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Recombinant human IgG1 isotype controls are available. Condition of sample preparation and optimal sample dilution should be determined experimentally by the investigator.
Price/availability/specifications subject to change without notice. Unless otherwise indicated, our catalog and customized products are for research use only and not intended for human or animal diagnostic or therapeutic use.
Phone: 1-617-401-8149
Fax: 1-617-606-5019
Email: message@sydlabs.com
Or leave a message with a formal purchase
order (PO) Or credit card.
Our Ultra-high Affinity Anti-FITC Antibody (Clone 4M5.3) is a sequence-defined, mammalian-expressed recombinant molecule engineered through directed evolution. Boasting an unprecedented equilibrium dissociation constant ($K_d$) of 270 fM, this femtomolar anti-FITC monoclonal antibody binds to fluorescein isothiocyanate (FITC) with near-irreversible tightness. This premium anti-FITC antibody clone 4M5.3 specifically recognizes FITC attached to proteins, peptides, nucleic acids, or solid matrices, making it the ultimate tool for advanced immunoassay signal amplification where conventional wild-type antibodies fail due to target dissociation.
The FITC/anti-FITC detection system provides a critical advantage over traditional biotin-streptavidin methods, particularly in single-molecule imaging and next-generation digital ELISAs. Endogenous biotin naturally present in mammalian tissues and cell lysates frequently causes high background noise, leading to false positives or reduced sensitivity in biotin-based assays. Because FITC is entirely absent in natural biological samples, utilizing our high-sensitivity recombinant anti-FITC antibody completely eliminates endogenous background interference. Combined with its 270 fM binding strength, this system ensures an exceptional signal-to-noise ratio for the most demanding ultra-sensitive analytical platforms.
Recombinant human IgG1 monoclonal antibody.
Clone: 4M5.3.
Isotype: Human IgG1 Kappa.
Source: The recombinant anti-FITC antibody (clone name: 4M5.3) was produced in mammalian cells.
Specificity/Sensitivity: The recombinant anti-FITC monoclonal antibody (clone: 4M5.3) specifically binds to FITC.
Applications: ELISA, Western Blot (WB), Flow Cytometry (FC).
Form of Antibody: 0.2 μM filtered solution of 1x PBS.
Purity: >95% by SDS-PAGE under reducing conditions and HPLC.
Shipping: The recombinant anti-FITC antibody of clone 4M5.3 is shipped with ice pack. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
12 months from date of receipt, -20 to -70°C as supplied.
1 month from date of receipt, 2 to 8°C as supplied.
The femtomolar-affinity mutant antibody (4M5.3) generated by directed evolution is interesting because of the potential of antibody engineering. In this study, the mutant and its wild type (4-4-20) were compared in terms of antigen-antibody interactions and structural flexibility to elucidate the effects of directed evolution. The pulling forces of SMD simulations elucidated the regions that form strong attractive interactions in the binding pocket. These landscapes were regarded as funnel-like binding free energy landscapes. It was shown that the fluctuations in the positions of the antigen and residues in contact with antigen tend to be smaller in the mutant than in the wild type.
Two anti-fluorescein single-chain antibodies, 4-4-20 and 4M5.3, were expressed as inclusion bodies in E. coli for use in a comparative refolding study. Although all three methods produced active protein for both 4-4-20 and 4M5.3, the extent of aggregation differed greatly among the methods. Genes for the anti-fluorescein single-chain antibodies 4-4-20 and 4M5.3 in pRS316 [17] were subcloned into pET-21a (Novagen) using the NheI and XhoI restriction sites. A significant decrease in the fluorescent intensity of a fluorescein solution is observed upon the binding of fluorescein to the active site of 4-4-20 and 4M5.3. The anti-fluorescein single-chain antibodies 4-4-20 and 4M5.3 were each expressed as inclusion bodies in E. coli as described in the Materials and methods section.
While standard wild-type clones (like 4-4-20) possess nanomolar affinity, our anti-FITC antibody clone 4M5.3 was generated via yeast display and directed evolution. It contains specific somatic mutations that form a tightly optimized aromatic slot around the fluorescein molecule. This results in a femtomolar anti-FITC monoclonal antibody with a $K_d$ of 270 fM, meaning it binds its target hundreds of times more tightly and practically never "lets go" during stringent washing steps.
Clone 4M5.3 is specifically recommended when your target biomarker abundance is extremely low (femtomolar or picomolar range) or when performing single-molecule detection. Its near-zero off-rate ensures that captured signals remain intact throughout complex multi-step protocols, providing unparalleled high sensitivity recombinant anti-FITC performance and powerful signal amplification.
Yes. This recombinant anti-FITC monoclonal antibody is fully validated for ELISA, Western Blot, and Flow Cytometry. It is highly effective as a second-step detection reagent for recovering or amplifying weak signals from FITC-conjugated primary antibodies, nucleic acids, or peptides.
Our recombinant anti-FITC antibody is expressed in mammalian cells (not E. coli inclusion bodies), ensuring proper folding, post-translational modifications, and low aggregation. The final product achieves >95% purity verified by both SDS-PAGE and HPLC, providing superior batch-to-batch consistency for reproducible research.
Yes. We provide extensive custom engineering services for the anti-FITC antibody mutants. You can request various heavy/light chain isotypes, engineered Fc regions (such as D265A or LALAPG), and specific affinity tags including Avi-tag, His-tag, or DYKDDDDK-tag. Biotinylated anti-FITC antibody options are also available upon request.
Syd Labs provides the following recombinant anti-Biotin/Digoxin/DNP/Fluorescein monoclonal antibodies:
Recombinant Anti-Biotin Monoclonal Antibodies (Clone F1)
Recombinant Anti-Biotin Monoclonal Antibodies (Clone M33)
Recombinant Anti-Digoxin Monoclonal Antibodies (Clone 5A7)
Recombinant Anti-Digoxin Monoclonal Antibodies (Clone 26-10)
Recombinant Anti-DNP Monoclonal Antibodies (Clone DNP.m3)
Recombinant Anti-Fluorescein Monoclonal Antibodies (Clone 4-4-20)
Recombinant Anti-Fluorescein Monoclonal Antibodies (Clone 4M5.3)
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