Abstract
Antigen Used for Immunization
Recombinant human PR3 proteins (rhuPR3) expressed in Escherichia coli were used as antigens. Human PR3 cDNA (Genbank accession no. NM 002777) was cloned from the THP-1 cell line using RT-PCR. The PCR product of mature human PR3 sequence was digested with EcoRI and XbaI and then inserted into the pPROEx HTa plasmid (Invitrogen Life Technologies, Carlsbad, CA) to generate huPR3/pPROEx HTa. The constructed huPR3/pPROEx HTa plasmid was transformed into DH5α competent cells for protein expression.
Recombinant huPR3 proteins expressed in E. coli DH5α cells were purified by using cobalt chelate affinity chromatography. The N-terminus six histidines were removed by TEV protease. The protein was then further purified by Superdex 75 HR 10/30 gel filtration (GE Healthcare Biosciences, Piscataway, NJ) using a FPLC system with buffer of 20 mM Tris-HCl (pH 8.0). The His6-tag removed rhuPR3 proteins with molecular weight of about 30 kDa and 20 kDa was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and identified by mass spectrometry.
Method of Immunization
Purified recombinant huPR3 proteins (rhuPR3) were used to immunize female BALB/c mice (6 weeks of age) by subcutaneous injections. The mixture of Freund's complete adjuvant (Sigma-Aldrich, St. Louis, MO) and rhuPR3 (30 μg/mouse) was used for the two immunizations with 2 week intervals. The mixture of Freund's incomplete adjuvant (Sigma-Aldrich) and rhuPR3 (30 μg/mouse) was used for the third immunization. Mice were bled 10 days after each boost. The titer of the mouse serum was determined by direct enzyme-linked immunosorbent assay (ELISA). The last boost of antigen was injected 3 days before a fusion.
Parental Cell Line Used for Fusion
BALB/c myeloma cell line FO was obtained from the ATCC and fused with splenocytes from immunized mice according to standard protocols.
Selection and Cloning Procedure
For screening of antibody producing hybridoma clones, recombinant huPR3 were used as antigens with direct ELISA. The hybridoma cells of positive wells with OD value (>3.00) were cloned by limiting dilution in aminopterin-free selection Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum. The single clones were screened by direct ELISA, dot blotting, and Western blotting for their specificity to rhuPR3 and neutrophil PR3. A single hybridoma clone was expanded for further purification. Anti-huPR3 MAb IgG was purified from hybridoma cell culture supernatants using protein G agarose (KPL, Gaithersburg, MD).
Heavy and Light Chains of Immunoglobulin
The immunoglobulin subclasses of all clones were identified as IgG1 subclass by direct ELISA using the subclass-specific secondary antibodies. Their light chains were identified as kappa chains by mouse immunoglobulin isotyping ELISA.
Specificity
By Western blots with recombinant huPR3 and human neutrophil proteinase 3
Method
Recombinant huPR3 and human neutrophil proteinase 3 (prepared from whole blood; Athens Research & Technology, Athens, GA) samples mixed with Laemmli sample buffer were boiled for 10 min and loaded on the 10% SDS-PAGE. Samples were electrotransferred onto a nitrocellulose membrane and blocked with 5% skim milk in PBS-T. Membrane was probed with protein G purified antibodies as primary antibodies, and the bound antibodies were detected by horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (Millipore, Temecula, CA) at 1:10,000 dilution followed by ECL substrate solutions (GE Healthcare).
Specificity
Clones 4, 27, 32, 36, and 38 sufficiently recognized both a few nanogram levels of recombinant huPR3 and human neutrophil proteinase 3 under reducing and non-reducing conditions whereas the MAb clone 19 detected only a few nanograms of recombinant PR3. The commercial PR3 (human neutrophil proteinase 3) is a monomeric form and is enzymatically active whereas rhuPR3 from E. coli is mostly a multimeric form with intra-disulfide bond.
By Western blots with intrinsic PR3 in human plasma and urine samples
Method
Whole blood samples from normal subjects were obtained in EDTA tubes. The whole blood was kept at 4°C and centrifuged within 3 h. The plasma was carefully aspirated with a Pasteur pipette and stored at −20°C until assayed. Individual human plasma (0.5 μL) was subjected in each lane under reducing conditions and transferred into the NC membrane. The membrane was probed with anti-huPR3 MAb clone 27 as primary antibodies.
Human urine sample was collected from normal subjects and centrifuged prior to use for Western blot. Individual human urine (25 μL) was subjected in each lane under reducing conditions and transferred into the NC membrane. The membrane was probed with the same MAb clone 27 as primary antibodies. The secondary antibody horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (Millipore, Temecula, CA) at 1:10,000 dilution was reacted with the NC membrane and then the Western blot membrane was analyzed by Bio-imaging using the LAS-4000 (Fujifilm Lifescience USA, Stamford, CT).
Specificity
MAb clone 27 could specifically and sufficiently detect endogenous PR3 in biological samples, such as human plasma and urine. There are variations in PR3 expression in human plasma and urine in individuals, and three distinct bands were detected in a very specific manner. The lowest molecular size band seems to be the mature form of circulating PR3 since its migration in 10% SDS-PAGE was similar to the mature form of human neutrophil proteinase 3. There were two high molecular bands detected in urine; however their molecular sizes slightly differ from the high molecular weight of plasma PR3. The two larger molecular sizes of PR3 is a complex form with other unknown molecules in human plasma and urine.
By immunofluorescent staining with native PR3 in human cell lines
Method
For indirect immunofluorescent staining, THP-1 cells grown on Permanox Labtek chambers (Nunc, Rochester, NY) were fixed with 100% acetone at RT for 20 min and then washed three times at RT. Cells were blocked with 3% BSA containing PBS for 1 h at RT and washed three times at RT. Then cells were incubated with mouse IgG1 control (eBioscience, San Diego, CA), biotinylated anti-rhuPR3 monoclonal IgG clone 32, and commercial mouse anti-PR3 monoclonal antibody (Biomeda, Foster City, CA). After extensive washing with PBS, cells were further incubated with secondary antibody (Streptavidin-FITC or anti-mouse-FITC, Pierce, Rockford, IL) at 1:1000 dilution in PBS at RT for 1 h and then incubated with DAPI (1:5000) in PBS at RT for 10 s. The samples were mounted with 5 μL medium, dried in the dark, and observed by fluorescence microscopy.
Specificity
We examined all six MAbs obtained from the hybridoma clones. The anti-hPR3 MAb clone 32 strongly reacted with endogenously synthesized PR3 proteins in THP-1 cells when used for immunofluorescent staining whereas the negative control mouse IgG showed no staining.
By flow cytometry analysis with cell surface-bound PR3 in human cell lines
Method
Two monocytic human cell lines (THP-1 and U937 cells) were used to analyze expression of PR3 on the cell surface. The membrane-bound PR3 proteins were detected by using flow cytometry with six different anti-huPR3 MAbs. Cells were incubated with anti-PR3 MAb (0.2 μg/mL) according to standard protocols. Fluorescence intensity profiles were obtained with various control conditions. A total of 10,000 cells were analyzed in each experiment.
Specificity
Endogenous PR3 exists in different locations of the cell. An enzymatically active mature form of PR3 usually resides in the secretory vesicle of neutrophils and monocytes whereas the immature form of PR3 exists on the cell surface of monocytes as a glycosyl-phosphatidylinositol-anchored membrane bound form.
We examined all six anti-PR3 MAbs to determine whether they recognize the membrane bound form on the cell surface PR3. The best result was obtained in FACS staining with MAb clone 38. Although MAb clones 4, 19, 27, 32, and 36 detected the membrane-bound form of PR3 in only THP-1 cells, its signal was very poor compared to MAb clone 38. Interestingly, MAb clone 19 detected a membrane-bound form of PR3 only from THP-1 cell although the signal was weaker than that of MAb clone 38.
Specific Antigen Identified
Not determined.