Papers by Keyword: Monoclonal Antibody

Abstract: To generate a group-specific monoclonal antibody (McAb) against danofloxacin (DANO), enrofloxacin (ENR) and ciprofloxacin (CIP), glutaraldehyde was used to link the presentative hapten of CIP to the immunogen and coating antigen, respectively, leaving the common structure of these 3 drugs exposed in both conjugates as a major antigenic site. Consequently, a McAb (6D3) with high cross-reactivity to this three antibiotics has been obtained by using hybridomas technique. In a biotin-avidin mediated enzyme-linked immunosorbent assay, the IC₅₀ values for DANO, ENR and CIP were 5.1, 4.5 and 4.2 ng mL^-1, respectively. The ELISA was used for the detection of spiked DANO and ENR+CIP in milk. The recoveries ranged from 74.1 to 92.4% and coefficients of variation were in a range of 6.6-11.9%. The accuracies and sensitivity of the method were good for simultaneous analysis of the 3 drugs in milk after a simple sample extraction process.

Abstract: Nanogold (NG) in size of 10 nm was prepared by the NaBH4 procedure. A new ligand 6-mercaptonicotinic acid (MNA) was used to couple both methylmercury chloride (CH3HgCl) and carrier protein to obtain an immunogen, it was immunized BALB/C mice, and the spleen cells of immunized mice were fused with myeloma cells. The monoclonal antibody (mAb) against mercury (II) ions was produced by the hybridoma technique. The mAb was labeled the NG to prepare an immunonanogold (ING) probe for Hg(II). In pH 5.4 Na2HPO4-citric acid buffer solution and under the condition of ultrasonic irradiation, the ING particles were aggregated un-specifically to form big particles that exhibited a strong resonance Rayleigh scattering (RRS) peak at 580 nm. When the Hg(II) was added, the specific immunoreaction of ING-Hg(II) take place, and the ING-Hg(II) immunocomplex dispersed in the solution that caused the RRS intensity decreasing linearly at 580 nm. The decreased intensity was linear to Hg(II) concentration in the range of 0.025-10 μmol/L, with a detection limit of 1.1 nmol/L Hg(II).

Abstract: Using PEG-10000 and sodium citrate as stabilizer, and NaBH4 as reducer, a stable nanosilvers (AgNPs) sol was prepared. In pH 6.6 phosphate buffer solution containing NaCl, the AgNPs were aggregated to large particles, which lead to resonance Rayleigh scattering (RRS) peak at 350 nm enhancement. Upon addition of cysteine, the peak decreased. The decreased value ΔI is linear to cysteine concentration in the range of 5-60×10^-8 mol/L. Thus, a new RRS method was proposed for detection of cysteine.

Abstract: Firstly, BPA structure was modified, then coupling BPA with BSA or OVA to prepare immunogen and coating antigen. Five Balb/C mice were immunized with BPA-BSA. Finally an antibody was prepared and the indirect competitive enzyme-linked immunosorbent assay was founded. Results:(1) The monoclonal antibody belongs to IgG1 subtype and К light chain.(2) The antibody titer is 1：256000, the most suitable concentration of coating antigen is 2μg/mL, and the optimal dilution of antibody and HRP are 1:16000 and 1:10000 respectively. (3)The linear regression line equation is y = 0.1139x + 0.1046, correlation coefficient is R2=0.97, the detection limit is 0.911ng/mL and IC50 is 2.454×103ng/mL. (4)The monoclonal antibody has high specificity for the cross reactivity with phenol, hydroquinone, and tert-butyl hydroquinone being lower than 0.01%, except ortho-hydroxybenzoic acid 2.1%. (5)The recovery range is 93%～116% and 89%～112% when adding BPA into black samples.（6）When the method was used in real materials to detect BPA residual, the results were proximate to the dates by HPLC.

Abstract: This study aimed to develop a method for the detection of tetrodotoxin (TTX) based on monoclonal antibody (McAb). The hapten TTX was linked to carrier protein keyhole limpet hemocyanin (KLH) as immunogen, and linked to ovalbumin (OVA) as coating antigen by the Mannich method. Then the 6~8 weeks Babl/c mice were immunized. After cell amalgamation, a cell line with high specificity and sensitivity was obtained, and McAb was produced. The indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed for detecting TTX. The optimized working conditions of the icELISA were 4.0 μg/mL coating antigen, 0.75 μg/mL McAb, 45 min competitive time, at room temperature (20~25 °C). The IC₅₀ value of this method was 24.0 ng/mL, the working ranges were 5.2~107.6 ng/mL, the intra-assay and inter-assay coefficient of variation (CV %) were 4.2 and 4.5, respectively. This investigation will benefit the assay kit development for detecting TTX.

Abstract: This study aimed to develop a monoclonal antibody based icELISA method for Ractopamine (Rac) residue. For this purpose, mixed anhydride method was employed to synthesize the immunogen of Rac-BSA and 1, 4-butanediol diglycidyl ether was used to prepare the coating antigen of Rac-OVA, thus pursue the heterologous sensitivity. Through cell fusion technology, four Hybridoma named R1-B5, R2-B3, R2-C6, and R4-C8 were screened out, and the Kas of all mAbs were between 2.7 and 4.8×109 L/mol. Based on the R1-B5 mAb, a heterologous icELISA standard curve was developed. The working range was from 0.013 to 33.7 ng/mL, with LOD and IC50 value of 0.007 ng/mL and 0.67 ng/mL, respectively. Therefore, this icELISA can be used for detecting Rac residue in animal products.

Abstract: Through cell fusion technology, five hybridoma lines of sarafloxacin (SAR), named S1-B2, S2-C6, S2-E7, S3-C5, and S3-E5, were identified and their corresponding mAbs were of the IgG1 isotype with a k light chain. The Kaffs of all mAbs were between 2.8 and 4.6×109 L/mol. The titers and IC50 values of purified ascite fluids were in the range of 0.512–2.56×106 and 0.32–0.48 ng/mL, respectively. The performances of S1-B2 and S2-C6 were more consistent in the stability experiments. Based on the S1-B2 hybridoma, an icELISA method was developed. The dynamic range was from 0.004 to 18 ng/mL, with a detection limit for the assay and IC50 values of 0.002 and 0.32 ng/mL, respectively. Therefore, the establishment of these hybridomas may provide an alternative method for the detection of SAR residues in food-original animals.

Abstract: This paper presents the generation of monoclonal antibodies (mAbs) with high specificity against enrofloxacin (ENR) through cell fusion procedures, and the development of a mAb-based indirect competitive ELISA (icELISA) method to detect ENR residue using one of these Hybridomas (clone 4B5-D6). Under the optimal experimental conditions, this assay exhibited a working range of 0.004-38 ng/mL with IC50 and LOD values of 0.4 and 0.002 ng/mL, respectively. Except for a high cross-reactivity (105.2%) to Ciprofloxacin, negligible cross-reactivity to the other compounds was observed. After optimization, 10% of methanol was used in the assay buffer and this ELISA system can tolerate acetonitrile not higher than 10%. Recovery studies indicate that an excellent correlation between concentration spiked and concentration determined was found, and the results also suggest this assay has the potential to be incorporated into a quantitative monitoring program for the rapid screening of ENR residue in poultry tissues.