Abstract
Antigen Used for Immunization
A recombinant Chaetomium globosum enolase that was produced in a bacterial expression system was used for immunizations.
Method of Immunization
Three 5- to 7-week-old female BALB/cJ mice were immunized via intraperitoneal (IP) injection with a 50:50 (v/v) emulsion of 25 μg of the purified recombinant C. globosum enolase protein and TiterMax® Gold Adjuvant. Five subsequent booster IP immunizations containing 5 μg recombinant C. globosum enolase in sterile PBS were administered at biweekly intervals. The final boost was administered 3 days before the monoclonal antibody fusion.
Parental Cell Line Used for Fusion
SP2/0-AG14 myeloma cells (ATCC# CRL-1581).
Selection and Cloning Procedure
Individual spleens were aseptically removed from each mouse and single cell suspensions of the splenocytes produced. Fusion of splenocytes with SP2/0-AG14 myeloma cells was performed as previously described,(1,2) and hybridoma cells were cultivated in Dulbecco's Modified Eagle Medium. The supernatant fluid from confluent hybridomas was diluted 1:2 (v/v) in PBS 0.05% Tween-20 containing 3% non-fat dry skim milk powder and tested using a recombinant C. globosum enolase indirect ELISA. Hybridomas that were confirmed to produce recombinant C. globosum enolase-specific MAb were selected, grown in bulk, and then transferred to a 96-well tissue culture plate and cloned twice by limiting dilution. Single positive clones were rescreened using the indirect ELISA, selected, grown in bulk, and purified according to the method of Kent.(3)
Heavy and Light Chains of Immunoglobulin
An MAb isotype ELISA was used to identify the isotype of the screened MAb. MAb 1C7 was identified to be IgG1 isotype.
Specificity
MAb 1C7 exhibited reactivity to immobilized recombinant C. globosum enolase in an indirect screening ELISA. The IgG1 isotype MAb was then further assessed in SDS-PAGE and Western blot studies. MAb 1C7 bound to immobilized recombinant C. globosum enolase in Western blotting. Additional Western blot studies were designed to evaluate the reactivity of MAb 1C7 to C. globosum protein extract fractions, including spore wash, homogenized spore, and mycelial extracts. The highest MAb 1C7 reactivity was observed to a ∼49 kDa band in the homogenized spore extract that corresponded to recombinant C. globosum enolase. In cross-reactivity studies, the greatest MAb 1C7 reactivity was observed to C. globosum UAMH strains 9683 and 841 as well as a putative enolase derived from C. atrobrunneum. Interestingly, MAb 1C7 did not react with enolase derived from C. indicum or Aspergillus fumigatus. Divergence with as few as two amino acids in the A. fumigatus enolase extract resulted in the loss of MAb reactivity. Based on these data, MAb 1C7 may additionally react with other closely related species within the fungal orders Sordariales and Hypocreales.
Specific Antigen Identified
Epitope mapping of MAb 1C7 demonstrated binding to the C. globosum enolase decapeptide, LTYEELANLY.