Monoclonal antibodies (mAbs) are an effective tool in therapeutics and diagnostics. A novel appro... more Monoclonal antibodies (mAbs) are an effective tool in therapeutics and diagnostics. A novel approach called the single-cellRT-PCR-linked in vitro expression system (SICREX) enables the high-hroughput generation and screening of mAbs from single B cells. In this paper, instead of using Bcells, cDNAs were synthesized from single plasma cells of animmunized mouse spleen.The light chain (Lc) and the Fd portion of the heavy chain (Hc) genes of each cell were amplified separately and followed by overlapping PCR to add a T7 promoter, a ribosome-binding site, and a T7 terminator. The paired Lc and Hc genes were simultaneously expressed by an Escherichia coli in vitro transcription and translation system followed by ELISA to measure their affinity for the antigen. A Fab fragment with affinity against the antigen was obtained from plasma cells of an immunized mouse with hepatitis B surface antigen (HBsAg).
pfmdr1 and its variants are molecular marker which are responsible for antibiotics resistance in ... more pfmdr1 and its variants are molecular marker which are responsible for antibiotics resistance in Plasmodium falciparum, a parasitic carrier for malaria dis-ease. A novel strategy to treat malaria disease is by disrupting parasite lactate dehydrogenase (pLDH), a crucial enzyme for Plasmodium survival during their erythrocytic stages. This research was aimed to investigate and characterize the pfmdr1 and pldh genes of P. falciparum isolated from Nusa Tenggara Indonesia. Genomic DNA of P.falciparum was isolated from malaria patients in Nusa Tenggara Indonesia. pfmdr1 was amplified using nested PCR and genotyped using Restriction Fragment Length Polymorphism (RFLP). pldh was amplified, sequenced, and analyzed using NCBI public domain databases and alignment using Clustal W ver. 1.83. Genotyping of the pfmdr1 revealed that sequence diversity was extremely high among isolates. However, a sequence analysis of pldh indicated that open reading frame of 316 amino acids of the gene showing 100% homology to the P. falciparum 3D7 reference pldh (GeneBank: XM_001349953.1). This is the first report which confirms the heterologous of pfmdr1 and the homologous sequences of P.falciparum pldh isolated from Nusa Tenggara Islands of Indonesia, indicating that the chloroquine could not be used effectively as antimalarial target in the region and the pLDH-targeted antimalarial compound would have higher chance to be successful than using chloroquine for curbing malaria worldwide.
The single-step PCR amplification of IgG Light chain (Lc) and Heavy chain (Hc) (Fd portion) from ... more The single-step PCR amplification of IgG Light chain (Lc) and Heavy chain (Hc) (Fd portion) from the cDNAs of a single cell was facilitated using a low concentration of cDNA-specific primerswith 5′ homotags in the presence of a homotag-specific primer. This method was found to be successful in generating a functional antibody with an antigen-binding activity and useful for the high-throughput generation or screening of monoclonal antibodies.
Expression of a functional antibody fragment (Fab) using an Escherichia coli cell-free expres-sio... more Expression of a functional antibody fragment (Fab) using an Escherichia coli cell-free expres-sion system has been reported previously [Jiang et al., FEBS Lett., 514, 290–294 (2002)]. The low yield of the synthesized antibody, however, limits the usefulness of the cell-free expression system, partly due to the degradation of product by endogenous proteases from the E. coli extract. To determine which proteases are responsible for the degradation, we compared the expression of a 6D9 Fab fragment under conditions whereby several protease inhibitors were added into the cell-free system. The addition of serine protease inhibitor increased the amount of the Fab fragment, indicating that serine proteases caused the antibody degradation. Therefore, several serine pro-tease-deficient mutants of E. coli BW25113 were constructed by targeted homologous recombination. The use of extract from a double protease-deficient mutant (degP-ompT) significantly increased the amount and antigen-binding activities of an anti-HSA scFv and a 6D9 Fab fragment.These results suggest that the DegP- and OmpT-deleted mutant is a useful source of S30 extractf or the production or screening of antibodies using the cell-free expression system.
Monoclonal antibodies (mAbs) are an effective tool in therapeutics and diagnostics. A novel appro... more Monoclonal antibodies (mAbs) are an effective tool in therapeutics and diagnostics. A novel approach called the single-cellRT-PCR-linked in vitro expression system (SICREX) enables the high-hroughput generation and screening of mAbs from single B cells. In this paper, instead of using Bcells, cDNAs were synthesized from single plasma cells of animmunized mouse spleen.The light chain (Lc) and the Fd portion of the heavy chain (Hc) genes of each cell were amplified separately and followed by overlapping PCR to add a T7 promoter, a ribosome-binding site, and a T7 terminator. The paired Lc and Hc genes were simultaneously expressed by an Escherichia coli in vitro transcription and translation system followed by ELISA to measure their affinity for the antigen. A Fab fragment with affinity against the antigen was obtained from plasma cells of an immunized mouse with hepatitis B surface antigen (HBsAg).
pfmdr1 and its variants are molecular marker which are responsible for antibiotics resistance in ... more pfmdr1 and its variants are molecular marker which are responsible for antibiotics resistance in Plasmodium falciparum, a parasitic carrier for malaria dis-ease. A novel strategy to treat malaria disease is by disrupting parasite lactate dehydrogenase (pLDH), a crucial enzyme for Plasmodium survival during their erythrocytic stages. This research was aimed to investigate and characterize the pfmdr1 and pldh genes of P. falciparum isolated from Nusa Tenggara Indonesia. Genomic DNA of P.falciparum was isolated from malaria patients in Nusa Tenggara Indonesia. pfmdr1 was amplified using nested PCR and genotyped using Restriction Fragment Length Polymorphism (RFLP). pldh was amplified, sequenced, and analyzed using NCBI public domain databases and alignment using Clustal W ver. 1.83. Genotyping of the pfmdr1 revealed that sequence diversity was extremely high among isolates. However, a sequence analysis of pldh indicated that open reading frame of 316 amino acids of the gene showing 100% homology to the P. falciparum 3D7 reference pldh (GeneBank: XM_001349953.1). This is the first report which confirms the heterologous of pfmdr1 and the homologous sequences of P.falciparum pldh isolated from Nusa Tenggara Islands of Indonesia, indicating that the chloroquine could not be used effectively as antimalarial target in the region and the pLDH-targeted antimalarial compound would have higher chance to be successful than using chloroquine for curbing malaria worldwide.
The single-step PCR amplification of IgG Light chain (Lc) and Heavy chain (Hc) (Fd portion) from ... more The single-step PCR amplification of IgG Light chain (Lc) and Heavy chain (Hc) (Fd portion) from the cDNAs of a single cell was facilitated using a low concentration of cDNA-specific primerswith 5′ homotags in the presence of a homotag-specific primer. This method was found to be successful in generating a functional antibody with an antigen-binding activity and useful for the high-throughput generation or screening of monoclonal antibodies.
Expression of a functional antibody fragment (Fab) using an Escherichia coli cell-free expres-sio... more Expression of a functional antibody fragment (Fab) using an Escherichia coli cell-free expres-sion system has been reported previously [Jiang et al., FEBS Lett., 514, 290–294 (2002)]. The low yield of the synthesized antibody, however, limits the usefulness of the cell-free expression system, partly due to the degradation of product by endogenous proteases from the E. coli extract. To determine which proteases are responsible for the degradation, we compared the expression of a 6D9 Fab fragment under conditions whereby several protease inhibitors were added into the cell-free system. The addition of serine protease inhibitor increased the amount of the Fab fragment, indicating that serine proteases caused the antibody degradation. Therefore, several serine pro-tease-deficient mutants of E. coli BW25113 were constructed by targeted homologous recombination. The use of extract from a double protease-deficient mutant (degP-ompT) significantly increased the amount and antigen-binding activities of an anti-HSA scFv and a 6D9 Fab fragment.These results suggest that the DegP- and OmpT-deleted mutant is a useful source of S30 extractf or the production or screening of antibodies using the cell-free expression system.
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