Abstract
Antigen Used for Immunization
The gene encoding human lipocalin 6 (hLCN6) was cloned from human epididymis. The antigen used for immunization was generated by delivering the gene coding mature hLCN6 protein into Escherichia coli host strain. The recombinant hLCN6 protein was expressed as a fusion protein containing an N terminal His tag mainly in inclusion body. After purification and refolding, the soluble concentrated fusion protein was used as antigen to immunize the animals.
Method of Immunization
Female BALB/c mice (7 weeks old) were immunized by subcutaneous (s.c.) injection with 100 μg of the purified hLCN6 protein in complete Freund's adjuvant. The subsequent three immunizations were carried out by s.c. injection at 2-week intervals with 100 μg of the same antigen mixed with the incomplete Freund's adjuvant. Three weeks later, a booster immunization was performed with an additional 100 μg of the same antigen in PBS by intraperitoneal injection.
Parental Cell Line Used for Fusion
Three days after the last injection, splenocytes from the mice were collected and fused with SP2/0 myeloma cells in the logarithmic growth phase at the ratio of 20:1.
Selection and Cloning Procedure
Positive hybridomas were identified by using indirect enzyme-linked immunosorbent assay, and cultured for a further 4 weeks, followed by subcloning by limiting dilution to obtain hybridoma cell lines stably secreting mAbs against human LCN6. The hybridoma clone with the highest ascite titer was expanded and the mAb was purified from the ascite by using protein A agrose resin.
Heavy and Light Chains of Immunoglobulin
The heavy chain (55 kDa) was IgG1 subtype and the light chain (22 kDa) was κ subtype.
Specificity
The specificity of anti-hLCN6 mAb was detected by Western blotting. Human epididymal tissue was fully triturated, extracted with Radio-Immunoprecipitation Assay (RIPA) lysis buffer containing phenylmethanesulfonyl fluoride for 30 min on ice, and centrifuged. The protein concentration in the supernatants was determined by using the bicinchoninic acid (BCA) Protein Assay kit. The protein samples (∼100 μg) were loaded and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and electrotransferred onto polyvinylidene fluoride membranes. After blocking at room temperature with 5% skim milk in phosphate-buffered saline with Tween-20 (PBST) for 2 h, the membranes were incubated with anti-hLCN6 mAb (1:10,000 dilution) at 4°C overnight. After washing, the membranes were incubated with horse-radish peroxidase-conjugated goat antimouse antibody secondary antibodies (1:2000 dilution) for 2 h at room temperature, and developed by using the enhanced chemiluminescent (ECL) reagents after extensive washing. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control.
Specific Antigen Identified
The antigen of human epididymal-specific lipocalin 6 protein was identified.