. A parallel comparison between the test strip, ELISA and HPLC were accomplished with swine muscle and liver samples. The results showed that the three methods had good linearity, the ELISA method and test strip were accuracy and reliable. The ELISA method and test strip were proved to be used for the rapid determination of DOX residues in animal food.In this research, an ELISA method and two test strips have developed firstly by anti-DOX mAb in the world for the detection of DOX, which provides a technical guidance for other small molecule compound. This method has high sensitivity, simple operation and large samples screening. So these methods are applicable for the rapid detection of DOX in porcine muscle and liver and it can effectively suspect and control DOX residue, ensure safety of animal production, avoid trade issue, improve the output, expand trade, increase export and guarantee human health.