Background of Recombinant Anti-Fluorescein Monoclonal Antibody (Clone: 4-4-20 (enhanced))
Our Recombinant Anti-Fluorescein Antibody (Clone 4-4-20 enhanced) is a sequence-defined, mammalian-expressed anti-FITC monoclonal antibody that specifically recognizes the fluorescein core structure attached to proteins, peptides, nucleic acids, or solid matrices. As a highly stable and reliable anti-fluorescein hapten antibody, this recombinant molecule serves as an essential second-step detection reagent for identifying fluorescein-labeled targets and amplifying signals across various standard in vitro immunoassays, completely avoiding the risk of hybridoma genetic drift.
The fluorescein/anti-fluorescein detection system offers a critical advantage over traditional biotin-streptavidin methods. Because biotin is naturally present in a large variety of mammalian tissues and cell lysates, biotin-based assays frequently suffer from high background noise and false-positive results. In contrast, fluorescein is completely absent in biological samples. Utilizing our recombinant anti-FITC monoclonal antibody entirely eliminates endogenous background interference. This makes our anti-fluorescein hapten antibody the ideal choice for achieving ultra-clean, accurate data in complex in vitro diagnostics, ELISA, and high-throughput screening platforms.
We offer comprehensive custom engineering and scale-up manufacturing specifically for the Clone 4-4-20 enhanced framework to support diagnostic kit developers and academic labs. Available configurations include:
- Isotype Switching & Fc Engineering: Various heavy/light chain isotypes and engineered Fc variants (such as effector-silent LALAPG mutants).
- Affinity Tags: Recombinant addition of Avi-tag, His-tag, or DYKDDDDK-tag for specific assay orientations.
- Conjugations: Biotinylated formats, as well as direct conjugations with a wide variety of fluorophores, dyes, and enzymes (HRP/AP).
Please contact our scientific support team at
message@sydlabs.com to request a quote for bulk orders or customized anti-fluorescein antibody formats.
PA007702.m1: Recombinant Anti-fluorescein Monoclonal Antibody (Clone: 4-4-20 (Enhanced)), Mouse IgG1 Kappa
Recombinant mouse IgG1 Monoclonal Antibody.
Clone: 4-4-20 (enhanced).
Isotype: Mouse IgG1 kappa.
Source: The recombinant anti-Fluorescein monoclonal antibody (clone: 4-4-20 (enhanced)) was produced in mammalian cells.
Specificity/Sensitivity: The recombinant anti-Fluorescein monoclonal antibody (clone: 4-4-20 (enhanced)) specifically binds to Fluorescein.
Applications: Western Blot, Flow Cytometry.
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-fluorescein monoclonal antibody of clone 4-4-20 (enhanced) 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.
References for Anti-Fluorescein Monoclonal Antibody
[1]
High-affinity rat anti-fluorescein monoclonal antibody with unique fine specificity properties including differential recognition of dynamic ligand analogues.
Miklasz, S. D., et al.
J Mol Recognit. 1995 Jul-Aug;8(4):258-69.
PMID: 8588943
The binding properties of a monoclonal rat anti-fluorescein antibody specific to a given isomer were quantitatively studied to determine the capacity to bind dynamic analogues of fluorescein which exists in multiple isomers. To generate monoclonal anti-fluorescein antibodies that reacted with specific dynamic analogues of fluorescein possessing unconjugated aromatic ring systems, immune splenocytes from Lou/M rats immunized with FITC(I)-KLH were fused with Balb/c SP2/0-Ag14 murine myeloma cells forming rat-mouse hybridomas. Thus, antibody reactivity of an anti-fluorescein antibody with phenol red and phenolphthalein was dictated more by ligand dynamics and aromatic orientation than by chemical structure similarities. P2A12-1-C8 exhibited significant reactivity with HPF and phenol red, which are dynamic structural analogues of the homologous fluorescein ligand. No reactivity was demonstrated with phenolphthalein, which based on relative chemical structures was expected to be more reactive than phenol red.
Tags: Anti-Fluorescein specificity; Fluorescein isomer recognition; Anti-Fluorescein rat hybridoma
[2]
Antibody networks and imaging: elicitation of anti-fluorescein antibodies in response to the metatypic state of fluorescein-specific monoclonal antibodies.
Cedergren, A. M., et al.
Mol Immunol. 1996 May-Jun;33(7-8):711-23.
PMID: 8760283
Affinity-labeled liganded murine monoclonal anti-fluorescein antibodies served as immunogens administered both in the syngeneic and xenogeneic modes to determine if the metatypic state elicited anti-hapten antibodies through imaging-like mechanisms. Polyclonal and monoclonal anti-Ab1 reagents in various hosts were assayed for anti-fluorescein and/or anti-metatype specificity. Significant anti-fluorescein responses were measured indicating that the metatypic state directly or indirectly stimulates an anti-hapten antibody population. Studies are described regarding generation of anti-hapten antibodies starting with a monoclonal Ig immunogen in the ligand-induced conformation or metatypic state. Liganded monoclonal Ab1 antibodies represent the unique feature of the study since previous reports investigating internal imaging in the original Idiotype Network Hypothesis [Jerne, 1974 (Ann. Immun. 125C, 373-389)] were based on the non-liganded or idiotypic state.
Tags: Anti-Fluorescein metatypic state; Ligand-induced conformation; Anti-hapten antibody elicitation
[3]
Pressure-induced dissociation of fluorescein from the anti-fluorescein single-chain antibody 4-4-20.
Coelho-Sampaio, T., et al.
Biochemistry. 1993 Oct 19;32(41):10929-35.
PMID: 8218158
Hydrostatic pressure was used to dissociate fluorescein (Fl) from the high-affinity anti-Fl single-chain antibody 4-4-20 (SCA 4-4-20). This difference is explained in terms of a higher overall flexibility of unliganded SCA and of a less stable binding site in SCA relative to mAb. Fl dissociation was monitored by measuring (1) the shift in the Fl absorption peak, (2) the recovery in Fl fluorescence intensity, which is quenched upon SCA binding, or (3) the decrease in Fl fluorescence polarization. Pressure effects were studied at two different Fl:SCA 4-4-20 molar ratios: 1:1, at which Fl fluorescence quenching was ca. The dependence of KD, and consequently of delta G of dissociation, on pressure permitted calculation of the magnitude of the standard volume change (delta V) involved in the dissociation process.
Tags: Anti-Fluorescein single-chain antibody; 4-4-20 SCA dissociation; Fluorescein fluorescence quenching
[4]
Preparation of Na,K-ATPase specifically modified on the anti-fluorescein antibody-inaccessible site by fluorescein 5'-isothiocyanate.
Lin, S. H., et al.
Anal Biochem. 2000 Dec 15;287(2):303-12.
PMID: 11112278
Removal of nonspecifically bound fluorescein was also confirmed by titration of the modified Na,K-ATPase with anti-fluorescein antibody and by time resolution of the fluorescence change when the modified enzyme was mixed with Na(+) in a stopped-flow instrument. The only fluorescence change when specifically modified Na,K-ATPase was mixed with Na(+) was the signal from fluorescein at the antibody-inaccessible, substrate-protectable site that reports the conformational change in unphosphorylated enzyme. The method should be generally applicable to the preparation of specifically labeled P-type pumps for use in kinetic and equilibrium titrations or energy transfer measurements. Specific labeling is required for energy transfer measurements and to avoid artifacts in the use of fluorophores as reporter groups. Sulfhydryl reagents protected against modification of cysteine residues, and treatment with dithiothreitol eliminated a slow doubling of the fluorescence of conventionally modified Na,K-ATPase upon dilution that is attributed to disappearance of self-energy transfer.
Tags: Na K-ATPase fluorescein modification; Antibody-inaccessible site; Fluorescein signal masking
[5]
Role of electrostatic interactions in the binding of fluorescein by anti-fluorescein antibody 4-4-20.
Omelyanenko, V. G., et al.
Biochemistry. 1993 Oct 5;32(39):10423-9.
PMID: 8399186
Anti-fluorescein antibodies are excellent model systems for studying the biochemical basis of molecular recognition because a prodigious amount of both physico-chemical and structural information is available for these antibodies. In previous studies, we determined the three-dimensional structure of an antigen-binding fragment of a high-affinity anti-fluorescein antibody (4-4-20) in complex with fluorescein. These studies showed that fluorescein binds tightly in an aromatic slot and participates in a network of electrostatic interactions. Furthermore, recombinant single-chain antibodies have been produced for several anti-fluorescein antibodies, and site-specific mutagenesis studies have defined the energetic contributions of a number of key active-site residues. The fluorescence lifetime of the 4-4-20/fluorescein complex was determined at both pH 8 and pH 10.6.
Tags: Anti-Fluorescein 4-4-20 structure; Electrostatic interactions in antibody binding; Antigen-binding fragment aromatic slot
For more technical references or data sheets regarding the Recombinant Anti-Fluorescein Monoclonal Antibody formats, please contact our scientific support team at
message@sydlabs.com.
Frequently Asked Questions (FAQ) about Anti-Fluorescein Antibody (Clone: 4-4-20 Enhanced)
Q: Is this anti-fluorescein antibody suitable for both free FITC and FITC-conjugated proteins??
Yes. This high-affinity anti-fluorescein hapten antibody (Clone 4-4-20 enhanced) is specifically raised against the fluorescein ring system. It demonstrates excellent cross-reactivity and tightly binds to both free fluorescein molecules and various FITC-conjugated proteins, antibodies, or dextran targets in solution.
Q: Why should I choose the recombinant 4-4-20 enhanced antibody over traditional hybridoma clones?
Our recombinant anti-FITC monoclonal antibody is sequence-verified and expressed in mammalian cells. Unlike traditional hybridomas that suffer from cell line drift or contaminating antibody light chains, this enhanced version guarantees 100% batch-to-batch reproducibility, ultra-pure target binding, and consistent signal backgrounds for routine in vitro screening.
Q: What are the primary validated applications for the anti-fluorescein monoclonal antibody?
This enhanced 4-4-20 recombinant antibody is fully validated for standard laboratory applications including Western Blotting (WB), Flow Cytometry (FC), Immunoprecipitation (IP), and competitive or sandwich ELISA assays. Its structured binding pocket ensures reliable primary or secondary detection of fluorescein-labeled targets.
Q: Why choose the recombinant version of Clone 4-4-20 over traditional hybridoma-derived antibodies?
Traditional hybridoma cell lines are prone to genetic drift, antibody expression loss, or contamination with unwanted light chains. Our recombinant Clone 4-4-20 is sequence-defined and expressed in mammalian cells, which guarantees 100% batch-to-batch consistency, ultra-high purity (>95% via SDS-PAGE), and a completely animal-free, reproducible manufacturing process.
Q: Are bulk quantities and custom configurations available for this anti-fluorescein antibody?
Yes. Syd Labs specializes in high-throughput scale-up production. We offer bulk quantities of the recombinant anti-fluorescein 4-4-20 antibody for assay kit manufacturing or high-screening diagnostic platforms. Custom services including specific isotype switching, Fc silencing (LALAPG variants), and biotin or enzyme conjugations are 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)