FPGS Recombinant Monoclonal Antibody
Description
Definition and Mechanism of Action
The FPGS recombinant monoclonal antibody is a bioengineered antibody produced via recombinant DNA technology. Unlike traditional antibodies derived from hybridoma cells or polyclonal sources, it is generated by cloning the heavy- and light-chain genes of FPGS-specific B cells into expression vectors, followed by transfection into mammalian host cells for production . This process ensures batch-to-batch consistency, high specificity, and reproducibility.
The antibody binds specifically to the FPGS protein, which catalyzes the sequential addition of glutamate residues to folates. This modification traps folates intracellularly, enhancing their participation in DNA synthesis, methylation, and amino acid metabolism . In cancer, elevated FPGS expression correlates with improved retention of antifolates (e.g., pemetrexed), making it a biomarker for chemotherapeutic response .
Production Process
The synthesis of FPGS recombinant monoclonal antibodies involves:
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Immunization: Rabbits are immunized with FPGS-derived peptides or recombinant proteins .
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B Cell Isolation: Antigen-specific B cells or plasma cells are isolated from immune tissues or peripheral blood .
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PCR Amplification: Heavy- and light-chain variable regions are amplified via nested PCR for cloning .
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Vector Cloning: Genes are inserted into plasmid vectors containing constant regions (e.g., human IgG1, κ/λ) .
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Transfection: Host cells (e.g., CHO or HEK293) express the antibody via transient or stable transfection .
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Purification: Affinity chromatography (e.g., Protein A/G) isolates the antibody for functional testing .
Key Advantages:
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Eliminates reliance on hybridoma cell lines, reducing genetic drift .
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Enables rapid generation (e.g., <10 days using single-cell ASCs) .
Applications in Research and Diagnostics
The FPGS recombinant monoclonal antibody is employed in:
Tumor-Specific Expression
Studies using the NN3.2 clone (generated via recombinant fusion protein immunization) revealed:
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Normal Tissue Expression: Strong staining in liver hepatocytes, colon crypts, and lymphoid cells .
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Cancer-Specific Patterns: Elevated FPGS in ovarian and colon adenocarcinomas compared to adjacent benign tissue .
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Diagnostic Potential: Suggests FPGS as a prognostic marker for antifolate therapy response .
Functional Validation
The 30C11 antibody (Invitrogen) demonstrates:
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Specificity: Binds exclusively to FPGS in ELISA and Western blotting .
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Sensitivity: Detects low-abundance FPGS in formalin-fixed paraffin-embedded (FFPE) samples .
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Consistency: Lot-to-lot reproducibility confirmed via Western blot across four production batches .
Therapeutic and Diagnostic Implications
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Cancer Therapy Monitoring: FPGS expression predicts tumor sensitivity to antifolates like pemetrexed .
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Drug Development: Used to screen inhibitors targeting FPGS for reducing antifolate efficacy in resistant cancers .
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Spatial Proteomics: Recombinant antibodies enable precise localization of FPGS in tumor microenvironments .
Challenges and Future Directions
Product Specs
The FPGS recombinant monoclonal antibody is produced through in vitro expression systems. This antibody is developed by cloning the DNA sequences of FPGS antibodies from immunoreactive rabbits. The immunogen used in this process is a synthetic peptide derived from the human FPGS protein. Subsequently, the genes encoding the FPGS antibodies are inserted into plasmid vectors, and these recombinant plasmid vectors are transfected into host cells for antibody expression. The resulting FPGS recombinant monoclonal antibody undergoes affinity-chromatography purification and is rigorously tested for functionality in ELISA and FC applications. These tests confirm its reactivity with the human FPGS protein.
The primary function of the FPGS protein is to regulate and enhance the cellular uptake and utilization of folate by converting it into polyglutamate forms. This modification facilitates folate retention within cells, storage for future use, and optimal utilization in essential biochemical processes, including DNA synthesis, amino acid metabolism, and methylation reactions.
Target Background
- Aberrant folylpolyglutamate synthetase splicing has been linked to ex vivo methotrexate resistance and clinical outcomes in childhood acute lymphoblastic leukemia. PMID: 27036162
- Low expression levels of FPGS have been associated with neuroendocrine lung tumors. PMID: 27064343
- Research has indicated a significant difference in overall survival between patients with AA genotype and GA + GG genotype (P<0.05). This study demonstrated a correlation between the FPGS rs1544105 polymorphism and MTX treatment efficacy, as well as overall survival, suggesting its potential use in predicting MTX treatment efficacy. PMID: 27987364
- Elevated expressions of the tumor-related genes FPGS/GGH and VEGF have been correlated with malignancy in thymic carcinoma and B3 thymoma tumors. PMID: 27387303
- A study involving 529 adults (n = 325 European-Americans, 204 Egyptians) on a stable warfarin dose was conducted to genotype GGCX rs12714145 and rs10654848, FPGS rs7856096, and STX1B rs4889606. PMID: 26751406
- Data suggests that FPGS modulation influences global and promoter CpG DNA methylation and the expression of several genes involved in significant biological pathways. PMID: 26895662
- Genetic polymorphisms in the FPGS coding region have been found to significantly influence the outcome of RA patients receiving MTX monotherapy. PMID: 26652611
- Genetic polymorphisms in the FPGS coding region are significantly associated with an increased risk of Acute Lymphoblastic Leukemia. PMID: 26107232
- A study revealed that the genetic polymorphism of FPGS rs10760502A > G is associated with susceptibility to primary retroperitoneal liposasrcoma. PMID: 25765001
- Antifolate-resistant leukemia cells harbor a heterozygous point mutation in exon12 of FPGS. This mutation disrupts FPGS activity by abolishing ATP binding and alters the binding pattern of transcription factors to the genomic region of exon12. PMID: 25229333
- Genome-wide association studies have identified 10 novel genetic loci as risk factors for methotrexate response in rheumatoid arthritis patients, in addition to polymorphisms in DHFR, FPGS, and TYMS. PMID: 24583629
- Mitochondrial FPGS is essential because folate polyglutamates are not substrates for transport across the mitochondrial membrane. PMID: 25164808
- The polymorphism of FPGS rs1544105 G>A has been associated with response to therapy in acute lymphoblastic leukemia. PMID: 24908438
- An interaction term, between FPGS rs7033913 heterozygotes and GGH rs11988534 homozygotes for the minor allele, exhibited a p-value <0.0001 and may contribute to methotrexate toxicity in rheumatoid arthritis. PMID: 24447348
- High Folylpoly-glutamate synthetase expression is associated with response to therapy in non-small-cell lung cancer. PMID: 22895141
- A novel missense mutation 502/490 T>C (L151/101P) in exon 5 of the FPGS gene was first identified in Chinese Han children. The cytosolic and mitochondrial variants display allelic frequencies of 0.70 % and 0.47 %, respectively. PMID: 22809608
- The mbr significantly enhanced the expression of FPGS and effectively increased the sensitivity of Jurkat cells to MTX. PMID: 22698741
- This study was one of the first to evaluate FPGS and GGH genetic variants in relation to plasma homocysteine. PMID: 22018726
- The expression of mFPGS varied in A549 cells exposed to different methotrexate enantiomers. PMID: 21418912
- Intratumor FPGS plays a significant role in the efficacy of oral fluoropyrimidine plus LV therapy for colorectal cancer. PMID: 21380490
- The FPGS gene has been proposed as a novel causative gene for cleft lip. PMID: 21093159
- Data revealed that high FPGS gene expression, low GGH gene expression, and low ABCC1 gene expression in CRC tissues were predictive factors for high reduced folate levels after LV administration. PMID: 19636555
- Low folylpolyglutamate synthase expression is associated with the development of aggressive metastatic behavior in colorectal cancer. PMID: 14676127
- Significantly higher expression levels of FPGS were observed in mucosa expressing the splice variant DCC342. PMID: 16122883
- Human mitochondrial FPGS has been found to be strongly associated with the inner mitochondrial membrane. PMID: 16169100
- Research has investigated the molecular regulatory mechanisms governing FPGS gene expression in B precursor-ALL and T-ALL cells. PMID: 16707018
- A study identified FPGS polymorphisms; results indicate that two of the cytosolic nonsynonymous coding SNPs affected protein expression, in vitro substrate kinetics, and folate and antifolate efficacy in cells expressing the polymorphisms. PMID: 17875718
- Results demonstrate that hFPGS can catalyze the processive addition of approximately four glutamate residues to DDAH 4PteGlu 1. PMID: 18672898
- Aberrant splicing of folylpolyglutamate synthetase is associated with antifolate resistance in leukemia. PMID: 19131550
