Prl Antibody, HRP conjugated

What is Prolactin antibody and what distinguishes HRP-conjugated versions from unconjugated variants?

Prolactin (PRL) antibodies are immunoglobulins that specifically recognize and bind to prolactin hormone or prolactin receptors. HRP (Horseradish Peroxidase) conjugation refers to the chemical linking of the HRP enzyme to the antibody molecule, creating a detection system that generates a measurable signal when exposed to appropriate substrates.

The primary advantage of HRP-conjugated PRL antibodies is direct detection capability without requiring secondary antibodies. For example, when used in Western blot applications, HRP-conjugated antibodies can directly catalyze chromogenic or chemiluminescent reactions upon addition of substrates, streamlining the detection process . Unconjugated PRL antibodies, like the Human Prolactin Antibody AF682, require a separate HRP-conjugated secondary antibody for visualization .

HRP-conjugated antibodies are particularly valuable in applications including:

• Western blotting

• ELISA

• Immunohistochemistry

• Dot blot assays

The conjugation typically does not interfere with antigen recognition when properly performed, but researchers should verify specificity through appropriate controls .

How should HRP-conjugated Prolactin antibodies be stored and handled to maintain optimal activity?

Proper storage of HRP-conjugated Prolactin antibodies is crucial for maintaining their functionality. According to product documentation, these antibodies should be stored at -20°C where they remain stable for approximately one year after shipment . Most commercial preparations are provided in storage buffers containing stabilizers such as:

To maintain antibody integrity, researchers should:

• Avoid repeated freeze-thaw cycles by preparing working aliquots

• Keep the antibody away from direct light exposure

• Maintain cold chain during handling

• Return unused portions to -20°C immediately after use

• Check for signs of precipitation or color change before use

What dilution ranges are typically effective for HRP-conjugated Prolactin antibodies in different applications?

Optimal dilution ranges for HRP-conjugated antibodies vary depending on the specific application and detection method employed. Based on available technical information, the following guidelines can be used as starting points:

These ranges should be considered initial guidelines, and as noted in product documentation: "Optimal dilutions should be determined by each laboratory for each application" . Factors affecting optimal dilution include:

• Target protein abundance

• Sample type and preparation method

• Detection system sensitivity

• Signal-to-noise requirements

• Incubation conditions and duration

How can researchers validate the specificity of Prolactin antibodies in their experimental systems?

Validating antibody specificity is critical for ensuring reliable experimental results. For Prolactin antibodies, several complementary approaches are recommended:

• Western blot validation: Human Prolactin Antibody AF682 has been validated in Western blot using human pituitary tissue lysate, where it detected a specific band at approximately 23 kDa under reducing conditions . Similar validation should be performed in your experimental system.

• Immunohistochemistry cross-validation: Comparing antibody staining patterns with known prolactin expression profiles in tissue sections. For instance, Prolactin was detected in human testis sections using the Human Prolactin Antibody, showing specific staining localized to the cytoplasm of sperm cells .

• Neutralization assays: Functional validation through neutralization studies, where antibody-antigen binding inhibits a measurable biological effect. The neutralization dose (ND₅₀) for Human Prolactin Antibody was found to be typically 0.02-0.05 μg/mL in the presence of 0.5 ng/mL Recombinant Human Prolactin .

• Negative controls: Testing the antibody on samples known not to express prolactin or in the presence of blocking peptides.

What are the key considerations when selecting between IgG isotypes for Prolactin antibodies in mechanistic studies?

The selection between different IgG isotypes (particularly IgG1 versus IgG4) has significant implications for research outcomes, especially in studies investigating Fc-mediated functions. Recent research has demonstrated that:

IgG1-based antibodies (like PL 200,031) demonstrate stronger binding to all FcγRs, particularly activating receptors FcγRIIA and FcγRIIIA, making them suitable for studies requiring robust effector functions .

In contrast, IgG4-based antibodies with S228P hinge stabilizing mutation (like PL 200,039) show significantly reduced binding to activating FcγRs while retaining binding to FcγRI, making them preferable when minimal effector function activation is desired .

Both formats can maintain comparable binding affinity to Prolactin. For example, the research showed humanized PRL antibodies in both IgG1 and IgG4 formats maintained sub-nanomolar affinity for human PRL (268 pM and 630 pM respectively) .

How do researchers optimize binding kinetics for HRP-conjugated Prolactin antibodies in time-sensitive assays?

When designing time-sensitive assays using HRP-conjugated Prolactin antibodies, understanding binding kinetics is crucial. Research on Prolactin neutralizing antibodies provides valuable insights into optimizing these parameters:

Recent studies characterized humanized Prolactin antibodies with the following kinetic properties:

For time-sensitive assays, researchers should consider:

• Association rate (kon): Higher kon values allow faster binding equilibrium, critical for short-incubation assays. Antibodies with kon values in the range of 10⁵-10⁶ M⁻¹·s⁻¹ (like those shown above) reach substantial binding within 30-60 minutes .

• Dissociation rate (koff): Lower koff values indicate greater binding stability over time, important for extended protocols with multiple wash steps. The exemplified antibodies show favorable koff values in the 10⁻⁴-10⁻⁵ s⁻¹ range .

• Antigen concentration: Higher antigen concentrations can partially compensate for lower affinity antibodies but may increase non-specific binding.

• Temperature effects: Kinetic parameters are temperature-sensitive; room temperature incubations typically enhance kon but may also increase koff.

For highest sensitivity in time-limited assays, antibodies with high kon and low koff (resulting in sub-nanomolar KD values) provide optimal performance .

How can researchers develop dual-function antibodies targeting both Prolactin Receptor and Growth Hormone Receptor?

The development of dual-function antibodies that target both Prolactin Receptor (PRLR) and Growth Hormone Receptor (GHR) represents an advanced research approach with potential applications in breast cancer research. According to recent literature, anti-idiotypic antibody strategy has proven effective for this purpose:

The methodology involves a multi-step process:

• Preparation of anti-GH antibodies (Ab1)

• Generation of monoclonal anti-idiotypic antibody to GH (Ab2)

• Screening for anti-idiotypic antibodies capable of binding both PRLR and GHR

• Identification of anti-idiotypic antibodies with antagonistic properties to both receptors (Ab2β)

This approach leverages Jerne's immune network theory, where the variable region of an antibody (Ab1) produced against an antigen can itself serve as an antigen to generate anti-antibodies (Ab2). The type Ab2β structurally mimics the initial antigen and is termed an "internal image" of the initial antigen .

Researchers have successfully used this approach to develop H53, a dual-function inhibitor that:

• Inhibits PRLR-mediated intracellular signaling

• Blocks GHR-mediated intracellular signaling in a dose-dependent manner

• Shows potential efficacy against breast tumors

This methodology is particularly valuable for studying cancers where both receptors play crucial roles, as the combined inhibition may produce superior anti-cancer effects compared to single-receptor targeting .

What analytical approaches best characterize the specificity of Prolactin neutralizing antibodies?

Characterizing the specificity of Prolactin neutralizing antibodies requires rigorous analytical approaches to ensure target selectivity and absence of cross-reactivity. Based on published research, a comprehensive characterization workflow should include:

• Direct binding assays via biolayer interferometry:
  Recent studies used this technique to determine binding kinetics and affinities of humanized antibodies for human PRL. This approach revealed strong, sub-nanomolar affinity for human PRL while confirming lack of significant binding to related hormones like human growth hormone (hGH) and human placental lactogen (hPL) .

• Cross-reactivity testing:
  Systematically evaluating antibody binding against related hormones and species orthologs. For example, PL 200,039 maintained strong affinity for non-human primate PRL but showed negligible binding to rat PRL, human growth hormone, and human placental lactogen .

• Functional inhibition assays:
  These measure the antibody's ability to neutralize PRL-mediated biological effects. Published data shows IC₅₀ values for different antibody formats:

  ID	Class	IC₅₀ (nM) for hPRL	IC₅₀ for hGH	IC₅₀ for hPL	IC₅₀ for nhpPRL	IC₅₀ for rPRL
  PL 200,019	Murine IgG1	2.7	>150	>2,600	3.2	>150
  PL 200,031	Humanized IgG1	2.8	>150	>2,600	3.0	>150
  PL 200,039	Humanized IgG4	3.6	>150	>2,600	5.3	>150

  These values demonstrate the antibodies' high selectivity for human and non-human primate PRL with minimal cross-reactivity to related hormones .

• Receptor binding competition assays:
  These evaluate the antibody's ability to prevent PRL from binding to its receptor, confirming the specific mechanism of neutralization .

• Epitope mapping:
  Determining the specific binding regions helps predict potential cross-reactivity and confirm the mechanism of action .

This systematic characterization approach ensures that neutralizing antibodies are both specific to PRL and functional in blocking its biological activity.

What are the key technical considerations when using HRP-conjugated antibodies for detecting low-abundance Prolactin in complex samples?

Detecting low-abundance Prolactin in complex biological samples presents significant technical challenges that require careful optimization of HRP-conjugated antibody detection systems:

• Signal amplification strategies:
  For Western blot applications, choosing the appropriate detection system is crucial. ECL (enhanced chemiluminescence) substrates provide significantly higher sensitivity compared to chromogenic substrates, requiring different dilution ranges of HRP-conjugated antibodies (1:2,000-1:10,000 for ECL versus 1:1,000-1:20,000 for chromogenic substrates) .

• Alternative detection platforms:
  Beyond traditional Western blotting, consider more sensitive platforms like Simple Western™, which has been validated for Prolactin detection. Research demonstrates that this platform detected Prolactin in human pituitary lysates at approximately 30 kDa using 2.5 μg/mL of Anti-Human Prolactin Antibody under reducing conditions .

• Sample enrichment techniques:
  When working with low-abundance targets, implementing immunoprecipitation before detection can concentrate the target protein and reduce background interference.

• Blocking optimization:
  For complex samples, blocking conditions should be carefully optimized. Most protocols use BSA (3 mg/ml is common in storage buffers) , but alternative blockers like milk proteins or commercial blockers may provide better background reduction depending on the sample type.

• Antibody validation in relevant matrices:
  Validation in matrices similar to experimental samples is essential. For example, Human Prolactin Antibody has been specifically validated in human pituitary tissue lysates and testis sections, demonstrating specific detection patterns .

• Pretreatment protocols:
  Sample preparation techniques like heat-induced epitope retrieval for tissue sections or deglycosylation for heavily glycosylated samples can significantly improve detection sensitivity.

• Consideration of physical state:
  The physical state of the antibody preparation (typically liquid, but sometimes lyophilized) can affect performance in low-abundance detection scenarios, with freshly reconstituted lyophilized antibodies sometimes offering improved sensitivity.

How can researchers evaluate the potential interference of HRP-conjugated Prolactin antibodies with cellular signaling in live-cell applications?

When using HRP-conjugated Prolactin antibodies in experiments involving live cells or signaling pathways, researchers must carefully assess potential interference with normal cellular processes:

• Neutralization potency assessment:
  Quantify the antibody's ability to inhibit Prolactin-mediated signaling using standardized assays. For example, the Neutralization Dose (ND₅₀) for Human Prolactin Antibody in the Nb2-11 rat lymphoma cell proliferation assay was found to be typically 0.0200-0.200 μg/mL . This measurement provides a baseline for understanding how the antibody might affect signaling pathways in experimental systems.

• Receptor binding competition analysis:
  Determine whether the antibody competes with natural ligands for receptor binding sites, which could directly impact downstream signaling. Recent research on Prolactin neutralizing antibodies demonstrated their ability to block Prolactin-Receptor interactions with nanomolar IC₅₀ values (2.7-3.6 nM range) .

• Pathway-specific signaling reporters:
  Employ reporter systems (e.g., luciferase-based) to specifically monitor the impact of antibodies on relevant signaling pathways like JAK/STAT, which is commonly activated downstream of Prolactin Receptor.

• Dual-function antibody considerations:
  When working with antibodies designed to affect multiple receptors, such as the GHR/PRLR dual-function antagonist H53, expect broader impacts on cellular signaling. These antibodies have been shown to inhibit both PRLR-mediated and GHR-mediated intracellular signaling in a dose-dependent manner , which may be advantageous or confounding depending on the research question.

• Control testing with non-HRP conjugated variants:
  Compare the effects of HRP-conjugated versus unconjugated versions of the same antibody clone to distinguish between effects caused by the antibody's binding specificity versus those potentially introduced by the HRP moiety.

• Cell viability monitoring:
  Prolonged exposure to HRP can potentially generate reactive oxygen species in live-cell applications, which may independently affect signaling pathways. Monitor cell viability and oxidative stress markers when using HRP-conjugated antibodies in extended live-cell protocols.

• IgG subclass selection:
  The choice between IgG1 and IgG4 formats can significantly impact cellular interactions through differential Fc receptor engagement. Research has shown that IgG4-based antibodies (like PL 200,039) demonstrate significantly reduced binding to activating FcγRs compared to IgG1 formats (like PL 200,031) , making IgG4 potentially preferable for applications where minimal cellular activation through Fc receptors is desired.