A high-sensitivity enzyme immunoassay for the quantification of soluble human semaphorin 4D in plasma

Area of Science:

• Biochemistry
• Immunology
• Clinical Chemistry

Background:

• Human semaphorin 4D (SEMA4D) is a glycoprotein involved in cellular functions, with its soluble form (sSEMA4D) circulating in blood.
• Previous studies on sSEMA4D levels used assays lacking thorough characterization and often employed serum, potentially leading to inaccurate results.
• Understanding sSEMA4D levels is crucial, but reliable quantification methods are needed.

Purpose of the Study:

• To develop and validate a robust sandwich ELISA for quantifying human soluble semaphorin 4D (sSEMA4D) in plasma.
• To assess the influence of protease activity on sSEMA4D levels in serum and plasma.
• To establish plasma as the recommended matrix for sSEMA4D quantification.

Main Methods:

• Developed a sandwich ELISA using two monoclonal antibodies targeting conformational epitopes in the sema domain of SEMA4D.
• Validated the assay for specificity, precision, accuracy, linearity, and robustness using human plasma (EDTA, citrate, heparin).
• Analyzed serum and plasma samples, including those collected with and without the protease inhibitor TAPI-1.

Main Results:

• The validated ELISA demonstrated a calibration range of 62.5–2000 fmol/L with a quantification limit of 31 fmol/L.
• Significantly higher sSEMA4D levels were observed in serum compared to plasma.
• Serum and plasma sSEMA4D levels showed no statistical difference when samples were collected in the presence of TAPI-1, indicating protease activity in serum.

Conclusions:

• The developed ELISA is a reliable tool for quantifying sSEMA4D in human plasma.
• Plasma is the recommended matrix for sSEMA4D analysis due to protease-induced artifactual increases in serum.
• Protease inhibition during sample collection is critical for accurate sSEMA4D measurements.