BH4 (Tetrahydrobiopterin) ELISA Kit

What it means: A Competitive ELISA uses a competitive inhibition format where unlabeled BH4 in your sample competes with HRP-labeled BH4 for antibody binding. Signal is inversely proportional to analyte concentration.

Why it matters:

• Well-suited for small molecules, haptens, and analytes that cannot support two-antibody sandwich formats.
• Particularly useful for hormones, lipid mediators, and small peptides.
• Requires careful sample dilution to stay within the competitive inhibition linear range.

How to interpret here: Read OD values from the standard curve in reverse — lower absorbance = higher BH4 concentration.

Tip: Dilute samples to fall in the mid-range of the curve for best accuracy.

What it means: This kit is validated for use with Serum as the primary sample matrix for BH4 quantification.

Why it matters:

• Sample matrix validation ensures the assay performs within its specified sensitivity and recovery range.
• Using non-validated matrices may introduce matrix interference, false-positive, or false-negative results.
• Sample preparation (dilution, pH adjustment) must be optimized for each matrix type.

How to interpret here: For this kit, Serum is the primary validated matrix. If using other matrices, perform a spike-and-recovery experiment to verify performance before generating reportable data.

What it means: The assay sensitivity is 18.75 pg/mL — defined as the minimum detectable concentration of BH4 distinguishable from zero (typically the mean OD of blank ± 2 SD mapped to the standard curve).

Why it matters:

• Determines whether low-abundance BH4 in your specific sample type can be reliably detected.
• Samples with BH4 below this limit should be concentrated or the assay should be optimized.
• Sensitivity may vary slightly between lots — always refer to the lot-specific certificate of analysis.

How to interpret here: A sensitivity of 18.75 pg/mL means this kit can reliably detect BH4 at 18.75 pg/mL or above. Plan sample dilutions to ensure values fall within the detection range.

Tip: Do not extrapolate below the lowest standard — report as <18.75 pg/mL instead.

What it means: The quantitative working range of this assay is 31.25-2000 pg/mL for BH4. Values outside this range cannot be accurately quantified.

Why it matters:

• Samples above the upper limit must be diluted before measurement; values below the lower limit are unreliable.
• Standard curves are validated within this range — interpolation is only valid within these bounds.
• Physiological concentrations of BH4 in your matrix should ideally fall in the mid-range for best precision.

How to interpret here: For BH4, the validated linear range is 31.25-2000 pg/mL. Prepare a two-fold dilution series of samples to bracket expected concentrations within this range.

What it means: This kit is validated for detecting BH4 from Universal (multi-species validated) biological samples. The capture and detection antibodies are raised and tested against Universal (multi-species validated)-derived BH4 protein.

Why it matters:

• Cross-species reactivity is not guaranteed unless explicitly validated — using an incorrect species can produce false-negative results due to epitope differences.
• Sequence identity between species determines the likelihood of cross-reactivity.
• For studies requiring multiple species, confirm cross-reactivity data in the kit datasheet.

How to interpret here: This kit is designed and validated for Universal (multi-species validated) samples. Contact technical support if you need to use samples from a different species not listed in the specification.

What it means: Total assay duration from sample addition to absorbance reading is approximately 2 h 30 min, including incubation, wash, substrate development, and stop steps.

Why it matters:

• Plan your experiment schedule to complete the assay in a single session — stopping and restarting mid-protocol can affect results.
• Hands-on time is typically much shorter than total assay time — most steps are passive incubation.
• Temperature control during incubation is critical for reproducibility; use a plate shaker if specified.

How to interpret here: Schedule approximately 2 h 30 min from sample plating to final read. Ensure all reagents are at room temperature before use.

What it means: This kit uses Colorimetric (TMB/HRP) detection — HRP enzyme conjugated to the detection antibody converts TMB substrate to a blue product (yellow after H₂SO₄ stop), measured at 450 nm on a standard microplate reader.

Why it matters:

• Colorimetric detection does not require specialized fluorescent readers — any standard 450 nm absorbance reader is compatible.
• TMB-HRP signal is stable for 30 minutes after the stop reaction — plan readings accordingly.
• Minimize light exposure after substrate addition to avoid photobleaching artifacts.

How to interpret here: Measure absorbance at 450 nm with a 570 nm or 630 nm reference wavelength to reduce background. Ensure the reader is calibrated and the plate is clean and free of condensation.