Preparation of monoclonal antibody and development of an indirect competitive enzyme-linked immunosorbent assay for ornidazole detection

Food Chemistry 229 (2017) 439–444

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Analytical Methods

Preparation of monoclonal antibody and development of an indirect competitive enzyme-linked immunosorbent assay for ornidazole detection Liang Zhao a,1, Jiaying Li a,1, Ye Li a, Tiangang Wang a, Xiaolu Jin b, Kai Wang c, Ebeydulla Rahman a, Yuan Xing d,e, Baoping Ji a, Feng Zhou a,⇑ a Beijing Key Laboratory of Functional Food from Plant Resources, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, People’s Republic of China b Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, People’s Republic of China c Institute of Apicultural Research, Chinese Academy of Agricultural Sciences, Beijing 100083, People’s Republic of China d Beijing Weifengyongzhu Technology Developing Co., Ltd., District A, Beijing 100020, People’s Republic of China e Pingyuan County Weifengyongzhu Technology Co., Ltd., Shandong Province, Dezhou City 252100, People’s Republic of China

a r t i c l e

i n f o

Article history: Received 27 September 2016 Received in revised form 6 January 2017 Accepted 20 February 2017 Available online 22 February 2017 Keywords: Ornidazole Monoclonal antibodies ELISA

a b s t r a c t A monoclonal antibody (mAb) and an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for ornidazole (ONZ) detection were developed. ONZ was conjugated with cationic bovine serum albumin as a hapten to generate the artificial immunogens and coating antigens. BALB/c mice were immunized, and mAbs were obtained. The competitive inhibition curve of ic-ELISA was y = 0.0438x2
0.2101x + 0.2925, with R2 = 0.9941. The 50% inhibition concentration, the limit of detection, and limit of quality for ONZ were 0.15, 0.01, and 0.05 mg/kg, respectively. The cross-reactivity of the mAbs to secnidazole was 0.33%. The recoveries were from 89.18% to 101.63% and the coefficient of variation was less than 7.15% in chicken, chicken liver, and honey samples, all of which had ONZ concentrations of 0.05 and 0.1 lg/kg. Results showed that the ic-ELISA based on mAb could be used for the rapid detection for ONZ. Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction Ornidazole (ONZ, Fig. 1A), whose systematic name is 1-(3chloro-2-hydroxypropyl)-2-methyl-5-nitroimidazole, is one of the third-generation nitroimidazoles and has been used since 1972 (Singh, Mittal, Sharma, Singh, & Singh, 2003). ONZ is traditionally used to treat the diseases caused by anaerobic bacteria and protozoa as veterinary medicine (Ahmad, Chaudhary, Soni, Payasi, & Dwivedi, 2010). ONZ has the properties of longer half-life and less dosage frequency compared with previous nitroimidazoles (Du et al., 2013). ONZ could also be chosen as a substitute of metronidazole (MNZ) if MNZ-resistance occurs (Wang et al., 2012). However, the side effects of ONZ mainly include genotoxicity, genomic instability (Ikbal, Yilmaz, Dogan, Alp, & Cebi, 2011), damage to the central nervous system (Taskapilioglu, Seferoglu, Kaygili, Hakyemez, & Zarifoglu, 2010), and ornidazole-induced ⇑ Corresponding author. 1

E-mail address: [email protected] (F. Zhou). The authors contributed equally to this work.

http://dx.doi.org/10.1016/j.foodchem.2017.02.100 0308-8146/Ó 2017 Elsevier Ltd. All rights reserved.

autoimmune hepatitis (Kosar et al., 2001). The chloroethanol in the structure is the basis of mutation (Rosenkranz, Carr, & Rosenkranz, 1974). Few papers have reported the detection methods of ONZ such as high-performance liquid chromatography (HPLC) (Dhandapani, Thirumoorthy, Rasheed, Rama kotaiah, & Anjaneyalu, 2010; Heizmann, Geschke, & Zinapold, 1990), high-performance thin layer chromatography (HPTLC) (Gandhimathi, Ravi, & Shukla, 2006; Puranik, Bhawsar, Rathi, & Yeole, 2010) and ultraviolet (UV) spectrophotometric method (Gandhi, Nair, Menezes, & Narayan, 2013). All these methods require high expense, complex operations, and professional operators. Based on the specific binding of antibody and antigen, the indirect competitive enzymelinked immunosorbent assay (ic-ELISA) has been proven to be an alternative screening method for rapid analysis of a relatively lower expense and a simpler operation than instrument detection methods mentioned above. Some ELISAs have been reported for the detection of nitroimidazoles in food products, but the sensibility and specificity for ONZ were not high, which should be further improved (Huet et al., 2005; Wang et al., 2011). Antibody played a

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Fig. 1. Chemical structures of ornidazole (A), and the preparation of immunogen/coating antigen A (B) and immunogen/coating antigen B (C).

central role in ELISA. The hapten is a critical step in the process of antibody production. ONZ has two coupling sites, hydroxyl and nitryl, which can be conjugated to prepare a specific antibody. This study described the preparation of a monoclonal antibody (mAb) with high specificity and good affinity and the development of an ic-ELISA for ONZ detection in chicken, chicken liver, and honey samples. We chose ONZ as the hapten and prepared immunogens by carbodiimide and glutaraldehyde methods. Cationic bovine serum albumin (cBSA) and ovalbumin (OVA) were chosen as the carrier proteins to conjugate the immunogens and coating antigens, respectively. cBSA has two advantages over bovine serum albumin (BSA). One is that cBSA possesses more free aminos, which can increase the combination probability between the haptens and the corresponding carrier proteins. The other merit is that the immunological competence of the immunogen is improved owing to the high positive charge of cBSA (Hermanson, 2013). 2. Materials and methods 2.1. Reagents and materials Ronidazole (RNZ) standard, dimetridazole (DMZ) standard, secnidazole (SCZ) standard, and ONZ standard were purchased from J & K chemical technology (Beijing, China). MNZ standard, N-(3-dimethylaminopropyl)-N0 -ethylcarbodiimide hydrochloride (EDCHCl), N-Hydroxysuccinimide (NHS), Freund’s complete adjuvants (FCA), Freund’s incomplete adjuvants, monoclonal goat anti-mouse immunoglobulin peroxidase conjugate (GAR-HRP), polyethylene glycol 4500, hypoxanthine-aminopterin-thymidine (HAT) and hypoxanthine-thymidine (HT) were purchased from

Sigma-Aldrich Chemical Co. (St. Louis, MO). Glutaric anhydride (GH) was purchased from Energy Chemical (Shanghai, China). RPMI 1640 and fetal bovine serum were purchased from Life Technologies. SP2/0 myeloma cells were obtained from America Type Culture Collection (Manassas, VA, USA). BSA were purchased from Biodee biotechnology Co., Ltd (Beijing, China). Other chemical reagents used in this study were of analytical grade or better and were obtained from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). BALB/c mice were purchased from Beijing Weitonglihua experimental animal technical Co., Ltd. Flat-bottom high-binding polystyrene ELISA plates were purchased from Xiamen Yijiamei experimental equipment Co., Ltd. (Xiamen, China). 2.2. Buffers and solutions PBS (0.01 M, pH 7.4) is a mixture of 10 mM sodium phosphate buffer and 140 mM NaCl. The washing solution is the PBS mentioned above that contains 0.05% (v/v) Tween-20 and 0.03‰ Proclin-300. Coating solution is a carbonate-bicarbonate buffer (50 mM, pH 9.6). Blocking solution is a 50 mM carbonatebicarbonate buffer mentioned above containing 0.5% (w/v) casein and 0.4‰ (v/v) Proclin-300. Stop solution is 2.0 M H2SO4. The substrate solution was purchased from Beijing Weifengyongzhu Technology Developing Co., Ltd. (Beijing, China). 2.3. Synthesis of cBSA (Hui et al., 2006) A total of 1.5 g of BSA was dissolved in 10 mL of PBS (0.01 M, pH 7.4) and added to an ethylene diamine solution (EDA) (27 mg of EDA dissolved in 10 mL of 0.01 M, pH 7.4 PBS) slowly.

L. Zhao et al. / Food Chemistry 229 (2017) 439–444

Approximately 84 mg of EDC was added to this solution, and the mixture was reacted for 2 h at room temperature. The protein was then dialyzed against PBS (0.01 M, pH 7.4) at 4 °C for 2 days. The protein solution was lyophilized and stored at
20 °C. The result of the synthesis was confirmed by trinitrobenzenesulphonic acid (TNBS) method (Spellman, McEvoy, O’Cuinn, & FitzGerald, 2003). TNBS reacted with primary amino groups on the surface of the protein to form a chromophore with a maximum absorbance measured at a wavelength of 335 nm. 2.4. Synthesis of the immunogens and coating antigens 2.4.1. Immunogen A and coating antigen A (Fig. 1B) Carbodiimide method: First, 219.6 mg of ONZ and 171 mg of GH were dissolved in 5 mL of pyridine. The mixture was stirred for 24 h at room temperature. The complete reaction was monitored by HPTLC. The solvent was later removed under the nitrogen in that the ONZ-GH was gained. Second, 23.36 mg of ONZ-GH, 21.73 mg of EDCHCl and 16.11 mg of NHS were dissolved in 1 mL of N,N-dimethylformamide and the mixture was stirred for 12 h at room temperature (Omidfar, Rasaee, Zaraee, Amir, & Rahbarizadeh, 2002; Wang et al., 2007). The reaction solution was added to the carrier protein solution (cBSA or OVA, the ratio of carrier protein and hapten was 1:80) dropwise, and the mixture solution was stirred at 4 °C for 12 h. Finally, the conjugates were dialyzed against PBS (0.01 M, pH 7.4) at 4 °C for 2 days and then lyophilized. The proteins were stored at
20 °C. 2.4.2. Immunogen B and coating antigen B (Fig. 1C) Glutaraldehyde method: First, 219.63 mg of ONZ was dissolved in 12.5 mL of glacial acetic acid and 0.185 g of excess iron dust was added to the solution, which was stirred at room temperature for 3 days. After the reaction, the iron and its oxidation products were filtered off, and the rest of the solution was diluted with 37.5 mL of water. The filtrate was extracted with 2.5 mL CHCl3 each time for 4 times, and the organic phase was dried over MgSO4. After removing the solvent, the crude product was purified by column chromatography on silica gel and crystallized from MeCN (Olender, _ Zwawiak, & Zaprutko, 2010). Hapten B was then gained. Second, 15.17 mg of hapten B was dissolved in 2 mL of DMSO, and the carrier protein solution (cBSA or OVA, the ratio of the carrier protein and the hapten was 1:80) was added to it dropwise. Approximately 25% (v/v) 0.2 mL of the glutaraldehyde solution was added to the reaction solution dropwise, and the mixture was stirred at 4 °C for 12 h. After the reaction, 3.0 mg of NaBH4 was added for another 2 h reaction. Finally, the conjugates were dialyzed in PBS (0.01 M, pH 7.4) at 4 °C for 2 days and then lyophilized. The proteins were stored at
20 °C. The coupling ratios of all hapten-carrier proteins were determined by UV–vis spectra (Yang, 1998). 2.5. Preparation of mAbs First, five 6–8-weeks old BALB/c female mice were prepared for Immunogen A and B. The mice were initially immunized with subcutaneous injection with 100 mg of immunogens dissolved in PBS emulsified with an equal volume of FCA. Three subsequent injections were taken at time intervals of 2, 4 and 6 weeks following the first injection, with the same dose of immunogen emulsified in IFA. Antiserums were collected from the retro-orbital plexus of each mouse and monitored by ic-ELISA 7–10 days after the fourth injection. Approximately 200 mg immunogen dissolved in PBS was injected into the peritoneal cavity of the mice, which showed higher titer 4 days before cell fusion based on Nowinski, Lostrom, Tam, Stone, and Burnette (1979). The splenocytes and the logarith-

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mical phase SP2/0 myeloma cells were fused at the ratio of 1:5. The fused cells were cultured in a HAT selection medium for 2 weeks, and the culturing condition was 37 °C in 5% CO2 atmosphere. The culture supernatants were collected and monitored by ic-ELISA. The hybridomas showing a positive response were subcloned according to the limiting dilution method and cultured in HT medium in the same culturing condition. The subcloned hybridomas were collected and stored in liquid nitrogen. Ascites from BALB/c female mice were purified using saturated ammonium sulfate precipitation (Wengatz, Stoutamire, Gee, & Hammock, 1998) and used to develop the following ic-ELISA. 2.6. ic-ELISA The ic-ELISA detection was carried out as follows (Crowther, 2009). First, the microplate wells were coated by coating antigens dissolved in a coating solution (100 mL/well) at 37 °C for 2 h. The plate was washed with a washing solution and blocked with a blocking buffer (150 mL/well) at 37 °C for 2 h. Second, antiserum diluted by PBS (50 mL/well) and the standard solutions with serial dilutions (50 mL/well) were added and incubated at 37 °C for 0.5 h. The plate was washed four times with washing solution, and GAR-HRP solution diluted at 1:750 (100 mL/well) was added. After incubation at 37 °C for 0.5 h, the plate was washed four times, and the substrate solution (100 mL/well) was added. The stop solution (50 mL/well) was added after incubation at 25 °C for 15 min. Finally, OD values were measured at wavelengths of 450 nm and 630 nm. 2.7. Cross-reactivity The specificity of antibody was determined by the crossreactivity (CR) with the structurally similar compounds to the target one. The IC50 value for each compound was measured by the inhibition curve. CR (%) = [IC50 (ONZ)/[IC50 (compound)]  100. 2.8. Sample preparation 2.8.1. Matrix effect The matrix effect of chicken and honey were accessed by preparing the ONZ standard curves in serially diluted extract buffer (0, 5, 10, 20 times). The parallelism of the curves was compared with that prepared in PBS to evaluate the extent of the interferences caused by the matrix. 2.8.2. Chicken and chicken liver samples First, 2.0 g of the minced chicken and chicken liver samples was homogenated with 8 mL of ethyl acetate in a 50 mL polypropylene centrifuge tube by the vortex mixer and then centrifuged at 4000g or 10 min. Second, the supernatant (4 mL) was separated, and the organic phase was evaporated under nitrogen flow at 50 °C. The residue was reconstituted by 1 mL of PBS (0.01 M, pH 7.4) and 1 mL of n-hexane. Finally, the solution was homogeneously mixed for 30 s by the vortex mixer and then centrifuged at 4000g or 10 min at room temperature. The water solution was diluted 20fold and analyzed by ic-ELISA procedure. 2.8.3. Honey sample First, 2.0 g of the honey sample was mixed with 2 mL of PBS (0.01 M, pH 7.4) and 6 mL of acetonitrile in a 50 mL polypropylene centrifuge tube by vortex mixer and then centrifuged at 4000g or 10 min. Second, the supernatant (2 mL) was separated, and the organic phase was evaporated under nitrogen flow at 50 °C. The residue was reconstituted by 0.5 mL of PBS (0.01 M, pH 7.4). The solution was diluted 20-fold and analyzed by ic-ELISA procedure.

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2.9. HPLC validation The HPLC validation of the ic-ELISA method was carried out according to the Sun’s study (Sun, Wang, & Ai, 2007). All samples were determined by ic-ELISA (mentioned above) and HPLC methods. The results analyzed by linear regression were used to verify the applicability of ic-ELISA method. A Shimadzu LC-10A HPLC system (Kyoto, Japan) was used for the detection of ONZ on a HydroC18 column (250 mm  4.6 mm i.d., 5 mm particle size). The mobile phase consisted of acetonitrile, methanol and water. The elution procedure is conducted according to the Sun’s study. The flow rate was 1.0 mL/min at 30 °C, and an aliquot of 20 mL of each sample filtered through a filter (pore size, 0.22 mm) was injected into the HPLC system. The absorption of analytes was detected at 320 nm. 2.10. Statistical analysis The results were expressed as mean ± standard deviation (SD). All samples and sample analysis were conducted at least in triplicate. Statistical analyses were used by GraphPad Prism (version 6.0) (GraphPad, San Diego, CA, USA). 3. Results and discussion 3.1. Synthesis of cBSA TNBS reacted with primary amino groups on the surface of BSA. The OD values of BSA and cBSA measured at a wavelength of 335 nm were 0.21 and 0.46, respectively. The changing of OD values illustrated the success of cBSA synthesis. 3.2. Synthesis and identification of protein conjugates ONZ-GH was prepared by linking ONZ with GH, and ONZderivative was prepared by transferring the nitro of ONZ to amino group. The substances were determined by LC–MS to confirm the structures (Fig. s1 and s2). The molecular weight of ONZ-GH was 333.07, and the mass result was 331.9 [H
]. The molecular weight of the ONZ-derivative was 189.07, and the mass result was 190.3 [H+]. The LC–MS results showed the success of chemical synthesis. ONZ-GH and ONZ-derivative were then coupled with carrier proteins according to the carbodiimide method and glutaraldehyde method separately. The haptens and the carrier proteins had UV scanning spectrums, and the UV scanning spectrums of the conjugates showed a superposition property of the two spectrums. This property indicated the success of conjugation, and the coupling ratio of the conjugates could also be calculated. The results showed that the haptens and carrier proteins were successfully coupled. The coupling ratio of Immunogen A and B was 16:1 and 12:1, respectively.

Fig. 2. Standard curve of the ic-ELISA for ornidazole. Each point represents the mean ± standard deviation from three determinations.

Finally, the inhibition standard curve of ONZ was obtained. Data were arranged with B/B0 (%) on the vertical axis and the logarithm of the concentration of ONZ (mg/kg) on the horizontal axis. The equation of the curve was y = 0.0438x2
0.2101x + 0.2925, with R2 = 0.9941 (Fig. 2). The limit of detection (LOD) and limit of quantity (LOQ) could be calculated according to the standard calibration curve. Moreover, 20 blank chicken samples were prepared based on the sample preparation. Then LOD was calculated by the average value plus three times the SD, and the LOQ was calculated by the average values plus 10 times the SD. According to the standard calibration curve, the IC50 value, LOD, and LOQ for ONZ were 0.14, 0.01, and 0.05 mg/kg, respectively.

Table 1 Cross-reactivity (CR) of analogues of ornidazole. Compound

Structure

OH

ONZ

IC50 (mg/kg)

CRs (%)

0.15

100

42.5

0.33

–

<0.1

–

<0.1

–

<0.1

Cl O2N

N

CH3 N OH

SCZ

CH3 O2N

N

CH3 N OH

MNZ

3.3. Antibody sensitivity The mice were immunized with Immunogen A and B. The optimal concentrations of the antibodies and the coating antigens were determined by checkerboard titration. The affinity of the antibodies was estimated by the IC50 values. However, Immunogen A elicited immune response, and Immunogen B failed to elicit a detectable one. Compared with Immunogen A, the feature structure of the nitroimidazoles may be destroyed by Immunogen B, which may decrease the chances to elicit the immune response. The IC50 values of the antiserum of the five mice immunized with Immunogen A were from 6.3 mg/kg to 12.3 mg/kg. Mouse number 3 was used to produce mAbs owing to its high sensitivity. After cell fusion and cloning, mAb 3C5 performed the lowest IC50 value.

O2N

N

CH3 N

CH3

RNZ O2N

N

O O

NH2

N

CH3

DMZ

O2N

N

CH3 N

ONZ, ornidazole; SCZ, secnidazole; MNZ, metronidazole; RNZ, ronidazole; DMZ, dimetridazole.

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Fig. 3. Comparison of ELISA curves for ONZ obtained from standards prepared in PBS, extract, 5-fold dilution, 10-fold dilution, and 20-fold dilution. Each point represents the mean ± standard deviation from three determinations.

Table 2 Recovery and coefficients of variation (CV) of ornidazole in spiked samples. Sample

Spiked (mg/kg)

Mean recovery (%)

CVs (%)

Chicken

0.05 0.1

101.63 ± 6.47 100.78 ± 7.19

6.36 7.13

Chicken liver

0.05 0.1

95.88 ± 6.61 96.60 ± 4.66

6.89 4.83

Honey

0.05 0.1

89.18 ± 6.38 91.25 ± 3.30

7.15 3.62

3.4. CR Table 1 shows that the mAb showed CR toward SCZ (CR = 0.33%) but did not exhibit measurable CR (CR < 0.1%) with other nitroimidazoles, such as MNZ, RNZ, and DMZ. The chlorine atom in ONZ was a strong antigenic determinant and determined the affinity specific of the resulting antibody to other nitroimidazoles (Esteve-Turrillas et al., 2015). The antibody obtained in this research is more sensitive and specific to ONZ compared with the study of Wang et al. (2011). 3.5. Matrix effect The results in Fig. 3 showed that extract buffer could affect the B/B0 and IC50 values. The result of the 20-fold dilution was comparable to that of the standard curve in PBS. The dilution of the extract buffer could decrease the matrix effect of samples after the 20-fold dilution of the extract buffer. 3.6. Validation of the ic-ELISA The accuracy of ELISA was measured by the recovery. The chicken, chicken liver, and honey samples were all spiked with ONZ with two concentrations of 0.05 and 0.1 mg/kg and then measured by the ic-ELISA procedure. The samples were prepared as previously described. Each sample was analyzed in triplicate. Table 2 presented the recoveries of different samples. The recoveries ranged from 89.18% to 101.63%, with the coefficient of variation ranging from 3.62% to 7.15%. The results showed that the ic-ELISA satisfied the accuracy requirement of residue detection in food samples. 3.7. Assay validation by HPLC Fig. 4 showed that the results measured by the ic-ELISA correlated well with those of HPLC method with the linear regression

Fig. 4. Correlation plots between ic-ELISA and HPLC results for samples spiked with ONZ.

equation of y = 0.9997x + 0.0055 (R2 = 0.9986). The results indicated that the two methods were highly comparable, and the icELISA could be used as an effective and accurate method for ONZ determination. Compared with Dhandapani’s study, ic-ELISA is more precise than the HPLC assay for detecting ONZ with low LOD (0.01 mg/mL vs. 10 mg/mL) and low LOQ (0.05 mg/mL vs. 25 mg/mL) (Sun et al., 2007). ic-ELISA for ONZ requires low expense, low consumption of reagents, simple equipment, and easy operations compared with HPLC method. The specific binding of antibody and antigen shows that ic-ELISA has more advantages than instrument detection methods for rapid analysis with the lower expense and simpler operation. 4. Conclusions A sensitive and specific mAb and ic-ELISA for ONZ detection were prepared and developed in this study. ONZ was conjugated with cBSA as hapten to generate the artificial immunogens and coating antigens. mAbs were obtained from the immunized BALB/c mice, and the IC50 value of the mAbs was 0.15 mg/kg. The competitive inhibition curve of ic-ELISA was y = 0.0438x2
0.2101x + 0.2925, with R2 = 0.9941. The LOD and LOQ for ONZ were 0.14, 0.01, and 0.05 mg/kg. The measurable CR of the mAbs to SCZ was 0.33%, and no measurable CR (CR < 0.1%) was detected

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with other nitroimidazoles, such as MNZ, RNZ, and DMZ. The recoveries were from 89.18% to 101.63%, and the CV value was less than 7.15% in chicken, chicken liver, and honey samples spiked with ONZ of tow concentrations of 0.05 and 0.1 mg/kg, which satisfied the residue detection requirement in food samples. The results showed that the ic-ELISA based on the mAb could be used for the rapid detection specific for ONZ with low expense and simple operation. Acknowledgement The authors declaim no conflict of interest. This work was financially supported by the Special Fund for Agro-scientific Research in the Public Interest of the Ministry of Agriculture, PRC (Grant No. 201303085), Natural Science Foundation of Shandong Province of China (Grant No. ZR2015BQ015). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2017. 02.100. References Ahmad, H., Chaudhary, M., Soni, A., Payasi, A., & Dwivedi, V. K. (2010). Comparative toxicity profile study of mebatic vs. ofloxacin, ornidazole and metronidazole drugs in rat model. Asian Journal of Biochemistry, 5(2), 78–88. Crowther, J. R. (2009). The ELISA guidebook (2nd ed.). America: Humana Press. Dhandapani, B., Thirumoorthy, N., Rasheed, S. H., Rama kotaiah, M., & Anjaneyalu, N. (2010). Method development and validation for the simultaneous estimation of ofloxacin and ornidazole in tablet dosage form by RP-HPLC. International Journal of Pharma Sciences and Research, 1(1), 78–83. Du, J., Ma, Z., Zhang, Y., Wang, T., Chen, X., & Zhong, D. (2013). Enantioselective determination of ornidazole in human plasma by liquid chromatography– tandem mass spectrometry on a Chiral-AGP column. Journal of Pharmaceutical and Biomedical Analysis, 86(1), 182–188. Esteve-Turrillas, F. A., Mercader, J. V., Parra, J., Agullo, C., Abad-Somovilla, A., & Abad-Fuentes, A. (2015). Ready access to proquinazid haptens via crosscoupling chemistry for antibody generation and immunoassay development. PLoS One, 10(7), 1–17. Gandhi, V., Nair, S., Menezes, C., & Narayan, R. (2013). Development of UVspectrophotometric method for the quantitative estimation of ofloxacin and ornidazole in combined liquid oral dosage form by simultaneous equation method. International Journal of Research in Pharmacy and Chemistry, 3(1), 6–11. Gandhimathi, M., Ravi, T., & Shukla, N. (2006). Validated high performance thin layer chromatography method for simultaneous estimation of ofloxacin and ornidazole in tablet dosage form. Indian Journal of Pharmaceutical Sciences, 68(6), 838. Heizmann, P., Geschke, R., & Zinapold, K. (1990). Determination of ornidazole and its main metabolites in biological fluids. Journal of Chromatography A, 534(1–2), 233–240.

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