Abstract
Antigen Used for Immunization
C. parapsilosis was isolated from patients with systemic candidiasis(1) based on morphology, biochemical tests, and molecular techniques. C. parapsilosis was maintained in YPD medium (2% peptone, 2% D-glucose, and 1% yeast extract) at 37°C for 72 h. The fungi were heat-shocked at 42°C for 48 h, harvested, resuspended in phosphate-buffered saline (PBS), and lysed with Microsmash in the presence of 1.0 mm glass beads until ≥50% of the fungi were broken without addition of any enzyme. The concentrations of the lysates were determined and used for the generation of monoclonal and polyclonal antibodies and for the development of immunoassays.
Method of Immunization
Balb/c mice (6 – 8 weeks old) were immunized subcutaneously with prepared antigen at 2 weekly intervals. The mice were given the first immunization dose containing antigen (100 μL of 1 μg/μL antigen) emulsified in equal volume (100 μL) of complete Freund's adjuvant. All booster injections consisted of antigen (100 μL of 1 μg/μL) and 100 μL of incomplete Freund's adjuvant and were given at 2 weekly intervals. The mice were given the final injection of 100 μL of 1 μg/μL of antigen (without any adjuvant) intravenously via tail vein 3 days before the mice were sacrificed.
Parental Cell Line Used for Fusion
NS1 myeloma cell line.
Selection and Cloning Procedure
The cells were grown in RPMI supplemented with HAT medium and 10% fetal bovine serum. After 1 week, the HAT medium was replaced by HT medium for 2 weeks. Aliquots of culture supernatant with growing hybridomas were screened for the presence of antigen-specific antibodies by dot-blot method. Positive hybrids were immediately subcloned by limiting dilution and the isotypes were determined by a mouse monoclonal antibody isotyping kit.
Heavy and Light Chains of Immunoglobulin
Not available.
Specificity
The 1B11 monoclonal antibody was found to cross-react with C. tropicalis but not with C. rugosa, C. albicans, C. glabrata, Malassezia furfur, Klebsiella pneumoniae, Bacillus cereus, β-Streptococci, Pseudomonas aeruginosa, and Escherichia coli. The 3D1 monoclonal antibody reacted with heat-shocked C. parapsilosis antigen but not with other antigens tested.
Specific Antigen Identified
The 1B11 clone secreted IgG2a monoclonal antibody that was reactive with the C. parapsilosis antigen at MW of 59 kDa. The 3D1 clone secreted IgG1 monoclonal antibody that was reactive with C. parapsilosis antigen at MW of 30 kDa. Both antigens did not show any positive reaction with the glycoprotein detection kit.