• The Journal of the Korean Society for Microbiology
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• v.22 no.4
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• pp.377-392
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• 1987

Monoclonal antibody to the Escherichia coli heat-stable enterotoxin(ST) was produced to develop a rapid and convenient diagnostic method to the ST. The toxin was purified from culture supernatant of enterotoxigenic E. coli O148H28($ST^+/LT^+$) and conjugated to bovine serum albumin(BSA). The ST-BSA conjugate was used to immunize BALB/c mice and the immune spleen cells from these mice were fused with $P3{\times}63$ Ag8.V653 plasmacytoma cells. Hybridomas were screened by ELISA and positive hybridomas were cloned by limiting dilution. Finally, one stable clone (AS36) specific to ST was selected for further growth and characterization. Antibody titers of culture supernatant and ascitic fluid from BALB/c mice were 1:1,024 and 1:20,480 respectively in ELISA. The isotype and subclass of monoclonal antibody was IgG1 in sandwich ELISA. To test the neutralizing effect of monoclonal antibody on toxin activity of ST, mixture of ascitic fluid and ST was assayed by infant mouse assay and this monoclonel antibody was proved to be a neutralizing antibody. The titer of ascitic fluid which completely neutralized biological activity of 4 units of ST was 1:4. Purified ST was quantitatively measured by competitive ELISA and minimum amount of ST detectable by this assay was 250pg, which was an amount six-fold smaller than that detectable by infant mouse assay. Four reference strains of enterotoxigenic E. coli from WHO were detected by competitive ELISA and highly specific, sensitive and reproducible result was obtained.

• Journal of Microbiology and Biotechnology
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• v.23 no.1
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• pp.69-75
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• 2013

Enrofloxacin is a fluoroquinolone antibiotic approved for the treatment of infections in animals. Because of the side effects to consumers of animal products, the maximum residue limits (MRLs) of enrofloxacin in animal tissues for consumption are regulated. In this study, a monoclonal antibody (mAb) against enrofloxacin was prepared and characterized for the development of a direct competitive enzyme-linked immunosorbent assay (ELISA). The obtained mAb, Enro44, was highly specific for enrofloxacin and had a 50% inhibition concentration ($IC_{50}$) of 1.99 ng/ml in a competitive ELISA, and the limit of detection (LOD) was 0.50 ng/ml. The cross-reactivity of the mAb with other quinolones and fluoroquinolones was lower than 0.01%. The subclass of the mAb Enro44 was identified as IgG1. The antigen (Ag)-captured direct competitive ELISA using the mAb Enro44 was tested on different spiked samples, including chicken muscle, cattle milk, and cattle urine, and the assay demonstrated recoveries of 82-112%, 80-125%, and 78-124%, respectively. Furthermore, the quantitation of enrofloxacin obtained from the ELISA and from high-performance liquid chromatography (HPLC) was in good agreement, with the linear regression coefficient between 0.933 and 1.056. The cDNAs encoding a heavy-chain Fd fragment (VH and CH1) and a light chain of the mAb Enro44 were cloned and sequenced. Taken together, the results obtained reveal a potential use of this mAb in an ELISA for the detection of enrofloxacin in food samples. The information of amino acid sequence of this mAb will be useful for further modification and production of the mAb in a bioreactor.

• Biomolecules & Therapeutics
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• v.29 no.2
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• pp.166-174
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• 2021

Multiple myeloma is a malignant cancer of plasma cells. Despite recent progress with immunomodulatory drugs and proteasome inhibitors, it remains an incurable disease that requires other strategies to overcome its recurrence and non-response. Based on the high expression levels of programmed death-ligand 1 (PD-L1) in human multiple myeloma isolated from bone marrow and the murine myeloma cell lines, NS-1 and MOPC-315, we propose PD-L1 molecule as a target of anti-multiple myeloma therapy. We developed a novel anti-PD-L1 antibody containing a murine immunoglobulin G subclass 2a (IgG2a) fragment crystallizable (Fc) domain that can induce antibody-dependent cellular cytotoxicity. The newly developed anti-PD-L1 antibody showed significant antitumor effects against multiple myeloma in mice subcutaneously, intraperitoneally, or intravenously inoculated with NS-1 and MOPC-315 cells. The anti-PD-L1 effects on multiple myeloma may be related to a decrease in the immunosuppressive myeloid-derived suppressor cells (MDSCs), but there were no changes in the splenic MDSCs after combined treatment with lenalidomide and the anti-PD-L1 antibody. Interestingly, the newly developed anti-PD-L1 antibody can induce antibody-dependent cellular cytotoxicity in the myeloma cells, which differs from the existing anti-PD-L1 antibodies. Collectively, we have developed a new anti-PD-L1 antibody that binds to mouse and human PD-L1 and demonstrated the antitumor effects of the antibody in several syngeneic murine myeloma models. Thus, PD-L1 is a promising target to treat multiple myeloma, and the novel anti-PD-L1 antibody may be an effective anti-myeloma drug via antibody-dependent cellular cytotoxicity effects.