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Related Experiment Videos

A specific and sensitive ELISA for laminin 5.

S Amano1, T Nishiyama, R E Burgeson

  • 1Skin Biology Research Laboratories, Shiseido Life Science Research Center, Yokohama, Japan. amano_satoshi@po.shiseido.co.jp

Journal of Immunological Methods
|June 5, 1999
PubMed
Summary

A new sandwich ELISA assay accurately detects laminin 5 (a key basement membrane protein) in human keratinocytes. This method quantizes laminin 5 synthesis, crucial for understanding skin development and disease.

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Area of Science:

  • Biochemistry
  • Cell Biology
  • Dermatology

Background:

  • Laminin 5 is a critical basement membrane protein involved in epithelial cell adhesion and tissue integrity.
  • Understanding laminin 5 synthesis and deposition is essential for studying skin development, wound healing, and diseases like squamous cell carcinoma.

Purpose of the Study:

  • To develop and validate a sensitive sandwich ELISA for quantifying human laminin 5.
  • To investigate the production and deposition of laminin 5 by human keratinocytes.
  • To determine factors influencing keratinocyte laminin 5 synthesis.

Main Methods:

  • Development of a sandwich ELISA using monoclonal antibodies BM165 and 6F12, specific for human laminin 5 alpha3 and beta3 chains.
  • Purification of laminin 5 from squamous carcinoma cell (SCC) 25-conditioned medium using 6F12-conjugated Sepharose for ELISA standardization.

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  • Quantification of both secreted and cell-associated laminin 5 from keratinocyte cultures.

    • Main Results:

    • The developed sandwich ELISA demonstrated high sensitivity, detecting as little as 125 pg of laminin 5.
    • The assay specifically detected laminin 5 produced by keratinocytes, distinguishing it from other laminins secreted by fibroblasts.
    • Laminin 5 was found to be both deposited around keratinocytes and released into the culture medium.
    • Keratinocyte laminin 5 synthesis was shown to be dependent on cell number, culture duration, and the underlying extracellular matrix.

    Conclusions:

    • A sensitive and specific sandwich ELISA for human laminin 5 has been successfully developed.
    • This assay enables accurate measurement of laminin 5 synthesis and deposition by keratinocytes.
    • The findings provide a valuable tool for research into laminin 5 function in normal and pathological conditions affecting the skin.