Monoclonal Antibody FPR92 Against Nuclear Mitotic Apparatus Protein

Abstract

Antigen Used for Immunization

Truncated recombinant nuclear mitotic apparatus protein (NuMA) was used for immunization. In this antigen the 300 amino acids of N terminus were cloned and expressed in Escherichia coli strain BL21.

Method of Immunization

BALB/c mouse was immunized with 20 μg of immunogen in complete Freund's adjuvant intraperitoneally, and booster injections were carried out with incomplete Freund's adjuvant biweekly—10 μg of immunogen for 3 months.

Parental Cell Line Used for Fusion

Sp2/0-Ag14 mouse myeloma cells.

Selection and Cloning Procedure

Hybridomas were cultured in HAT medium and subcloned thrice by limiting dilution.

Heavy and Light Chains of Immunoglobin

The heavy chain is IgG2a; the light chain is kappa.

Specificity

The cross-reactivity of the monoclonal antibody with NuMA, Ck19, and CD147 recombinant proteins, or Ck19, CD147, and EGFRvIII-related peptides, was determined using ELISA. The selected clone toward NuMA was reactive with NuMA recombinant protein and with EGFRvIII-related peptide but not with other mentioned recombinant proteins or their related synthetic peptides.

Specific Antigen Identified

Nuclear mitotic apparatus protein.

Availability

Tissue culture supernatant	Yes ✓	No
Ascitic fluid	Yes	No ✓
Hybridoma cells	Yes ✓	No

References

Harborth

, Weber

, and Osborn

: Epitope mapping and direct visualization of the parallel, in-register arrangement of the doublestranded coiled-coil in the NuMA protein. EMBO J, 1995; 14(11):2447–2460.