Abstract
Antigen Used for Immunization
Recombinant human CXCL4 (rhCXCL4) was used as an immunogen.
Method of Immunization
Sprague-Dawley (SD) rats (female, 8–10 weeks old) were immunized at five sites with 200 μg recombinant human CXCL4 (rhCXCL4) in Freund's complete adjuvant at the ratio (1:1) and reimmunized using the same protocol but with the antigen in Freund's incomplete adjuvant once a week for three times. Testing bleed was performed until serum became positive to the antigen in enzyme-linked immunosorbent assays (ELISAs) against rhCXCL4.
Parental Cell Line Used for Fusion
Mouse myeloma cell line (SP2/0) was used for fusion with rat splenocytes.
Selection and Cloning Procedure
The fusion cells were cultured with HAT-1640 medium containing 20% FBS to select hybrid clones. Several hybridoma clones were isolated and established with ELISA against both human and mouse recombinant CXCL4. The positive clones were subcloned at least three times using the limiting dilution method. Furthermore, we excluded the His-tag provoked immunogenicity by rescreening the clones, which were not recognizing recombinant mouse CXCL14 protein (rmCXCL14) with His-tag. rmCXCL4 also shares 39% amino acid identity with rmCXCL14, which provided additional high specificity to the positive clones. We calculated the ratio of the absorbance of samples and the negative control (P/N), and chose the P/N value of 2 for cutoff baseline.
Heavy and Light Chains of Immunoglobulin
50 kDa heavy chain and 25 kDa light chain, identified by SDS-PAGE gel.
Specificity
16D6-3 was sequenced and characterized by Western blot and BIAcore assays. With a typical characteristic of rat CDRs, variable fragments containing four frame regions (FRs), three CDRs, and two cysteine residues were identified. 16D6-3 recognized both human and mouse CXCL4 with high affinity and neutralized the effect of rhCXCL4 in ACHN cells.
Specific Antigen Identified
Both recombinant human and mouse CXCL4 were recognized.