FGF19 Antibody, Biotin conjugated

What is FGF19 and why is it significant in biological research?

FGF19 (Fibroblast Growth Factor-19) is a secreted heparin-binding protein that belongs to the endocrine subfamily of fibroblast growth factors, which also includes FGF-21 and FGF-23. Unlike classic FGFs, FGF19 functions primarily as an endocrine hormone, circulating in the bloodstream to act on distant target tissues. It is produced by epithelial cells of the ileal intestine in response to bile acids and signals through a receptor complex of FGF R4 and Klotho-beta predominantly in the liver . FGF19 plays crucial roles in regulating bile acid synthesis, enhancing hepatic protein synthesis, and modulating glycogen metabolism in an insulin-independent manner . Given its involvement in metabolic regulation and association with diseases including hypercholesterolemia, gallstones, bile acid-induced diarrhea, hepatocellular carcinoma (HCC), colon cancer, and metabolic syndrome, FGF19 represents an important research target for both basic and translational studies .

How do biotin-conjugated FGF19 antibodies enhance detection sensitivity compared to unconjugated antibodies?

Biotin-conjugated FGF19 antibodies leverage the strong avidin-biotin interaction (one of the strongest non-covalent biological interactions known) to significantly amplify detection signals in various immunoassays. The conjugation process typically involves attaching biotin molecules to specific lysine residues or N-terminal amino groups on the antibody without interfering with its antigen-binding capacity. When using biotin-conjugated antibodies, researchers can employ secondary detection systems with avidin, streptavidin, or neutravidin conjugated to enzymes (like HRP or AP), fluorophores, or gold particles, creating a detection system with enhanced sensitivity. This biotin-streptavidin system provides greater signal amplification than direct conjugation methods because multiple biotin molecules can be attached to each antibody molecule, and each streptavidin molecule can bind four biotin molecules, creating a cascading effect that increases detection sensitivity by orders of magnitude . For FGF19 detection in samples with low abundance, such as clinical specimens or complex tissue samples, this enhanced sensitivity is particularly valuable for accurate quantification and reliable results.

What are the molecular characteristics of FGF19 relevant to antibody design and selection?

FGF19 is a 24 kDa protein comprising 216 amino acids, with specific structural domains that influence antibody targeting strategies. The protein contains distinct N-terminal and C-terminal regions that serve different functions in its biological activity . The N-terminus of FGF19 appears to be particularly important for its tumorigenic activities in hepatocellular carcinoma, while other regions may be more involved in its physiological bile acid regulatory functions . When selecting or designing antibodies against FGF19, researchers must consider these domain-specific functions. For instance, antibodies targeting the N-terminus, such as the G1A8 and HS29 antibodies described in research literature, have shown promise in selectively inhibiting FGF19's tumorigenic activity while preserving its beneficial bile acid regulatory function . Understanding these structure-function relationships is critical when selecting biotin-conjugated FGF19 antibodies for specific research applications, as epitope specificity may significantly impact experimental outcomes and interpretations.

How does FGF19 signaling work at the molecular level?

FGF19 mediates its biological effects through a complex signaling cascade that begins with binding to its specific receptor complex. Unlike paracrine FGFs that require heparan sulfate as a cofactor, FGF19 binds preferentially to fibroblast growth factor receptor 4 (FGFR4) in conjunction with the co-receptor β-Klotho, which provides signaling specificity . This interaction triggers receptor dimerization and autophosphorylation, activating multiple downstream signaling pathways including the ERK1/2 MAPK pathway, the PI3K/Akt pathway, and the STAT3 pathway. In the liver, FGF19 signaling leads to suppression of CYP7A1, the rate-limiting enzyme in bile acid synthesis, through a mechanism involving the SHP (small heterodimer partner) nuclear receptor . FGF19 also influences hepatic metabolism by enhancing protein and glycogen synthesis through pathways distinct from insulin signaling. Dysregulation of these pathways has been implicated in various pathological conditions, including the development of hepatocellular carcinoma, where FGF19 can drive cell proliferation and tumor growth through sustained FGFR4 activation . Understanding these signaling mechanisms is essential for designing antibody-based experiments to probe FGF19 function or modulate its activity.

What are the optimal sample preparation methods when using FGF19 biotin-conjugated antibodies?

Sample preparation is critical for successful detection of FGF19 using biotin-conjugated antibodies. For serum and plasma samples, proper collection and storage protocols must be followed to preserve protein integrity and prevent degradation of FGF19. Blood should be collected in appropriate tubes (EDTA for plasma, or serum separator tubes), allowed to clot for serum (30 minutes at room temperature), and then centrifuged at 1000-2000×g for 10 minutes . Samples should be aliquoted to avoid freeze-thaw cycles and stored at -80°C until analysis. For tissue samples, rapid freezing in liquid nitrogen followed by homogenization in a suitable lysis buffer containing protease inhibitors is recommended to preserve FGF19 antigenicity. Cell culture supernatants should be cleared by centrifugation and may require concentration if FGF19 levels are low. When preparing cell lysates, such as from HT-29 cells which express FGF19, use a complete lysis buffer with protease inhibitors and perform the lysis on ice to prevent protein degradation . Prior to immunoassays, all samples should be clarified by centrifugation (12,000×g for 10 minutes at 4°C) to remove particulates that might interfere with antibody binding or cause high background signals.

What ELISA configurations are most effective for FGF19 detection using biotin-conjugated antibodies?

Several ELISA configurations can be employed for FGF19 detection, with sandwich ELISA being the most widely used for its specificity and sensitivity. In a typical sandwich ELISA using biotin-conjugated antibodies, the microplate is first coated with a capture antibody specific to a different epitope of FGF19 than the biotin-conjugated detection antibody . After sample addition and incubation, the biotin-conjugated anti-FGF19 antibody is applied, followed by streptavidin-HRP to form the detection complex. This approach minimizes cross-reactivity and increases specificity compared to direct ELISA methods. Alternatively, researchers may use a capture antibody format where the biotin-conjugated FGF19 antibody serves as the capture antibody immobilized on streptavidin-coated plates, followed by a different labeled detector antibody. For multiplex detection, bead-based assays using distinct sets of colored beads coated with capture antibodies can be employed, with biotin-conjugated FGF19 antibodies serving as one of the detection antibodies in the panel. The colorimetric sandwich ELISA format, such as the OmniKine™ Human FGF-19 ELISA Kit, is particularly suitable for quantifying FGF19 concentrations in supernatants, sera, and plasma samples for research purposes .

How can researchers validate the specificity of FGF19 biotin-conjugated antibodies?

Validation of antibody specificity is a critical step before using biotin-conjugated FGF19 antibodies in research applications. A comprehensive validation approach should include multiple complementary methods. First, Western blotting should be performed using positive control samples known to express FGF19 (such as HT-29 cells) and negative controls, verifying that the antibody detects a protein of the expected molecular weight (approximately 24 kDa for FGF19) . Researchers should also test the antibody against recombinant FGF19 protein and against closely related family members (FGF21 and FGF23) to assess cross-reactivity. Knockdown or knockout validation experiments using siRNA or CRISPR techniques to reduce or eliminate FGF19 expression in positive control samples provide strong evidence of specificity when the signal is correspondingly reduced. Immunoprecipitation followed by mass spectrometry can confirm that the antibody is capturing the intended protein. For tissue or cell experiments, immunostaining patterns should match the known biological distribution of FGF19, with appropriate negative controls (including isotype controls and blocking peptide experiments). Finally, antibody specificity should be confirmed through binding kinetics analysis using techniques such as surface plasmon resonance to characterize the interaction with FGF19 and potential cross-reactants .

What are the recommended protocols for using FGF19 biotin-conjugated antibodies in Western blot analyses?

When using biotin-conjugated FGF19 antibodies for Western blot analyses, several protocol optimizations are essential for reliable results. Begin with proper sample preparation by lysing cells (such as HT-29) in a buffer containing protease inhibitors, followed by protein quantification to ensure equal loading . Proteins should be separated on 12-15% SDS-PAGE gels to optimally resolve the 24 kDa FGF19 protein, followed by transfer to a PVDF or nitrocellulose membrane. After transfer, block the membrane thoroughly with 5% non-fat dry milk or BSA in TBST to prevent non-specific binding, with added avidin (10-50 μg/mL) if endogenous biotin is a concern in your samples. Incubate the membrane with biotin-conjugated anti-FGF19 primary antibody at an optimized dilution (typically starting at 1:2000-1:10000) overnight at 4°C . After washing, detect using streptavidin-HRP or streptavidin conjugated to a fluorophore for chemiluminescent or fluorescent detection, respectively. Always include positive controls (recombinant FGF19 or HT-29 cell lysate) and molecular weight markers. For multiplex detection, use streptavidin conjugated to a fluorophore with a distinct emission spectrum from your other detection antibodies. When optimizing, test different blocking agents, antibody concentrations, and incubation times to achieve the best signal-to-noise ratio while maintaining specificity for the 24 kDa FGF19 band.

How can FGF19 biotin-conjugated antibodies be used to investigate FGF19's role in hepatocellular carcinoma?

FGF19 biotin-conjugated antibodies offer powerful tools for investigating FGF19's role in hepatocellular carcinoma (HCC) through multiple experimental approaches. Researchers can use these antibodies in immunohistochemistry or immunofluorescence studies of HCC patient samples to quantify FGF19 expression levels and correlate them with clinical outcomes, tumor stage, and other molecular markers. In cell-based assays, biotin-conjugated antibodies enable the tracking of FGF19 secretion, cellular localization, and receptor interactions in HCC cell lines like Hep3B, providing insights into autocrine/paracrine signaling mechanisms . Co-immunoprecipitation experiments using biotin-conjugated anti-FGF19 antibodies can identify novel protein interaction partners specific to HCC cells versus normal hepatocytes, potentially revealing cancer-specific signaling networks. For functional studies, these antibodies can be used to neutralize FGF19 activity in cell proliferation assays, xenograft models, and patient-derived xenograft (PDX) models to assess the dependency of tumor growth on FGF19 signaling . Recent research has shown that antibodies specifically targeting the N-terminus of FGF19 (such as G1A8 and HS29) effectively inhibit FGF19-induced HCC cell proliferation and suppress tumor growth in mouse models without disrupting FGF19's normal bile acid regulatory function, highlighting the potential of selective antibody targeting approaches for therapeutic development .

What approaches can be used to study FGF19 interactions with its receptor complex using biotin-conjugated antibodies?

Studying FGF19 interactions with its receptor complex (FGFR4 and β-Klotho) requires sophisticated methodologies where biotin-conjugated antibodies offer significant advantages. Biotin-conjugated anti-FGF19 antibodies can be employed in pull-down assays to isolate intact receptor complexes from cell membranes, enabling the identification of additional complex components through proteomics analysis. In situ proximity ligation assays (PLA) using biotin-conjugated FGF19 antibodies in combination with antibodies against FGFR4 and β-Klotho can visualize and quantify receptor complex formation in fixed cells or tissues, providing spatial information about where these interactions occur within the cellular environment. Surface plasmon resonance (SPR) or bio-layer interferometry (BLI) techniques can utilize immobilized biotin-conjugated FGF19 antibodies to capture FGF19 in a defined orientation, allowing detailed kinetic analysis of its interactions with soluble receptor components . Researchers can also develop competitive binding assays where biotin-conjugated FGF19 antibodies are used to measure displacement by potential therapeutic compounds, as demonstrated in studies showing that G1A8 efficiently competed with FGFR4 for binding to FGF19 . For in vivo studies, biotin-conjugated antibodies can be used to track the biodistribution of FGF19 and evaluate how potential therapeutic antibodies might alter FGF19's interaction with its receptor complex in different tissues.

How can researchers differentiate between FGF19 and other FGF family members in experimental settings?

Differentiating between FGF19 and other closely related FGF family members, particularly FGF21 and FGF23 which share the endocrine subfamily classification, requires careful experimental design and antibody selection . Biotin-conjugated antibodies specifically raised against unique epitopes of FGF19 that are not conserved in FGF21 or FGF23 provide the foundation for selective detection. Researchers should verify antibody specificity through cross-reactivity testing against recombinant FGF21 and FGF23 proteins using Western blot or ELISA methods. For complex samples where multiple FGF family members may be present, sandwich ELISA configurations using a capture antibody against one epitope and a biotin-conjugated detection antibody against a different FGF19-specific epitope can provide the necessary specificity . Immunoprecipitation followed by mass spectrometry can confirm the identity of the captured protein based on unique peptide sequences. At the functional level, selective receptor binding assays can distinguish between these family members, as FGF19 preferentially signals through FGFR4/β-Klotho, while FGF21 primarily uses FGFR1c/β-Klotho and FGF23 utilizes FGFR1c/α-Klotho . Gene expression analysis following stimulation with recombinant FGFs can also help differentiate their activities, as FGF19 specifically suppresses CYP7A1 expression in hepatocytes, a response not triggered by FGF21 or FGF23 .

What considerations are important when designing multiplex assays incorporating FGF19 biotin-conjugated antibodies?

Designing multiplex assays that include biotin-conjugated FGF19 antibodies requires careful consideration of several technical factors to ensure reliable results. Antibody compatibility is paramount—researchers must select antibodies against different targets that do not cross-react or sterically hinder each other's binding when used in combination. The detection system must be carefully planned, particularly when incorporating biotin-conjugated antibodies, as endogenous biotin in biological samples can interfere with specific signal detection; pre-blocking with free avidin or using the Biotin Blocking System can minimize this interference. Signal separation strategies must be implemented, such as using streptavidin conjugated to spectrally distinct fluorophores when combining biotin-conjugated FGF19 antibodies with other biotin-conjugated antibodies in multiplex fluorescence assays. Dynamic range considerations are essential, as FGF19 concentrations may differ significantly from other analytes in the multiplex panel; careful optimization of antibody concentrations and detection parameters for each analyte is necessary to ensure all targets fall within the quantifiable range . Validation of the multiplex assay should include comparison with single-plex measurements for each analyte to confirm that multiplexing does not compromise sensitivity or specificity. Researchers should also account for potential biological interactions between analytes in the sample preparation phase, as certain treatments might affect multiple analytes differently.

What are common pitfalls when using biotin-conjugated antibodies for FGF19 detection, and how can they be addressed?

Several common pitfalls can complicate experiments using biotin-conjugated antibodies for FGF19 detection. Endogenous biotin interference is perhaps the most prevalent issue, particularly in samples derived from biotin-rich tissues such as liver, kidney, and brain. This interference can lead to false positive signals or elevated backgrounds. Researchers can address this by using commercial biotin blocking kits prior to adding biotin-conjugated antibodies or by pre-clearing samples with streptavidin-coated beads . Over-conjugation of biotin to antibodies is another potential problem that can reduce antibody affinity or cause steric hindrance at the antigen-binding site. Working with optimally conjugated antibodies (typically 3-8 biotin molecules per antibody) or performing titration experiments to determine the optimal working concentration helps mitigate this issue. Hook effects can occur in samples with very high FGF19 concentrations, where excess antigen paradoxically leads to decreased signal; performing sample dilutions in a series can identify and overcome this phenomenon. Non-specific binding, particularly in complex biological matrices, can be reduced by optimizing blocking conditions and including appropriate blocking agents such as normal serum from the same species as the secondary reagent. Signal dropout in multiplex assays may occur if FGF19 concentrations are substantially lower than other analytes; adjusting detector sensitivity or using amplification steps specifically for low-abundance analytes can help balance signal detection across all targets.

What controls are essential when using FGF19 biotin-conjugated antibodies in various experimental settings?

A robust experimental design using FGF19 biotin-conjugated antibodies requires comprehensive controls to ensure reliable data interpretation. For all applications, positive controls using samples with confirmed FGF19 expression (such as HT-29 cells or recombinant FGF19 protein) should be included to verify detection system functionality . Negative controls are equally important and should include samples known to lack FGF19 expression or samples where FGF19 has been knocked down/out using siRNA or CRISPR technologies. Isotype controls using biotin-conjugated antibodies of the same isotype but irrelevant specificity help distinguish between specific binding and Fc receptor-mediated or other non-specific interactions. In multiplexed assays, single-analyte controls should be run in parallel to confirm that detection of FGF19 is not affected by the presence of other targets or detection reagents. For biotin-specific concerns, include avidin/streptavidin-only controls (without biotin-conjugated primary antibody) to assess endogenous biotin levels and potential direct binding of detection reagents to the sample. Blocking peptide controls, where the biotin-conjugated antibody is pre-incubated with excess FGF19 protein to block specific binding sites, can confirm signal specificity. When performing quantitative analyses, standard curves using purified recombinant FGF19 at known concentrations are essential for accurate quantification. For functional studies evaluating antibody effects on FGF19 activity, include controls testing the antibody's effect on related FGF family members to confirm specificity of inhibition.

What methodological approaches can help resolve discrepancies in FGF19 detection across different experimental platforms?

Resolving discrepancies in FGF19 detection across different experimental platforms requires systematic troubleshooting and method harmonization. Begin by standardizing sample collection and processing protocols across all platforms to minimize pre-analytical variables; this includes standardized collection times (important for FGF19 due to its circadian and postprandial regulation), consistent anticoagulants for blood samples, and uniform processing delays . Perform cross-platform calibration using identical reference standards, ideally including recombinant FGF19 and well-characterized control samples that can be measured on all platforms. Epitope mapping of the antibodies used on different platforms can identify whether discrepancies arise from detection of different forms of FGF19 (e.g., fragment vs. full-length, or differentially post-translationally modified forms) . Interference testing should be conducted to identify platform-specific susceptibilities to common interferents such as heterophilic antibodies, autoantibodies, or high lipid content in samples. Matrix effect evaluation using spike-and-recovery experiments, where known amounts of recombinant FGF19 are added to real samples and measured across platforms, can reveal sample-dependent factors affecting detection. Consider developing a bridging study design where a subset of samples is analyzed on all platforms to develop conversion factors if systematic biases exist. Finally, implement a tiered approach to FGF19 measurement where screening is performed on one platform and confirmatory testing on another, with discrepant results resolved by a third orthogonal method.

Research Applications Table