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Construction and preliminary screening of a human phage single-chain antibody library associated with gastric cancer

Jin He1, Ge Zhou, Kang-Da Liu

  • 1Department of Surgery, Fudan University, Shanghai 200032, People's Republic of China.

The Journal of Surgical Research
|February 7, 2002
PubMed
Abstract

Insights

Researchers created a human phage antibody library from gastric cancer patient lymph nodes to find CEA-binding single-chain variable fragments (scFv). This library is effective for generating tumor-antigen-specific antibody fragments for cancer research.

Area of Science:

  • Immunotechnology
  • Oncology
  • Molecular Biology

Background:

  • Gastric cancer research requires specific diagnostic and therapeutic tools.
  • Human single-chain variable fragments (scFv) offer potential for targeted therapies.
  • Phage display technology enables the selection of high-affinity antibodies.

Purpose of the Study:

  • To construct a human phage antibody library from gastric cancer patient samples.
  • To screen this library for scFv that bind to Carcinoembryonic antigen (CEA).
  • To validate the utility of the library for identifying tumor-antigen-specific antibodies.

Main Methods:

  • mRNA from metastatic lymph nodes/spleen of gastric cancer patients was used for RT-PCR to create a cDNA library.
  • Antibody variable regions were assembled into single-chain format and cloned into a phagemid.
  • Phage display screening involved panning against CEA and CEA-positive gastric cancer cells (MKN-28).
  • Specificity was confirmed using ELISA, immunofluorescence, and FACS analysis.

Main Results:

  • A library of 2.5 x 10^7 cfu/microg ampicillin-resistant clones was generated.
  • Sequencing revealed V(H) genes from V(H) III and V(L) genes from V(kappa) III subgroups.
  • Four rounds of panning increased binding phage titer 135-158 fold.
  • 20/95 clones bound CEA, and 47/95 bound fixed tumor cells, confirmed by immunofluorescence and FACS.

Conclusions:

  • A human phage antibody library was successfully constructed from gastric cancer patient lymph nodes.
  • The constructed library is a valuable tool for generating tumor-antigen-specific human antibody fragments.
  • This approach facilitates the discovery of novel diagnostic and therapeutic candidates for gastric cancer.

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