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Four specific hapten conformations dominating antibody specificity: quantitative structure-activity relationship analysis for quinolone immunoassay Jiahong Chen, Lanteng Wang, Lanlan Lu, Xing Shen, Xinan Huang, Yingju Liu, Xiulan Sun, Zhanhui Wang, Sergei Alexandrovich Eremin, Yuanming Sun, Zhenlin Xu, and Hongtao Lei Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 17 May 2017 Downloaded from http://pubs.acs.org on May 18, 2017
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Analytical Chemistry
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Four specific hapten conformations dominating antibody
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specificity: quantitative structure-activity relationship analysis
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for quinolone immunoassay
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Jiahong Chena#, Lanteng Wanga#, Lanlan Lua, Xing Shena, Xin-an Huangb, Yingju Liuc,
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Xiulan Sund, Zhanhui Wange, Sergei Alexandrovich Ereminf, Yuanming Suna, Zhenlin
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Xua, Hongtao Leia*
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a
Guangdong Provincial Key Laboratory of Food Quality and Safety, South China Agricultural
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University, Guangzhou 510642, China
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b
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Guangzhou University of Chinese Medicine, Guangzhou 510405, China
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c
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University, Guangzhou 510642, China
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d
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University, Wuxi, Jiangsu 214122, China
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e
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Agricultural University, Beijing 100094, China
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f
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119991 Moscow, Russia
Tropical Medicine Institute &South China Chinese Medicine Collaborative Innovation Center,
Department of Applied Chemistry, College of Materials and Energy, South China Agricultural
State Key Laboratory of Food Science and Technology, School of Food Science of Jiangnan
Department of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine, China
Faculty of Chemistry, M.V. Lomonosov Moscow State University, Leninskie gory 1, Building 3,
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* Corresponding author. Phone: +8620-8528 3448. Fax: +8620-8528 0270. E-mail: hongtao@scau.edu.cn (Hongtao Lei).
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#
Equal contribution
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Notes
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The authors declare no conflict of interest.
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Analytical Chemistry
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Abstract: Antibody-based immunoassay methods have been important tools for
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monitoring drug residues in animal foods. However, due to limited knowledge of the
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quantitative structure-activity relationship between a hapten and its resultant antibody
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specificity, it is still a huge challenge for antibody production with a desired
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specificity. In this study, the three-dimensional quantitative structure-activity
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relationship (3D QSAR) was analyzed in accordance with the cross-reactivity of
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quinolone drugs reacting with the antibody raised by pipemidic acid as the
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immunizing hapten, as well as comparing with the reported cross-reactivity data and
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their hapten structures. It was found that the specificity of quinolone antibody was
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strongly related to the conformation of the used hapten, and the hapten conformations
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shaped like “I”, “P” and “ф” were essential for the desired a high specificity with low
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cross-reactivity, but the hapten conformation shaped like “Y” led to a broad
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specificity antibody with high cross-reactivity. Almost all the antibodies against
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quinolones could result from these four hapten conformations. It was firstly found that
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the concrete conformations dominated the specificity of the antibody to quinolone,
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which will be of significance for the accurate hapten design, predictable antibody
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specificity and better understanding the recognition mechanism between the hapten
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and the antibodies for immunoassay.
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INTRODUCTION Quinolones are a class of antimicrobial drugs widely used in the prevention and
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treatment of animal diseases. However, their residues in animal foods have raised a
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series of health issues, including skin reactions, phototoxicity, and hyperglycemia1-3.
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In order to effectively monitor the quinolones abuse in animal-derived food products,
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it is important to develop a rapid screening method for monitoring quinolone residues.
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Compared with the traditional instrumental methods4-8, immunoassays relying on
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antigen−antibody interaction are favored by analytical chemists due to their
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convenient manipulation, simple sample treatment, low-cost and easy automation.
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Some immunoassays have been developed for the detection of quinolone residues9-11.
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It is well-known that an antibody with the desired broad or high specificity (high
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or low cross-reactivity) is crucial to develop an immunoassay12. High specificity
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means low cross-reactivity to structure-related compounds13,14, which is traditionally
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favored by a single analyte analysis in one test. On the contrary, broad specificity
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means that the high cross- reactivity to structure-related compounds is useful for the
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monitoring a series of compounds to finish multianalyte analysis in one test15. Now
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the broad-specificity, multianalyte recognition in one assay, seems to be a focus of
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antibody production and immunoassay development for quinolone drugs16-18. There
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have been a few of reports about the broad specificity of quinolone antibodies based
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on molecule modeling. Wang et al developed a generic immunoassay using
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ciprofloxacin antibodies for 12 fluoroquinolone antibiotics, and the obtained
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cross-reactivity data showed the ethyl group of the piperazinyl ring exerted a limited
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effect on antibody binding but appeared to be important during antibody production18.
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In our previous study, pazufloxacin was employed as hapten to produce a broad
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specific antibody recognizing 23 quinolones19, it was found that the quinolones could
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interact with the antibody with different binding positions, and cross-reactivity was
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mainly positively correlated with the bulky substructure containing an electronegative
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atom at position 7, while it was negatively associated with the large bulky
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substructure at position 1 of quinolones19. However, the topological properties of
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haptens provided a rich structural information which could be helpful for 4
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Analytical Chemistry
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understanding the specificity of antibody19, the reasonably predicting and designing a
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resultant antibody specificity are still a great challenge due to the unclear
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structure-activity relationship and the limited knowledge of the recognition
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mechanism19.
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In this study, pipemidic acid, a quinolone drug with a flat conformation shaped
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like “I” at position 7, was used as a hapten to produce a polyclonal antibody, a highly
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sensitive competitive indirect ELISA (ciELISA) was successfully constructed in a
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heterologous coating format. Using the comparative molecular field analysis (CoMFA)
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based on the obtained cross-reactivity of the antibody raised by pipemidic acid, the
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three-dimensional quantitative structure-activity relationship (3D QSAR) was
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constructed among quinolone hapten structures and the specificity of pipemidic acid
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antibody. Moreover, comparing with the reported cross-reactivity data and typical
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quinolone hapten structures including clinafloxacin, ofloxacin, pazufloxacin and
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ciprofloxacin, the optimal hapten conformations for the corresponding antibody
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specificity were investigated for the first time.
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EXPERIMENTAL SECTION
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Reagents
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Pipemidic acid, 3,3’,5,5’-tetramethylbenzidine (TMB), bovine serum albumin
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(BSA), ovalbumin (OVA), complete and incomplete Freund’s adjuvants,
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1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), Tween-20,
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N,N-dimethylformamide (DMF) were purchased from Sigma (St. Louis, MO, USA).
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HRP-conjugated goat-anti-rabbit IgG was obtained from Boster Biotech Corporation
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Limited. (Wuhan, China). Rufloxacin, prulifloxacin, norfloxacin, pefloxacin,
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enrofloxacin, oxolinic acid, racemic ofloxacin, ciprofloxacin, lomefloxacin,
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danofloxacin, garenoxacin, pazufloxacin, clinafloxacin, gatifloxacin, marbofloxacin,
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difloxacin, sarafloxacin, sparfloxacin and tosufloxacin were purchased from
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Veterinary Medicine Supervisory Institute of China (Beijing, China). S-(-)-ofloxacin
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and R-(+)-ofloxacin were purchased from Daicel Chiral Technologies Company
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(Figure 1). All of the chemicals and organic solvents, which were analytical grade or 5
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better, were obtained from a local chemical supplier (Yunhui Trade Co., Ltd.,
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Guangzhou, China). Coating buffer, washing solution, blocking solution, substrate
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buffer, stopping reagent and TMB solution used in this study were prepared as
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previous work in our laboratory20. Standard stock solution (1 mg/mL) was prepared
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by dissolving an appropriate amount of each standard in 0.03 mol/L sodium hydroxide
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solution and kept at 4 °C until use. Working standard solutions (0.038, 0.31, 2.44,
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19.53, 156.25, 1250, 10000 ng/mL) were prepared by diluting the stock solution in
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Phosphate-buffered saline with 0.1% Tween-20 (PBST).
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Instruments
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UV-visible absorption measurement was performed on a UV-3010
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spectrophotometer (Hitachi, Japan). ELISA plates were washed by a microtiter plate
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washer DEM-3 (Tuopu, China). Absorbance was measured at a wavelength of 450 nm
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using a Multiskan MK3 microplate reader (Thermo Labsystems, USA).
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Preparation of hapten-protein conjugates
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Pipemidic acid was coupled to BSA via EDC for the immunogen, and both
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pipemidic acid and quinolone drugs (norfloxacin-OVA, pazufloxacin-OVA,
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ciprofloxacin-OVA, gatifloxacin-OVA, lomefloxacin-OVA, sarafloxacin-OVA and
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garenoxacin-OVA) were coupled with OVA via EDC for the use of the coating
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antigens according to previous work with modification21.
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Antibody production
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Animal treatments were conducted in accordance with the guidelines of the
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Chinese Association for Laboratory Animal Sciences. Two New Zealand rabbits
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(1.5-2.0 kg), supplied by the Guangdong Medical Laboratory Animal Center, were
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immunized using pipemidic acid-BSA as the immunogen to generate the polyclonal
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antibody against pipemidic acid according to our previous work with modification20.
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The obtained antisera from rabbits were purified by caprylic acid-saturated
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ammonium sulfate precipitation, and the purity was confirmed by sodium dodecyl
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sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and then divided into
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aliquots, labeled, and stored at -20 °C until use22.
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ELISA procedure 6
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Analytical Chemistry
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The ELISA was established on the basis of the common procedure of competitive
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indirect enzyme linked immunosorbent assay (ciELISA)20. Calibration curves were
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obtained by plotting the normalized signal B/B0 against the logarithm of analyte
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concentration. The logarithm of pipemidic acid concentration served as X-axis,
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whereas B/B0 (B, the average absorbance of the wells in the presence of a competitor;
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B0, the average absorbance of the well without analyte) served as Y-axis. The 50%
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inhibition value (IC50) were obtained using a four-parameter logistic equation that was
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used to fit the sigmoidal curve by OriginPro 8.5 software (OriginLab Corporation,
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Northampton, MA)23 . The equation is Y = (A–D)/[1+(X/C)E]+D
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where A is the maximum response at high asymptotes of the curve, D is the
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minimum response at low asymptotes of the curve, C is the concentration of the
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analyte that results in 50% inhibition and E is the slope of sigmoidal curve. The limit
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of detection (LOD) was defined as the concentration of analyte that inhibited 10%
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(IC10)24. The dynamic working range was defined as the lower and upper
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concentration that inhibited 20% ~ 80%25.
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Specificity
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Specificity of the antibody was evaluated by measuring the cross-reactivity (CR)
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using a group of structurally related quinolone drugs. 22 compounds were selected for
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this test (Figure 1), the obtained IC50 values were used to calculate cross-reactivities
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as follows:
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CR (%) = [IC50 (pipemidic acid) / IC50 (structurally related quinolone drugs)] × 100%
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QSAR
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CoMFA for pipemidic acid immunoassay
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The molecular modeling was conducted using SYBYL-X 2.1 program package.
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These 22 dataset molecules were constructed using the “SKETCH” option function;
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then they were energy minimized using the Powell method with MMFF94 force field
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and MMFF94 charges. The criteria of the termination and max iterations were set to
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be 0.005 kcal/(mol×Å) and 1000, respectively. The other parameters were the defaults.
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The molecular alignment was carried out using pipemidic acid as the template 7
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molecule and its C-4a, C-5, N-8 and C-8a as the common core structure. Pipemidic
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acid, prulifloxacin, rufloxacin, norfloxacin, pefloxacin, enrofloxacin, lomefloxacin,
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danofloxacin, garenoxacin, S-(-)-ofloxacin, clinafloxacin, gatifloxacin, marbofloxacin,
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difloxacin, sarafloxacin, sparfloxacin and tosufloxacin were classified into the
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training set, and the others of oxolinic acid, R-(+)-ofloxacin, ciprofloxacin and
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pazufloxacin were treated as the test set. The converted pIC50 (-log IC50) values were
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used in analysis.
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CoMFA steric and electrostatic interaction fields of each molecule were
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calculated on a 3D cubic lattice. A sp3 carbon probe atom with Van der Waal radius of
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1.52 Å and +1 charge was used to generate the steric and electrostatic filed energies.
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The cross-validated correlation coefficient R2 (q2) and the optimum number of
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components (ONC) were obtained using the partial least square (PLS) method with
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leave one out (LOO) option. Using the obtained ONC, the final non-cross-validated
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model was created. Except for using the MMFF94 charges, the other parameters or
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options of the CoMFA were the defaults.
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Conformation comparing analysis
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Previously, we raised a clinafloxacin polyclonal antibody and developed a rapid,
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specific immunoassay for clinafloxacin21. In this work, the converted pIC50 (-log IC50)
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values of cross-reactivity data of clinafloxacin were directly used for molecular
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modeling. The molecular alignment was carried out using clinafloxacin as the
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template molecule and its C-4a, C-5, N-8 and C-8a as the common core structure.
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clinafloxacin, gatifloxacin, ciprofloxacin, S-(-)-ofloxacin, rufloxacin, enrofloxacin,
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marbofloxacin, lomefloxacin, prulifloxacin, pefloxacin, nalidixic acid tosufloxacin
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and difloxacin were classified into the training set, and the others of danofloxacin,
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R-(+)-ofloxacin, sparfloxacin and sarafloxacin were treated as the test set. The
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molecular modeling was conducted using the same method and parameter as above.
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The CoMFA of other quinolones, including pazufloxacin, ciprofloxacin and ofloxacin,
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were conducted by literature and used to discuss in this study18,26,27.
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RESULTS AND DISCUSSION 8
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Analytical Chemistry
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Immunoreagent preparation
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The pipemidic acid bearing a carboxylic acid group at the end of the spacer was
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covalently coupled with a carrier protein (BSA or OVA) by the direct EDC method.
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UV spectra were measured to assure the successful conjugation. Figure S1 shows the
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UV spectra of BSA, OVA, pipemidic acid, pipemidic acid-BSA and pipemidic
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acid-OVA. BSA/OVA showed a characteristic absorption peak at 280 nm, while
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pipemidic acid had the peak at 332 nm. The absorption spectra of pipemidic acid
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-BSA/OVA conjugates contained both absorption peaks of pipemidic acid and
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BSA/OVA, but with somewhat blue shift. This indicated that the coupling of
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pipemidic acid to BSA and OVA was successful28. Similar results were obtained with
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other quinolone drugs-OVA conjugate (data not shown). The SDS-PAGE result
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showed that the purified pipemidic acid antibody had a heavy chain observed at 50
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kDa and a light chain at about 25 kDa. There was no superfluous band (Figure S2).
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Thus, the purified antibody was ideal for the further investigation.
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Optimization of ciELISA
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To obtain the optimal sensitivity of ciELISA, the concentration of coating
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antigen and antibody dilution time were optimized to obtain a maximum absorbance
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(Amax) for the zero standard concentration (blank) in the range of 1.0-1.5 and the best
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sensitivity (minimum IC50 value)29.
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For homologous assay format (pipemidic acid-OVA for the use of the coating
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antigen) , a poor binding affinity with a low inhibition rate of 48.05% was obtained.
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In addition, the standard curve for pipemidic acid (Figure 2) was constructed in the
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concentration range from 6.53 to 144.87 ng/mL and the values of LOD at 10%
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inhibition was within 2.6 ng/mL. The results indicated that the sensitivity had 14.4
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times lower than that of the UPLC–MS/MS method7 and the inhibition rate (48.05%)
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was undesirable, which failed to meet the purpose of this study.
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In order to obtain the better sensitivity, a series of coating antigens
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(norfloxacin-OVA, pazufloxacin-OVA, ciprofloxacin-OVA, gatifloxacin-OVA,
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lomefloxacin-OVA, sarafloxacin-OVA and garenoxacin-OVA) were compared at the
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same coating concentration (1µg/mL) (Table S1). Surprisingly, although homologous 9
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assay format demonstrated a better titer (titer=32000) than any one of heterogeneous
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formats (norfloxacin-OVA, pazufloxacin-OVA, ciprofloxacin-OVA,
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gatifloxacin-OVA, lomefloxacin-OVA, sarafloxacin-OVA, garenoxacin-OVA for the
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use of the coating antigens, respectively), it was found that lomefloxacin-OVA
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showed the highest inhibition rate (86.7%) with a titer of 8000.
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The calibration curve for pipemidic acid in homologous assay and heterogeneous
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assay format were constructed(Figure 2). Compared with the IC50 (30.76 ng/mL) and
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LOD (2.64 ng/mL) in homologous assay format (Table S1 and Figure 2), the IC50 and
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LOD in heterogeneous assay format (lomefloxacin-OVA as the coating antigen) were
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5.99 ng/mL and 0.31 ng/mL, respectively, indicating that the sensitivity was better
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than that of the homologous combination and that of the reported physio-chemical
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method7. As a result, the combination of pipemidic acid antibody and
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lomefloxacin-OVA was used for the further investigation due to its better sensitivity.
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Specificity
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The cross-reactivities of 22 kinds of quinolones were determined using the
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developed ELISA with pipemidic acid as the reference compound. Except for
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rufloxacin and prulifloxacin, the cross-reactivity values of other quinolones are lower
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than 15% (Table 1). Only 9% of the quinolones in this study displayed that their
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cross-reactivities were over 15%, thus, it could be regarded as high specificity of the
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pipemidic acid antibody.
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Generally, the high cross-reactivity of haptens could be directly ascribed to the
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similarity of the molecular structure, and the low cross-reactivity are possibly related
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to their structure difference of the cross-reactants30. In this study, norfloxacin had ever
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been proved to be a good hapten candidate resulting to a broad-specificity antibody
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that could recognize 13 quinolones31. The structure difference between norfloxacin
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and pipemidic acid was at position 6 and 8, the atoms of norfloxacin were carbon at
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position 6 and C-F group at position 8, however, the atoms of pipemidic acid were
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nitrogen. Such difference seems to be a little, but their cross-reactivities were
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significantly different (14.1% for norfloxacin, 100% for pipemidic acid). This
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suggested that the position 6 and 8 were likely crucial to the desired hapten 10
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conformation for antibody recognizing. Besides, the structures of rufloxacin and
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ofloxacin are similar except for the ring formed by the group at position 1 and 8, this
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implied that the ring also played an important role in the hapten-antibody binding.
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Additionally, comparing with the structure of pipemidic acid, difloxacin had one
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supplemented benzene ring at position 1, however, the cross-reactivity of difloxacin
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dramatically dropped from CR 100% for pipemidic acid to CR 0.5%. Moreover, the
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cross-reactivities of sarafloxacin (CR=0.3 %) and tosufloxacin (CR=0.3%) were also
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very low due to added benzene ring at position 1. These dramatic changes in
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cross-reactivity induced the importance of the groups at position 1, 6 and 8 for a
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desired binding conformation. To better understand the high specificity of the
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obtained pipemidic acid antibody, CoMFA was used for the further investigation.
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CoMFA analysis
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The CoMFA models exhibited the rational q2 values greater than 0.5 and the S
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and E fields offered 58.2% and 41.8% contribution to the affinity (Table S2). The
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pIC50 values of the molecules in the test set had been perfectly predicted in the
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CoMFA model (Figure S3).
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The contour maps reflected the desired/undesired steric/electrostatic features for
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the molecular binding affinity in the CoMFA model, in which the green and yellow
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contours represent the favorable and unfavorable steric regions, while the blue and red
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contours represent the favorable and unfavorable electropositive regions, respectively.
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The piperazine ring at position 7 was nearly in the same plane with the basic structure,
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1,4-dihydro-4-oxo-3-quinolinecarboxylic acid shared by almost all quinolones (Figure
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3a) and the conformation of pipemidic acid shaped like a letter “I”. Thus, from the
280
view of looking down, the piperazine ring formed a flat antibody binding cavity that
281
could not accommodate a large group.
282
And the green contour is near the position 4’ of pipemidic acid (Figure 3a), this
283
implied that the antibody could not recognize the group at both sides of position 7.
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However, the piperazine ring at position 7 of norfloxacin was almost perpendicular to
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the basic structure (Figure 3b). As a result, in order to accommodate a large group, it
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seemed to be essential for hapten owning a large antibody binding cavity at this 11
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position. The piperazine ring at position 7 in other quinolones was also basically
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perpendicular to the basic structure and was consistent with norfloxacin (Figure 3 c-f).
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These could contribute to the main reason that the resultant antibody against
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pipemidic acid exhibited low cross-reactivity to quinolones.
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The hydrogen atom at the oxazine ring of S-(-)-ofloxacin interacted with the little
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yellow contour and the methyl group were near the big yellow contour, however, the
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hydrogen atom at the oxazine ring of R-(+)-ofloxacin interacted with the green
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contours and the methyl group deviated from the little yellow contour due to their
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opposite conformations (Figure 3c, d). This could be the reason that S-(-)-ofloxacin
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(experimental pIC50=9.585) showed a lower binding activity to the pipemidic acid
297
antibody than that of R-(+)-ofloxacin (experimental pIC50= 10.000) (Table S3).
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Compared the conformation of S-(-)-ofloxacin with that of norfloxacin, the group at
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position 8 in S-(-)-ofloxacin was bigger and closer to yellow contours than that of
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norfloxacin, this could be ascribed to the lower activity of S-(-)-ofloxacin
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(experimental pIC50=9.585) (Figure 3b, d). In addition, comparison of the CoMFA
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maps of pefloxacin and difloxacin (Figure 3e, f), the yellow contours near the benzene
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ring of difloxacin indicated the bulky group at position 1 decreased the binding
304
activity, therefore, the activities of difloxacin (experimental pIC50=8.827) were lower
305
than that of pefloxacin (experimental pIC50=10.252) (Table S3).
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Besides the major contribution of steric field, the electrostatic field also played
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an important role in the quinolone-antibody binding conformation (Figure 3g, h). The
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difference between the norfloxacin and sarafloxacin was the group at position 1, and
309
the group was interacted with blue contour of a rhombus. However, the
310
electronegative fluorine atom of sarafloxacin was near these regions, this additionally
311
led to lower binding activity of sarafloxacin. It indicated that electronegative groups
312
at position 1 decreased the binding activity, which was consistent with the conclusion
313
of steric effect above.
314
Conformation comparing analysis
315
Table 2 showed 22 kinds of models of the minimum energy conformations of the
316
quinolones from the immunoassay for pipemidic acid in this study and the previously 12
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reported immunoassay for clinafloxacin21, they were divided into four groups in
318
accordance with their conformation features at minimum energy. The first group
319
contained pipemidic acid, nalidixic acid and oxolinic acid, their conformations shaped
320
like a letter “I”. The second group contained clinafloxacin, danofloxacin and
321
tosufloxacin, and their conformations shaped like a letter “P”. The third group
322
contained difloxacin, enrofloxacin, gatifloxacin, lomefloxacin, pefloxacin, rufloxacin,
323
R-(+)-ofloxacin, S-(-)-ofloxacin, garenoxacin, prulifloxacin, sparfloxacin and
324
marbofloxacin, and their conformations shaped like “ф”,. The fourth group contained
325
ciprofloxacin, norfloxacin, pazufloxacin and sarafloxacin, and their conformations
326
formed “Y” shape (or a lollipop).
327
In the first group, the conformation features of nalidixic acid and oxolinic acid
328
were also shaped like “I” and consistent with pipemidic acid. Thus, the antibody
329
derived from nalidixic acid or oxolinic acid as a hapten could be of high specificity.
330
In the second group, CoMFA was used for the further investigation of
331
clinafloxacin immunoassay. The CoMFA models exhibited the rational q2 values was
332
0.587 and the S and E fields offered 53.4% and 46.6% contribution to the affinity
333
(Table S2), respectively. The pIC50 values of the molecules in the test set had been
334
perfectly predicted (Figure S3). Therefore, the contour analysis of the model was
335
further carried out.
336
The group at position 7 of clinafloxacin was on the side of the basic structure
337
plane and the conformation of clinafloxacin shaped like a letter “P” (Figure 4a). And
338
the green contour near the amino group at position 3’ in clinafloxacin (Figure 4a), this
339
implied that the antibody to clinafloxacin could not recognize the group on the side
340
near the position 5’. However, the piperazine ring at position 7 in ciprofloxacin was
341
basically perpendicular to the basic structure (Figure 4b). And other quinolones with
342
piperazine ring at position 7 were also basically perpendicular to the basic structure
343
and were consistent with that of ciprofloxacin (Figure 4c-f). Moreover, the amino
344
group and 5-membered ring at position 7 seems to form a small antibody binding
345
cavity that would not accommodate a large group. For example, a piperazine ring is a
346
big group that could not allow to be accommodated in the small antibody binding 13
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347
cavity formed by the amino group and 5-membered ring of clinafloxacin. However,
348
the ciprofloxacin shaped like “Y” and the piperazine ring in ciprofloxacin could form
349
a bigger antibody binding cavity, and thus the bigger antibody binding cavity should
350
contribute to the broad specificity of the corresponding antibody to cipfloxacin, and
351
then the high specificity of clinafloxacin antibody would also be understandable. The
352
previous study of clinafloxacin also showed that all the CRs for structure-related
353
quinolones were lower than 15% (Table S5) 21.
354
In addition, when comparing the contour maps of gatifloxacin, lomefloxacin and
355
S-(-)-ofloxacin (Figure 4c, d, f), it could be found that the group at position 8 of
356
gatifloxacin was bigger and closer to the green contour over the structure than those
357
of S-(-)-ofloxacin. But the group at position 8 in S-(-)-ofloxacin was bigger and closer
358
than those of lomefloxacin, this could be ascribed to the highest activity of
359
gatifloxacin (experimental pIC50=6.076), followed by S-(-)-ofloxacin (experimental
360
pIC50=5.440), and lomefloxacin (experimental pIC50=4.828) (Table S4).
361
The green contour at the map bottom interacted with the methyl at the oxazine
362
ring of S-(-)-ofloxacin, however, the methyl at the oxazine ring of R-(+)-ofloxacin
363
were brought near the yellow contour in the upper portion of map due to their
364
opposite conformations (Figure 4e, f). It revealed the mechanism that S-(-)-ofloxacin
365
had higher binding affinity than R-(+)-ofloxacin. When the antibody distinguishing
366
the chiral isomer of quinolones, it showed that the bulky group near the green contour
367
at position 8 played a critical role. Comparison of the CoMFA maps of sarafloxacin
368
and ciprofloxacin , the yellow contours near the benzene ring of sarafloxacin
369
indicated the bulky group at position 1 decreased the binding activity (Figure 4b, g),
370
therefore, the activities of sarafloxacin (experimental pIC50=4.628) were lower than
371
that of ciprofloxacin (experimental pIC50=5.640) (Table S4). Since the yellow
372
contours near the benzene ring of sarafloxacin, it is understandable that the activity of
373
sarafloxacin (experimental pIC50=4.628) was lower, and the predicted pIC50 value of
374
sarafloxacin in the test set was lower than the experimental value (Table S4).
375
The electrostatic contour maps also reflected the molecular binding affinity in the
376
CoMFA model (Figure 4). The amino group at position 7 in clinafloxacin near the 14
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blue contour was a strong electron donor group, while the groups of gatifloxacin and
378
lomefloxacin were weak electron donor groups. As a result, the binding activity of
379
gatifloxacin or lomefloxacin was much lower than that of clinafloxacin, this was
380
consistent with the steric effect above. The conformations of danofloxacin and
381
tosufloxacin in the second group also like “P” and similar with clinafloxacin, as a
382
result, their antibodies also would show high-specificity as clinafloxacin. This had
383
been proved by a previous study using anti-danofloxacin antibody (Table S5)32. The
384
conformation of tosufloxacin demonstrated high similarity with that of clinafloxacin
385
and could be a suitable hapten for the production of highly specific antibody.
386
The third group consisted of 12 kinds of quinolones in this study, they all shaped
387
like “ф”, and it could be predicted that their antibodies would be predicted to
388
accordingly be high-specific, this have also been confirmed by experiments (Table 2
389
and Table S5)10,11,27,33-37. For example, ofloxacin contains a substituent on the
390
piperazine ring at position 7, and the previous QSAR study showed that the green
391
contour was near the substituent, while two yellow contours distributed in both sides
392
of piperazine ring27. This implied that the antibody could not recognize quinolones
393
without a substituent on the piperazine ring or with a substituent on other site of
394
piperazine ring. And the experimental cross-reactivity had already proved that only
395
rufloxacin, garenoxacin and marbofloxacin had high cross reactivity values
396
(cross-reactivity>15%)27. Therefore, it could be concluded that hapten quinolone
397
shaped like “I” or “P” or “ф” would result in an antibody with low cross-reactivity
398
and high specificity.
399
In the fourth group (Table 2), different from the structures shaped like “I”, “P”
400
and “ф”, all the proposed compounds shaped like “Y” or a lollipop, their
401
conformations could be predicted to produce a large antibody binding cavity to
402
accommodate the piperazinyl group at position 7 (Table 2). For example,
403
pazufloxacin contained a 1-aminocyclopropyl group at position 7 but not a
404
piperazinyl group shared by many quinolones, both the amino and cyclopropyl groups
405
around position 7 of pazufloxacin formed “Y” shape or a lollipop, and the green
406
contour also lay on both sides of the position 726, it implied that the resultant antibody 15
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407
to pazufloxacin would broadly recognize other quinolones. In fact, the experimentally
408
raised pazufloxacin antibody with high sensitivity and broad specificity could
409
recognize 24 quinolones as expected (Table S5)26, this is a broad specificty, but not a
410
high specificity, because pazufloxacin shaped unlike any one of three conformations
411
“I” or “P” or “ф”.
412
Ciprofloxacin is another good example to prove the three essential conformations
413
for a high specificity18. The piperazine ring at position 7 in ciprofloxacin was almost
414
perpendicular to the basic structure (Figure 4b and Table 2), the conformation shaped
415
liked a lollipop, and thus the ciprofloxacin antibody should be able to accommodate a
416
large group and demonstrated a broad specificity according to the QSAR analysis
417
above in this study. Actually this has also been verified again by a previously reported
418
monoclonal antibody and a developed broad-specificity immunoassay for 12
419
quinolones (Table S5)18. The conformations of norfloxacin and sarafloxacin in the
420
fourth group also like “Y” and were similar with ciprofloxacin, as a result, their
421
antibodies also would show broad specificity as ciprofloxacin. The structure-related
422
quinolones of norfloxacin and sarafloxacin whose cross-reactivity values were over
423
15%, were 13 kinds and 10 kinds, respectively (Table S5)9,31.
424
Therefore, it could be concluded that the shape like “I”, “P” and “ф” was the
425
essential conformation feature of quinolone hapten used to produce antibody with
426
high specificity, while the conformation of quinolone hapten like “Y” shape (or a
427
lollipop) could be used to produce antibody with broad specificity.
428 429
CONCLUSIONS
430
In summary, 3D QSAR studies were performed to investigate the molecular
431
recognition between the antibody and quinolone drugs, the CoMFA model revealed
432
that the quinolones shaped like “I”, “P” and “ф” formed a flat conformation, which
433
could not accommodate a large group, and thus the resultant antibody would be high
434
specificity. But the hapten conformation shaped like “Y” will lead to a broad
435
specificity antibody with the high cross-reactivity. Almost all the antibodies against
436
quinolone could result from these four hapten conformations. This finding is the first 16
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437
time to systemically and effectively elucidate and predict the high specificity of
438
quinolone antibodies, it will be of significance of the accurate hapten design,
439
predictable antibody specificity and better understanding of the recognition
440
mechanism of hapten and the antibody specificity.
441 442 443
ASSOCIATED CONTENT Supporting Information. Description of the optimized ciELISA conditions, the
444
summary of the calculated parameters of the CoMFA model, the predicted values
445
derived from the CoMFA models of pipemidic acid, the predicted values derived from
446
the CoMFA models of clinafloxacin, a review of immunoassay for the determination
447
of quinolones in recent years, UV spectra of BSA, OVA, pipemidic acid, pipemidic
448
acid-BSA and pipemidic acid-OVA, SDS-PAGE pattern using purified pipemidic acid
449
polyclonal antibody, the scatter plots of predicted versus experimental pIC50.
450 451 452
ACKNOWLEDGMENTS This work was supported by Natural Science Foundation of China (U1301214,
453
30700663, 21475047), Guangdong and Guangzhou Planned Program in Science and
454
Technology (S2013030013338, 2016201604030004, 2014TX01N250,
455
2013B051000072, and 2014A030306026). Guangdong Natural Science Foundation
456
(2015A030313366), the support of 2016KYDT04 and 2016KYDT06 by Guangzhou
457
University of Chinese Medicine.
458 459
REFERENCES
460
(1) Van Bambeke, F.; Michot, J. M.; Van Eldere, J.; Tulkens, P. M. Clin. Microbiol.
461
Infec. 2005, 11, 256-280.
462
(2) Andriole, V. T. Clin. Infect. Dis. 2005, 41, S113-S119.
463
(3) Mehlhorn, A. J.; Brown, D. A. Ann. Pharmaco. 2007, 41, 1859-1866.
464
(4) Hernández-Arteseros, J.; Barbosa, J.; Compano, R.; Prat, M. J. Chromatog. A
465
2002, 945, 1-24.
17
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
466
(5) Espinosa-Mansilla, A.; Jiménez Girón, A.; Muñoz de la Peña, A. Czech J. Food
467
Sci. 2012, 30, 74-82.
468
(6) Pecorelli, I.; Galarini, R.; Bibi, R.; Floridi, A.; Casciarri, E.; Floridi, A. Anal. Chim.
469
Acta 2003, 483, 81-89.
470
(7) Chang, C. S.; Wang, W. H.; Tsai, C. E. J. Food Drug Anal. 2010, 18, 87-97.
471
(8) Dorival‐García, N.; Zafra‐Gómez, A.; Cantarero, S.; Navalón, A.; Vílchez, J.
472
Microchem. J. 2013, 106, 323-333.
473
(9) Huet, A. C.; Charlier, C.; Tittlemier, S. A.; Singh, G.; Benrejeb, S.; Delahaut, P. J
474
Agr. Food Chem. 2006, 54, 2822-2827.
475
(10) Lu, S. X.; Zhang, Y. L,; Liu, J. T.; Zhao, C. B.; Liu, W.; Xi, R. M. J Agr. Food
476
Chem. 2006, 54, 6995-7000.
477
(11) Sheng, W.; Xia, X. F.; Wei, K. Y.; Li, J.; Li, Q. X.; Xu, T. J Agr. Food Chem.
478
2009, 57, 5971-5975.
479
(12) Wang, Z. H.; Kai, Z. P.; Beier, R. C.; Shen, J. Z.; Yang, X. L. Int. J. Mol. Sci.
480
2012, 13, 6334-6351.
481
(13) Cui, J. L.; Zhang, K.; Huang, Q. X.; Yu, Y. Y.; Peng, X. Z. Anal. Chim. Acta 2011,
482
688, 84-89.
483
(14) Zhang, H. Y.; Wang, S.; Fang, G. Z. J. Immunol. Methods 2011, 368, 1-23.
484
(15) Li, Y. F.; Sun, Y. M.; Beier, R. C.; Lei, H. T.; Gee, S.; Hammock, B. D.; Wang, H.;
485
Wang, Z.; Sun, X.; Shen, Y. D.; Yang, J. Y.; Xu, Z. L. TrAC. Trends Anal. Chem. 2017,
486
88, 25-40.
487
(16) Leivo, J.; Lamminmäki, U.; Lövgren, T.; Vehniäinen, M. J Agr. Food Chem.
488
2013, 61, 11981-11985.
489
(17) Cao, L. M.; Kong, D. X.; Sui, J. X.; Jiang, T.; Li, Z. Y.; Ma, L.; Lin, H. Anal.
490
Chem. 2009, 81, 3246-3251.
491
(18) Wang, Z. H.; Zhu, Y.; Ding, S. Y.; He, F. Y.; Beier, R. C.; Li, J. C.; Jiang, H. Y.;
492
Feng, C. W.; Wan, Y. P.; Zhang, S. X. Anal. Chem. 2007, 79, 4471-4483.
493
(19) Xu, Z. L.; Shen, Y. D.; Zheng, W. X.; Beier, R. C.; Xie, G. M.; Dong, J. X.; Yang,
494
J. Y.; Wang, H.; Lei, H. T.; She, Z. G. Anal. Chem. 2010, 82, 9314-9321.
495
(20) Luo, L.; Xu, Z. L.; Yang, J. Y.; Xiao, Z. L.; Li, Y. J.; Beier, R. C.; Sun, Y. M.; Lei, 18
ACS Paragon Plus Environment
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Analytical Chemistry
496
H. T.; Wang, H.; Shen, Y. D. J Agr. Food Chem. 2014, 62, 12299-12308.
497
(21) Chen, J. H.; Lv, S. W.; Wang, Q.; Xu, Z. L.; Yang, J. Y.; Shen, Y. D.; Wang, H.;
498
Sun, Y. M.; Lei, H. T. Food Anal. Methods 2015, 8, 1468-1476.
499
(22) Page, M.; Thorpe, R. In The Protein Protocols Handbook; Humana Press: 2002,
500
p 983-984.
501
(23) Oubiña, A.; Barceló, D.; Marco, M. P. Anal. Chim. Acta 1999, 387, 267-279.
502
(24)Yuan, Y. L.; Hua, X. D.; Li, M.; Yin, W.; Shi, H. Y.; Wang, M. H. RSC Adv. 2014,
503
4, 24406-24411.
504
(25)Yu, F. Y.; Vdovenko, M. M.; Wang, J. J.; Sakharov, I. Y. J Agr. Food Chem. 2011,
505
59, 809-813.
506
(26) Chen, J. H.; Lu, N.; Shen, X.; Tang, Q. S.; Zhang, C. J.; Xu, J.; Sun, Y. M.;
507
Huang, X. A..; Xu, Z. L.; Lei, H. T. J Agr. Food Chem. 2016, 64, 2772-2779.
508
(27) Mu, H. T.; Lei, H. T.; Wang, B. L.; Xu, Z. L.; Zhang, C. J.; Ling, L.; Tian, Y. X.;
509
Hu, J. S.; Sun, Y. M. J Agr. Food Chem. 2014, 62, 7804-7812.
510
(28) Zhang, H. T.; Jiang, J. Q.; Wang, Z. L.; Chang, X. Y.; Liu, X. Y.; Wang, S. H.;
511
Zhao, K.; Chen, J. S. J. Zhejiang Univ. Sci. B 2011, 12, 884-891.
512
(29) Han, D.; Yu, M.; Knopp, D.; Niessner, R.; Wu, M.; Deng, A. P. J Agr. Food Chem.
513
2007, 55, 6424-6430.
514
(30) Lei, H. T.; Su, R.; Haughey, S. A.; Wang, Q.; Xu, Z. L.; Yang, J. Y.; Shen, Y. D.;
515
Wang, H.; Jiang, Y. M.; Sun, Y. M. Molecules 2011, 16, 5591-5603.
516
(31) Jiang, W. X.; Wang, Z. H.; Beier, R. C.; Jiang, H. Y.; Wu, Y. N.; Shen, J. Z. Anal.
517
Chem. 2013, 85, 1995-1999.
518
(32) Liu, Z. Q.; Lu, S. X.; Zhao, C. H.; Ding, K.; Cao, Z. Z.; Zhan, J. H.; Ma, C.; Liu,
519
J. T.; Xi, R. M. J. Sci. Food Agric. 2009, 89, 1115-1121.
520
(33) Li, C.; Jiang, J.; Qi, X. H. In 2011 Eighth International Conference on Fuzzy
521
Systems and Knowledge Discovery (FSKD) 2011; Vol. 3, p 1446-1449.
522
(34) Zeng, H. J.; Yang, R.; Liu, B.; Lei, L. F.; Li, J. J.; Qu, L. B. J. Pharmaceut. Anal.
523
2012, 2, 214-219.
524
(35) Zhao, C. B.; Liu, W.; Ling, H. L.; Lu, S. X.; Zhang, Y. L.; Liu, J. T.; Xi, R. M. J
525
Agr. Food Chem. 2007, 55, 6879-6884. 19
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526
(36) Zhao, Y. L.; Zhang, G. P.; Liu, Q. T.; Teng, M.; Yang, J. F.; Wang, J. H. J Agr.
527
Food Chem. 2008, 56, 12138-12142.
528
(37) Zhi, A. M.; Li, B. B.; Liu, Q. T.; Hu, X. F.; Zhao, D.; Hou, Y. Z.; Deng, R. G.;
529
Chai, S. J.; Zhang, G. P. Food Agr. Immunol. 2010, 21, 335-345.
530
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531
Figure Captions
532
Figure 1 The structures of quinolones
533
Figure 2 ciELISA calibration curves for pipemidic acid with different coating
534
antigens. Each point represented the mean ± standard deviation of three replicates.
535
Figure 3 CoMFA contour maps of pipemidic acid. (a-f) CoMFA streic contour maps
536
together with (a) embedded pipemidic acid, (b) embedded norfloxacin, (c) embedded
537
R-(+)-ofloxacin, (d) embedded S-(-)-ofloxacin, (e) embedded pefloxacin and (f)
538
embedded difloxacin. (g-h) CoMFA electrostatic contour maps together with (g)
539
embedded norfloxacin and (h) embedded sarafloxacin. The energies of all fields were
540
calculated with the weight of the standard deviation and the coefficient. Green, yellow,
541
blue, and red contours represent steric bulk desirable, steric bulk undesirable, positive
542
charge desirable and negative charge desirable, respectively.
543
Figure 4 CoMFA contour maps of clinafloxacin. (a-f) CoMFA streic contour maps
544
together with (a) embedded clinafloxacin, (b) embedded ciprofloxacin, (c) embedded
545
gatifloxacin, (d) embedded lomefloxacin, (e) embedded R-(+)-ofloxacin, (f)
546
embedded S-(-)-ofloxacin and (g) embedded sarafloxacin. CoMFA electrostatic
547
contour maps together with (h) embedded clinafloxacin, (i) embedded gatifloxacin, (j)
548
embedded lomefloxacin. The energies of all fields were calculated with the weight of
549
the standard deviation and the coefficient. Green, yellow, blue, and red contours
550
represent steric bulk desirable, steric bulk undesirable, positive charge desirable and
551
negative charge desirable, respectively.
552
21
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553
Table 1 Cross-reactivity of pipemidic acid and 22 quinolone drugs based on coating
554
antigen lomefloxacin –OVA. IC50
CR
IC50
CR
Name
Name (nmol/mL)
(%)
(nmol/mL) (%)
Pipemidic acid
0.0076
100.0
Danofloxacin
0.21
3.6
Rufloxacin
0.026
29.5
Garenoxacin
0.23
3.3
Prulifloxacin
0.026
28.8
S-(-)-ofloxacin
0.26
2.9
Norfloxacin
0.054
14.1
Pazufloxacin
0.27
2.8
Pefloxacin
0.056
13.4
Clinafloxacin
0.49
1.5
Enrofloxacin
0.071
10.7
Gatifloxacin
0.50
1.5
Oxolinic acid
0.082
9.2
Marbofloxacin
0.96
0.8
Ofloxacin
0.090
8.4
Difloxacin
1.49
0.5
R-(+)-ofloxacin
0.10
7.3
Sarafloxacin
2.32
0.3
Ciprofloxacin
0.14
5.4
Sparfloxacin
2.53
0.3
Lomefloxacin
0.21
3.7
Tosufloxacin
2.91
0.3
22
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Analytical Chemistry
Table 2 Models of the minimum energy conformations of the quinolones from the immunoassay for pipemidic acid and for clinafloxacin. The elements are represented in the following manner: oxygen, red; nitrogen, navy blue; hydrogen, baby blue; fluorine, green; carbon, white.; sulphur, yellow. Shape Characteristic Models of the minimum energy conformations of the quinolones a I High-specificity Nalidixic acid P
ф
Y
Oxolinic acid
Pipemidic acid
High-specificity Clinafloxacin
Danofloxacin
Tosufloxacin
Difloxacin
Enrofloxacin
Gatifloxacin
Lomefloxacin
Pefloxacin
Rufloxacin
R-(+)-ofloxacin
S-(-)-ofloxacin
Garenoxacin
Prulifloxacin
Sparfloxacin
Marbofloxacin
Ciprofloxacin
Norfloxacin
Pazufloxacin
Sarafloxacin
High-specificity
b
Broad-specificity
558
a
High-specificity means the structurally related quinolones whose cross-reactivity values over 15% were less than 3 kinds.
559
b
Broad-specificity means most of the structurally related quinolones whose cross-reactivity values over 15%.
23
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560
TOC
561
24
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Analytical Chemistry
Figure 1 The structures of quinolones 209x120mm (300 x 300 DPI)
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Figure 2 ciELISA calibration curves for pipemidic acid with different coating antigens. Each point represented the mean ± standard deviation of three replicates. 296x209mm (300 x 300 DPI)
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Analytical Chemistry
Figure 3 CoMFA contour maps of pipemidic acid. (a-f) CoMFA streic contour maps together with (a) embedded pipemidic acid, (b) embedded norfloxacin, (c) embedded R-(+)-ofloxacin, (d) embedded S-(-)ofloxacin, (e) embedded pefloxacin and (f) embedded difloxacin. (g-h) CoMFA electrostatic contour maps together with (g) embedded norfloxacin and (h) embedded sarafloxacin. The energies of all fields were calculated with the weight of the standard deviation and the coefficient. Green, yellow, blue, and red contours represent steric bulk desirable, steric bulk undesirable, positive charge desirable and negative charge desirable, respectively. 147x147mm (300 x 300 DPI)
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Analytical Chemistry
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Figure 4 CoMFA contour maps of clinafloxacin. (a-f) CoMFA streic contour maps together with (a) embedded clinafloxacin, (b) embedded ciprofloxacin, (c) embedded gatifloxacin, (d) embedded lomefloxacin, (e) embedded R-(+)-ofloxacin, (f) embedded S-(-)-ofloxacin and (g) embedded sarafloxacin. CoMFA electrostatic contour maps together with (h) embedded clinafloxacin, (i) embedded gatifloxacin, (j) embedded lomefloxacin. The energies of all fields were calculated with the weight of the standard deviation and the coefficient. Green, yellow, blue, and red contours represent steric bulk desirable, steric bulk undesirable, positive charge desirable and negative charge desirable, respectively. 198x286mm (300 x 300 DPI)
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