Sandwich ELISA Using a Mouse/Human Chimeric CSLEX-1 Antibody.

Area of Science:

• Biotechnology and Immunology
• Cancer Biomarker Development

Background:

• The mouse anti-sialyl Lewis X (sLeX) antibody CSLEX-1 is clinically used for breast cancer monitoring in Japan.
• The IgM isoform of CSLEX-1 has limitations in assay performance due to its bulkiness, hindering antigen accessibility.
• A novel mouse/human chimeric IgG antibody, CH-CSLEX-1, was developed to address these limitations.

Purpose of the Study:

• To develop and evaluate a mouse/human chimeric IgG antibody (CH-CSLEX-1) for improved sialyl Lewis X (sLeX) detection.
• To establish a homologous sandwich ELISA using CH-CSLEX-1 and assess its performance characteristics.
• To compare the specificity and reliability of the CH-CSLEX-1 assay against the conventional CSLEX-1 assay.

Main Methods:

• CH-CSLEX-1 was generated using a GAL4/UAS binary gene expression system in transgenic silkworms.
• Reactivity and specificity of CSLEX-1 and CH-CSLEX-1 were assessed using Biacore analysis against sLeX, sLeA, and Lewis Y (LeY) antigens.
• A homologous sandwich ELISA format was optimized using CH-CSLEX-1 for both capture and probing.

Main Results:

• CH-CSLEX-1 correctly formed as an IgG molecule, exhibiting high specificity for sLeX and not cross-reacting with sLeA or LeY.
• The homologous sandwich ELISA using CH-CSLEX-1 demonstrated excellent concordance (r = 0.98) with the conventional CSLEX-1 assay in human samples.
• The CH-CSLEX-1 assay showed no interference from human anti-mouse IgG (HAMA IgG) or HAMA IgM antibodies.

Conclusions:

• The development of the mouse/human chimeric antibody CH-CSLEX-1 enables a highly specific sandwich ELISA for sLeX detection.
• This novel assay overcomes the limitations associated with the IgM isoform of CSLEX-1, offering improved performance.
• The CH-CSLEX-1 assay is robust and unaffected by HAMA, making it suitable for clinical applications in breast cancer screening and monitoring.