LIMM0882-01391532-4311Immunological Investigations, Vol. 39, No. 2, Dec 2009: pp. 0–0Immunological InvestigationsProduction and Characterization of Monoclonal Antibodies against the Extracellular Domain of CA 125Production of anti-CA 125 Monoclonal AntibodyS. Shojaeian et al.
Sorour Shojaeian,1 Abdolamir Allameh,1 Amir Hassan Zarnani,2,3 Mahmood Chamankhah,4 Roya Ghods,5 Ali Ahmad Bayat,5 and Mahmood Jeddi-Tehrani5
1Department of Biochemistry, Faculty of Medical Sciences, Tarbiat ModaresUniversity, Tehran, Iran2Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR,Tehran, Iran3Immunology Research Center, Faculty of Medicine, Iran University of MedicalSciences, Tehran, Iran4Nanobiotechnology Research Center, Avicenna Research Institute, ACECR, Tehran,Iran5Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran,Iran
Carcinoma antigen 125 (CA 125) is overexpressed in ovarian cancer and antibodiesagainst it are widely employed for diagnostic purposes. The rarity of CA 125 antigenicdomains and its highly glycosylated structure, however, is a problem that may preventimmunized mice from developing a diversified population of anti-CA 125 antibodies. Inthis study a prime-boost strategy, which potentially could augment the humoralimmune responses against rare and poorly immunogenic determinants, was used forimmunization of mice and monoclonal antibodies (mAbs) were produced by hybridomatechnology. Reactivity of mAbs was then assessed by ELISA, western blotting, immuno-precipitation, immunohistochemistry and immunofluorescence staining of OVCAR-3cell line. Altogether, 10 clones were produced, 3 of which had IgG isotype and the restwere IgM. Two-third of clones recognized cognate antigen in fixed and living cells andhad strong immunoreactivity in IHC staining. In Western blotting, our antibodies rec-ognized CA 125 as high molecular weight antigen mostly migrated in the 3% stackinggel. Immunoprecipitation of OVCAR-3 cell lysate by mAbs resulted in a very similar
Address correspondence to Mahmood Jeddi-Tehrani, Monoclonal Antibody ResearchCenter, Avicenna Research Institute, ACECR, P.O. Box 19615-1177, Tehran, Iran;E-mail: [email protected]
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Production of anti-CA 125 Monoclonal Antibody 115
migration pattern that reconfirmed their specificities. The mAbs produced in thisstudy are invaluable tools in diagnosis and research fields for assessment of CA 125expression in cancerous ovarian tissues.
Ovarian cancer is a relatively asymptomatic entity until later stages (Reynoldset al., 2006). Carcinoma antigen 125 (CA 125) is the most widely used markerfor diagnosis of ovarian cancer which is often considered as the gold standard(Bast et al., 1983; Bon et al., 1996; Hogdall, 2008). Serum levels of CA 125 areused to monitor responses to chemotherapy, relapse and disease progressionin ovarian cancer patients (Dorigo et al., 2007). CA 125 was first identified byBast, Knapp and colleagues in 1981 by a monoclonal antibody (OC 125) thathad been developed from mice immunized with an ovarian cancer cell line(Bast et al., 1981).
Nagata et al. (1991) suggested that CA 125 is a membrane-bound glyco-protein complex with a glycophosphatidylinositol anchor. This mucin-like proteinhave high molecular mass, estimated from 200 to 2000 KD, although smallersubunits have been reported (Davis et al., 1986; Matsuoka et al., 1987; de losFrailes et al., 1993; Kobayashi et al., 1993; O’Brien et al., 1998). CA 125 ispoorly immunogenic and the rarity of CA 125 antigenic domains may be due toits unusual structure which consists of more than 60 repeat units of 156 aminoacids (O’Brien et al., 2001; Yin et al., 2002), hyperglycosylated state and to itsimmunosuppressive effects (Patankar et al., 2005) that may prevent immunizedmice from developing a diversified population of anti-CA 125 antibodies.
Since 1981, numerous mAbs detecting CA 125 have been developed. Aninternational workshop divided the antibodies into three major epitopegroups: OC 125-like antibodies, M 11-like antibodies and OV 197-like antibodies(Nustad et al., 1996). This report describes production and characterization ofanti-CA 125 monoclonal antibodies against extracellular domain of antigenand discusses their potential application for assessment of CA 125 expressionin cancerous ovarian tissues.
MATERIALS AND METHODS
Generation and Purification of Monoclonal AntibodiesAll work with animals has been approved by the ethical committee of the
Avicenna Research Institute. Two protocols were used for NMRI mouse(Pasteur Institute, Tehran, Iran) immunization. In the first protocol, micewere immunized three times by intraperitoneal injection of 100 μg purified CA
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125 (Biodesign, USA) on the first day and 50 μg purified CA 125 on days 21and 45. After completion of immunization schedule, mouse sera were testedfor anti-CA 125 antibody by indirect enzyme-linked immunosorbent assay(ELISA) (see below) and four mice with the highest titers were selected forfusion.
In the second protocol, mice were immunized three times by intraperitonealinjection of 5 × 106 OVCAR-3 living cells. After completion of immunizationschedule, ELISA assay was performed as described later and two mice with thehighest titers were selected for fusion. Booster immunization was performed byintravenus injection of 66 μg purified CA 125, 4 and 3 days before fusion. Hybri-domas were produced by the standard method (Stähli et al., 1980).
Culture supernatants of hybridoma clones were screened by ELISA againstpurified CA 125 and the positive clones were subcloned by limiting dilution.Subcloning was performed four times to ensure monoclonality and stability ofthe clones. To be used for immunocytochemical stainings, final clones werescreened for reactivity with CA 125 positive human ovarian cancer cell line,OVCAR-3, and the CA 125 negative fibroblast cell line, HFFF-PI6, served asnegative control. Accordingly, three clones were selected for purification.
mAbs, produced by the selected clones, were isotyped by isostrip (Roche,Germany) and the corresponding clones were grown in RPMI 1640 mediumcontaining 10% fetal bovine serum (FBS) and the culture supernatants werecollected. Given the IgM isotype of selected clones, a rabbit anti-mouse IgMaffinity chromatography column was set up for IgM antibody purification.Purified antibodies were quantified by spectrophotometry at 280 nm. Purity ofantibodies was tested by sodium dodecylsulfate-polyacrylamide gel electro-phoresis (SDS-PAGE). The antibodies further characterized by dot blotting,ELISA, immunofluorescent staining of living and fixed OVCAR-3 cells, westernblotting and immunoprecipitation.
Dot BlottingOVCAR-3 cell lysate was prepared in radio immunoprecipitation assay
buffer (RIPA buffer). Briefly, 1.5×107 cells were incubated in 1 ml RIPA buffer(150 mM NaCl, 1% Triton X-100, 0.1% SDS, 50 mM Tris-HCl PH 7.5) in thepresence of 1% protease inhibitor cocktail (Sigma, USA) at 4 °C for 15 min.Two fold serial dilutions of the cell lysate were dot blotted on nitrocellulosemembrane each spot containing 5 μl of corresponding dilution. The membranewas blocked with 5% non-fat skim milk in Tris-buffered saline containing0.1% Tween-20 and 0.4% Triton X-100 (TBS-TT).
The membrane was then incubated with 10 μg/ml purified monoclonalantibodies for 1 h at room temperature, washed 3 times with TBS-TT, fol-lowed by incubation with horse raddish peroxidase (HRP) conjugated-sheepanti-mouse immunoglobulin (Ig) (Avicenna Research Institute, Tehran, Iran)
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Production of anti-CA 125 Monoclonal Antibody 117
(1:1000) for 1 h at room temperature. The membrane was washed 3 times andthe detection was performed by chemiluminescence using enhanced chemilu-minescence (ECL) (GE-Healthcare, Sweden) system.
ELISAThe same ELISA protocol was used for measurement of anti-CA 125
antibody titers in mouse sera, hybridoma culture supernatants and purifiedantibodies. Ninety-six well plates (Nunc, Denmark) were coated with optimalconcentration of CA 125 (3 μg/ml) in PBS overnight at 4°C. After washing withPBS containing 0.05% Tween-20 (PBS-T), plates were blocked with 2.5% BSAfor 1 h at 37°C.
As primary antibody, two-fold serial dilutions (from 1:500) of the mousesera, hybridoma culture supernatants or two fold dilution series (from 40 μg/ml)of the purified antibodies were used and incubated for 1.5 h at 37°C.After washing with PBS-T, HRP-conjugated sheep anti-mouse (Ig) (1:1000)(Avicenna Research Institute) was added to each well and incubated for 1 h at37°C. Finally, color was developed after adding the substrate, 3,3′,5,5′ tetra-methylbenzidine (TMB) (US Biological, USA) for 15 min. The reaction wasstopped by 20% H2SO4 and the absorbance was measured at 450 nm.
The negative controls included omission of first layer (antigen), secondlayer (anti-CA 125 antibodies) or combination of both aforesaid controls. Theresult of such tests was always negative showing that there is no non-specificbinding of our anti-CA 125 antibodies.
Indirect Immunlofluorescent StainingFor staining living OVCAR-3 cells, 1 × 106 cells were incubated with 100
μl of 5 μg/ml purified antibodies for 30 min on ice, washed twice with PBS andstained with fluorescent isothiocyanate (FITC)-conjugated sheep anti-mouseIg (1:50) (Avicenna Research Institute) for 30 min on ice. After two washes withPBS, stained cells were examined under fluorescent microscope (Olympus, BX51, Japan). Human fibroblastic cell line, HFFF-PI6, served as negative cellcontrol. For negative reagent control, primary antibody was substituted bynormal mouse Ig (5 μg/ml). In some settings, cytospinned OVCAR-3 cells werefixed by ice-cold acetone for 2 min on glass slides and stained as above.
ImmunohistochemistryFor further characterization of our monoclonal antibodies, immunohis-
tochemical analysis was performed on formalin-fixed paraffin-embeded(FFPE) ovarian cancer tissue samples and normal ovarian tissue as negativetissue control according the protocol we published elsewhere (Jeddi-Tehrani et al.,2009). Briefly, 3 μm sections were prepared and subjected to heat-activated
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antigen retrieval in citrate buffer (10mM, pH 6) at 98 °C for 30 min. Followingthree washes with Tris-buffered saline, pH 7.4, containing 0.1% bovine serumalbumin (TBS-BSA), endogenous peroxidase activity were quenched by theaddition of 0.3% H2O2 in TBS for 15 min.
Slides were then washed three times with TBS-BSA and endogenousbiotin was blocked with biotin blocking system (Dako, Sweden) according tomanufacturer’s instruction. Sections were then washed three times withTBS-BSA and incubated for 15 min with 5% normal rabbit serum. At the nextstep, slides were tilted and incubated for 90 min with anti-CA 125 mAb at aconcentration of 2.5 μg/ml. After washing with TBS-BSA, biotin-labeled rabbitanti-mouse antibody (Avicenna Research Institute, Iran) at a concentration of2.5 μg/ml were added for 45 min, the slides were then washed three times withTBS-BSA and incubated for 30 min with 1: 500 dilution of HRP-conjugatedstreptavidin (Biosource, USA). Finally, after three washes, the substrate/chromogen, 3, 3′ diaminobenzidin (Roche, Germany), was added on each slidefor 15 min. The slides were then washed with deionized water, dehydratedwith increasing graded ethanol, counterstained with Harris hematoxylin, andmounted with Enthelan (Merck, Germany). In negative reagent control slides,primary antibody was omitted. In positive control slides, OC 125 was used asprimary antibody.
Western BlottingThe same Western blot analysis protocol was used for OVCAR-3 cell lysate
and purified CA 125 (US Biological, USA). OVCAR-3 cell lysate was preparedas described above in RIPA buffer. Sample (30 μl/well of cell lysate or 2.5 μg/well of purified CA 125) was resolved by SDS-PAGE in 7% gel under non-reducing condition by the Leammli method (Laemmli, 1970). Proteins weretransferred to PVDF (polyvinylidine difluoride) membrane using the methoddescribed by Towbin et al. (1979). After blocking with 5% non-fat skim milkin TBS-TT, the membrane was incubated with 10 μg/ml purified antibodies for1 h at room temperature followed by three washes with TBS-TT and thenincubated with HRP-conjugated sheep anti-mouse Ig (1:1000) for 1 h at roomtemperature. After three washes, detection was performed by chemillumines-cence using ECL (GE-Healthcare, Sweden) system according to manufacturer’sinstruction.
ImmunoprecipitationFor immunoprecipitation study, purified antibodies (10A6 and 7A3 mAbs)
were incubated with Protein L-Agarose beads (Sigma, USA) for 2 h at 4°C,separately and then washed two times with RIPA buffer. OVCAR-3 cell lysateprepared as before was added to Protein-L antibody complex and incubatedfor 2 h at 4°C under continuous mixing. The immunoprecipitates were washed
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Production of anti-CA 125 Monoclonal Antibody 119
four times with RIPA buffer. After adding of sample buffer containing2-mercaptoethanol, the immunoprecipitates were boiled for 10 min. Thesamples were resolved by SDS-PAGE in 7% gel and then transblotted toPVDF membrane; after then corresponding monoclonal antibodies or OC 125were used as primary antibodies for detection of specific bands.
Antibody Competitive ELISAAntibody competitive ELISA was performed by labeled streptavidin-
biotin (LSAB) method. To this end, anti-CA 125 mAbs were biotinylated byN-hydroxysuccinimidobiotin (NHS-Biotin) linker according to a standardprotocol and used in competitive ELISA as follows: Ninety-six well plateswere coated with optimal concentration of CA 125 (3 μg/ml) in PBS over-night at 4 °C. After washing with PBS-T, plates were blocked with 3% BSA inPBS-T for 1.5 h at 37 °C.
As primary antibody, optimized concentration (2.5 μg/ml) of commercialmouse anti-CA 125 mAb (Invitrogen, USA, Clone: OC 125, Isotype: IgG1) inPBS was used and incubated for 1 h at 37 °C. After washing with PBS-T,plates were blocked with PBS-FBS 10% for 15 min at 37 °C and washed withPBS-T. At the next step, optimized concentration of biotinylated anti-CA 125mAbs (5 μg/ml for 10A6 or 10 μg/ml for 7A3 and 9F7) in PBS-FBS 5% wereadded and incubation was continued for 1 h at 37 °C. After washing 1 : 50,000dilution of HRP-conjugated streptavidin (Biosource, USA) was added to thewells for 30 min at 37 °C followed by 5 times washes with PBS-T. Finally,color was developed after adding TMB substrate for 15 min. The reaction wasstopped by 20% H2SO4 and optical densities were measured at 450 nm.
The negative controls included omission of CA 125, biotinylated mAbs orcombination of the aforementioned controls. In reference wells, OC 125 mAbwas omitted and served as positive control where the ODs of all wells werecompared with. To study the probable competition between our mAbs and OC125 for the same epitope, the ODs of competitive wells were compared withthat of positive control of the same antibody. Lack of a significant differencebetween the ODs was interpreted as no competition.
RESULTS
Production of Monoclonal AntibodiesTwo protocols were applied for mouse immunization. In the first protocol,
immunization was performed by purified CA 125 and mouse immunizationwas confirmed by ELISA method. However, all fusions failed to produce stableclones. In the second protocol, immunization was performed by intraperitonealinjection of OVCAR-3 cell line. Living cells were injected into mouse peritonealcavity. Mouse immunization was confirmed by ELISA.
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120 S. Shojaeian et al.
It is noteworthy that the latter procedure led to more efficient immunizationreflected by higher titers of specific antibodies in mouse sera (Data not shown).After fusion, a total of 780 clones were screened for the presence of anti-CA125 specific antibody and 34 clones were selected by ELISA. Finally, 10 stableclones were obtained. Isotyping of mAbs revealed that three clones have IgGisotype while the rest were IgM.
Purification of AntibodiesBased on ELISA and immunofluorescent staining of OVCAR-3 cells by
hybridoma culture supernatants, three mAbs named 10A6, 7A3 and 9F7 (allIgM) were selected for purification and further analysis.
Antibody purification was performed by a house-made Rabbit anti-mouseIgM affinity chromatography column. The purity of antibodies was confirmedby SDS-PAGE and comassie blue staining which showed a single band at thesame position as IgM marker under non-reducing conditions (Fig. 1). Thisdata was confirmed by silver staining (Data not shown). Purification yield wascalculated for each clone. It was approximately 18 mg, 14 mg and 7 mg per literof 10A6, 7A3 and 9F7 supernatants, respectively.
Dot Blot AnalysisDot blot analysis was performed to confirm reactivity of purified mAbs with
CA 125 expressed by OVCAR-3 cells. Purified mAbs were clearly reactive withCA 125 expressed by as few as about 5×103 cell (1/16 dilution of cell lysate, see
Figure 1: SDS-PAGE analysis of purified IgM monoclonal antibodies. Monoclonal anti-CA 125antibodies were purified over anti-mouse IgM affinity column and separated by 7% SDS-PAGEunder non-reducing condition. Lane 1: IgM Marker, Lane 2–4: purified monoclonal antibodiesof 7A3, 9F7 and 10A6 clones (5 μg/well), respectively.
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Production of anti-CA 125 Monoclonal Antibody 121
methods). Reactivity of the purified antibodies was confirmed by OC 125 com-mercial antibody, which served as positive control (Data not shown).
ELISA
mAbs were titrated with two fold serial dilutions from 40 μg/ml to 0.16 μg/ml usingindirect ELISA (Fig. 2). The clone 10A6 showed a more persistent reactivitywith CA 125 than the other two clones which may reflect the higher affinity ofthis clone to the antigen.
Immunofluorescent StainingTo confirm the reactivity of purified mAbs with native form of extracellular
domain of CA 125, immunofluorescent staining of living OVCAR-3 wasperformed. All mAbs showed reactivity with living OVCAR-3 cells as judgedby OC 125 antibody as positive control (Fig. 3) but failed to react with CA 125negative HFFF-PI6 cell line (Data not shown). The mAbs reacted with the rimof living OVCAR-3 cells, which is characteristic of surface staining pattern.However, the fluorescence intensity of 10A6 clone was clearly higher than theother two clones which is reminiscent of its higher reactivity in ELISA assay.mAbs were also able to react strongly with fixed OVCAR-3 cells and again10A6 showed higher reactivity compared to the other clones (Fig. 4).
Figure 2: Titration of purified anti-CA 125 monoclonal antibodies by ELISA. CA 125 antigen wascoated in ELISA plate. The purified IgM mAbs were titrated in two fold dilution series from40 μg/ml to 0.16 μg/ml. NC, negative control.
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122 S. Shojaeian et al.
ImmunohistochemistryTo address whether our anti-CA 125 mAbs are able to detect in situ
expression of CA 125, immunohistochemical analysis was performed onFFPE ovarian cancer tissues. As depicted in Fig. 5a, 10A6 had strongimmunoreactivity with CA 125 expressing epithelial cells with comparableintensity with that of OC 125 (Fig. 5d) at the same concentration. Anti-CA125 immunoreactivity was predominantly observed in the cytoplasm ofpositive cells. Mucins accumulated in the lumen of epithelial glands alsoshowed strong immunoreactivity. Normal ovarian tissues did not show anysign of immunostaining when subjected to the same procedure (Fig. 5c,f)pointing to specificity of our mAbs. Negative reagent control slides werealways shown to be negative (Fig. 5b,e).
Western Blot AnalysisIt is known that CA 125 exists in various molecular weight forms
from greater than 1000 KD to lower molecular mass species of approxi-mately 200–600 KD (Davis et al., 1986). Due to this very high molecularmass of CA 125, the major part of the antigen usually migrates in the 3%
Figure 3: Immunofluorescence staining of living OVCAR-3 cells with anti-CA 125 monoclonalantibodies. Surface immunostaining of CA 125 positive cells, OVCAR-3, was performed byincubation of living cells with appropriate dilution of purified anti-CA 125 mAbs, OC 125 (positivecontrol) or normal mouse immunoglobulin (negative control), followed by FITC-conjugatedanti-mouse antibody. Magnification: 400×.
Figure 4: Immunofluorescence staining of fixed OVCAR-3 cells with anti-CA 125 mono-clonal antibodies. Acetone-fixed CA 125 positive cells, OVCAR-3, were incubated withpurified anti-CA 125 mAbs, OC 125 (positive control) and normal mouse serum (negativecontrol) and specific signal was traced by FITC-conjugated anti-mouse antibody. Magnifi-cation: 200×.
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Production of anti-CA 125 Monoclonal Antibody 123
acrylamide stacking gel and does not enter the separating gel (Lloydet al., 1997).
This phenomenon was also observed in our experiments where Westernblot analysis of OVCAR-3 cell lysate by our mAbs revealed a variety of highmolecular weight species of CA 125 that mostly migrated in the 3% stackinggel and this pattern was also observed when OC 125 was used as primaryantibody.
In addition, we observed a band at the interface of stacking and separatinggels (Fig. 6A). A band just beneath the interface was detected by 10A6 mAband OC 125 but not by other clones. A very faint band of about 200 KD couldbe traced by all clones as well as OC 125 which might represent smallerversion of CA 125 (Fig. 6A). We also detected lower molecular weight forms ofCA 125 (around 42 KD and two smaller bands) in western blot analysis usingour mAbs and OC 125 as well (Data not shown). The 42 KD band was verysimilar with what has been reported by Wang et al. (2007).
Purified CA 125 (purity≥99%) (US Biological) showed a similar pattern,namely a part of antigen migrated in the 3% acrylamide stacking gel and did notenter the separating gel and a band at the interface of stacking and separatinggels (Fig. 6B).
ImmunoprecipitationTo further characterize the specificity of the anti-CA 125 antibodies,
immunoprecipitated CA 125 from OVCAR-3 cell lysate were resolved by SDS-PAGE and then transblotted to PVDF membrane. Immunoprecipitated CA125 antigen by each mAb was detected by corresponding monoclonal antibody
Figure 5: Confirmation of anti-CA 125 mAb reactivity by immunohistochemistry. Expression ofCA 125 was assessed in FFPE sections of ovarian cancer tissue by 10A6 anti-CA 125 mAb usinglabeled-biotin-streptavidin procedure (a). As positive reagent control, OC 125 was applied asprimary antibody (d). All negative reagent (b, e) and tissue (c, f) control slides were shown tobe negative. Magnification: 200×.
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124 S. Shojaeian et al.
or OC 125 as positive control. We observed a very similar migration patterncompared to that of western blot (Fig. 7).
as did OC 125, antibody competitive ELISA was performed. The resultsclearly showed that no competition occurred and OC 125 could not block theepitopes of our antibodies (Fig. 8). Thus none of our antibodies were OC 125-likeantibodies.
DISCUSSION
Epithelial ovarian carcinoma presents a vast clinical challenge. It is oftenreferred to as the “silent killer” because symptoms do not develop or are not
Figure 6: Western blot analysis of OVCAR-3 cell lysate (A) and purified CA 125 (B) by purifiedanti-CA 125 monoclonal antibodies. OVCAR-3 cell lysate (A) and purified CA 125 (B) wereseparated by SDS-PAGE under non-reducing condition and analyzed using purified mono-clonal antibodies (10 μg/ml) after blotting on PVDF membrane. Lane1: 10A6, Lane 2: 7A3,Lane 3: 9F7, Lane 4: OC 125 (positive control), Lane 5: normal mouse Ig (5 μg/ml, negativecontrol). Arrows indicate the interface between stacking and separating gel.
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Production of anti-CA 125 Monoclonal Antibody 125
Figure 7: Immunoprecipitation analysis of CA 125 by anti-CA125 monoclonal antibodies. CA125 was immunoprecipitated from OVCAR-3 cell lysate by its treatment with anti-CA 125mAbs, 10A6 (Lane 1,3,5) and 7A3 (Lane 2,4,6), separately. Immunoprecipitated antigen waselectrophoresed and blotted on PVDF membrane and corresponding monoclonal antibod-ies were used as primary antibody for detection of specific bands including 10A6 (Lane 1),7A3 (Lane 2). Positive control (Lane 3,4) and negative control lanes (Lane 5,6) were tracedwith OC 125 and normal mouse serum, respectively. Arrow indicates the interface betweenstacking and separating gel.
Figure 8: Competitive ELISA. For detection of a probable competition between our antibodiesand the commercial anti-CA 125 antibody (OC 125), antibody competitive ELISA was per-formed. To this end, the ODs of competitive wells were compared with that of positive controlof the same antibody. The results clearly showed that no competition occurred and OC 125could not block the epitopes of our antibodies. Each bar represents the results (Meam ± SD)of three experiments.
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126 S. Shojaeian et al.
recognized until the disease is in its advanced stages (Reynolds et al., 2006).CA 125 has been used as a useful tumor marker in the detection, vaccinationand therapy of ovarian cancer (Cannon et al., 2004; Wang et al., 2004). Serumassay of CA 125 has become the gold standard for monitoring patients withovarian carcinoma (Bast et al., 1983; Bon et al., 1996).
Over the years a total of 32 anti-CA 125 antibodies have been submittedto the TD-1 workshop and these have been categorized into three majorepitope groups: OC 125-like antibodies, M 11-like antibodies and OV 197-like antibodies (Rye et al., 2003). In this study, two protocols were appliedfor mouse immunization. The first protocol, using purified CA 125 failed toproduce stable clones. Notability CA 125 is poorly immunogenic and scarcityof its antigenic nature may be due to its unusual structure, which consists ofmore than 60 repeat units of 156 amino acids (O’Brien et al., 2001; Yin et al.,2002), hyperglycosylated state and to its immunosuppressive effects (Patan-kar et al., 2005) which may prevent immunized mice to elicit a strong immuneresponse. Interestingly, the second protocol, using OVCAR-3 cell line asimmunogen, yielded ten stable clones that produced specific anti-CA 125monoclonal antibodies.
In most circumstances, booster antigens are identical to the primingantigens. However, for some antigens this strategy fails to produce sufficientamount of high affinity antibodies. Prime-boost approach, in which the natureor composition of antigens in primary and boosting immunization are different,is the method of choice which markedly enhances antibody production. Oursecond protocol of immunization benefits from advantages of such strategywhich yielded stable clones. Just why this mixed modality works well is notentirely clear, but we think that high protein content of cell preparation in theinitial immunization may have a helper effect on promoting antigen specificB cell proliferation (Hu et al., 1991).
Among 10 stable clones we produced in this study, three clones were IgGand the rest were IgM. Based on the results of ELISA and immunifluorescentstaining of living OVCAR-3 cell line, three IgM clones were selected for fur-ther studies. Immunofluorescence staining of fixed and living OVCAR-3 cellswith selected IgM antibodies revealed that all IgM antibodies have consider-able immunoreactivity with both cell types without non-specific reaction. The10A6 clone, however, showed stronger reaction compared to the other clones.This result was confirmed by other assays including ELISA, western blot andimmunoprecipitation in which 10A6 was more reactive than two other IgMs.This may reflect higher affinity of 10A6 to CA 125.
Immunohistochemistry (IHC) and in situ hybridization are common detectionmethods in pathology and clinical diagnosis in which dye or small moleculeorganic fluorescent probes are used to detect and localize proteins and nucleicacids, respectively. Accordingly, CA 125 antigen in different specimens (fixedcell, tissue sections and tissue pieces) has been detected by specific anti-CA
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Production of anti-CA 125 Monoclonal Antibody 127
125 monoclonal antibodies (Davis et al., 1986; Wang et al., 2004). The applica-tion of 22 antibodies in immunohistochemistry was reported in the second reportfrom TD-1 workshop (Nap et al., 1996).
The results of immunofluorescence staining of ovarian cancer cell line,OVCAR-3, depicted that our antibodies have excellent capacity for in situdetection of CA 125. Based on these results, these antibodies seemed to besuitable candidates to be used in IHC assays for in situ detection of CA 125 inovarian cancer tissues. To address this issue, immunohistochemical analysis ofovarian cancer tissues were performed and the results showed that our mAbshave strong and specific immunoreactivity with CA 125 expressing epithelialcells as judged by the same immunostaining pattern with that of OC 125.
Indeed, reactivity of our antibodies with extracellular domain of CA 125was confirmed by immunofluorescence staining of living OVCAR-3 cells.Although our IgM antibodies were able to recognize CA 125 in its native formon the cell surface, it seems they are too big to be useful for in vivo applica-tions and need to be monomerized before such application. Anti-idiotype andanti-CA 125 antibodies have been recently used for immunotherapy ofpatients with ovarian cancer. Using a monoclonal antibody (oregovemab)against CA 125 for consolidation in ovarian cancer patients after first linetreatment, Berek et al. reported that a subgroup of patients with favoriteprognosticators had a significantly longer time to relapse compared topatients in the placebo group. Another clinical strategy has employed an anti-idiotype vaccine approach in patients with relapsed ovarian cancer using amonoclonal antibody (abagovomab) that mimics CA 125 antigen. The initialresults of these trials as reported by Sabbatini et al. showed that all patientsdeveloped an anti-anti-idiotypic antibody response. In addition, five patientsshowed the generation of T cell immunity to CA 125 (Dorigo et al., 2007).
Previous work on the biochemical structure of the CA 125 antigen hadresulted in a confusing and inconsistent picture of its molecular composition.While most of these studies agreed that CA 125 was a high molecular weightglycoprotein, many studies also detected smaller reactive species (Davis et al.,1986; de los Frailes et al., 1993; Kobayashi et al., 1993; Lloyd et al., 1997). Lloydet al. by pulse-chase experiments demonstrated that CA 125 antigen was syn-thesized through a glycosylated 400 KD precursor species that did not appear inthe culture supernatant. On the other hand, no smaller species of the matureform of CA 125 antigen were detected in this study (Lloyd et al., 2001).
Nustad et al. studied epitopes of CA 125 using six new anti-CA 125 anti-bodies. Radioimmunoprecipitation analysis of in vivo labeled CA 125 antigenpresent in OVCAR-3 cell supernatant by SDS-PAGE gave the same patternfor all tested antibodies, namely a major antigen band in the 3% stacking geland three faint bands of about 200 KD in the 7% separating gel. A major bandat the interface between the stacking and separating gel was non-specific as itwas present also in the negative control (Nustad et al., 2002). The antibodies
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128 S. Shojaeian et al.
presented here gave the similar pattern in Western blot analysis, namely amajor diffused band in the 3% stacking gel and a band at the interface ofstacking and separating gels.
Notably, in our hand, this band appeared to be specific as such band wasnot detected in negative control lanes. Indeed, a band just beneath the inter-face was detected by 10A6 mAb and OC 125 but not by other clones. A veryfaint band of about 200 KD could be traced by all clones as well as OC 125,which may represent smaller version of CA 125. It is possible that dependingon the source of the antigen in western blot analysis, different patterns areachieved and this may explain partial discrepancies between our results andwhat Nustad reported previously. He used OVCAR-3 cell supernatant, whilewe used OVCAR-3 cell lysate as the source of antigen.
To clarify further antigen-specificity of the antibodies produced in this study,purified CA 125 (purity ≥ 99%) was used as source of antigen in Western blotanalysis. The pattern of bands observed in such experiment was the same com-pared to condition in which OVCAR-3 cell lysate was used as antigen source.This data clearly showed that our antibodies are exactly directed against CA125. More importantly 9F7 which has little or no affinity for the purified CA125preparation (see Figure 6B Lane3) showed an extraordinary reactivity withCA125 antigen derived from the OVCAR-3 cell lysate (see Figure 6A, Lane 3).
Thus it would seem that the two antibodies 10A6 and 7A3 are directedtoward the repeat domain of CA125. But the 9F7 antibody does not recognizeany low molecular bands (see Figure 6A, Lane 3) which are likely produced byproteolysis of the parent molecules repeat domain. But instead 9F7 recognizesa high molecular species not recognized by the other two antibodies or byOC125. Furthermore this high molecular species is only present in the celllysate preparation and not in the purified CA125 preparation (compare Fig-ures 6A, Lane 3 and 6B Lane 3). Therefore it is likely that this antibody recog-nizes the amino terminal domain of CA 125, a 10,000 amino acid domainwhich is not known to be recognized by other CA 125 antibodies. This doesnot, however, rule out the possibility that this clone may recognize CA 125-binding molecules like galectin.
Moreover Singleton et al. demonstrated that M 11 and OC 125 clearlyreact with a very high molecular weight species of shed CA 125. In contrast776.1 and 368.1 anti-CA 125 antibodies showed little or no binding to a simi-lar species (Singleton et al., 2006). On the other hand, Lloyd et al. purified CA125 antigen from OVCAR-3 cells supernatant by applying a column of mAbVK-8. This purified CA 125/VK-8 antigen was used in subsequent character-ization studies. Western blot analysis of purified CA 125/VK-8 antigen withmAb VK-8 gave similar bands compared to OC 125 but these were muchweaker, possibly due to the greater susceptibility of the VK-8 epitope to SDStreatment (Lloyd et al., 1997). So, it may be speculated that different anti-CA125 antibodies behave differentially at the same experimental conditions.
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Production of anti-CA 125 Monoclonal Antibody 129
The result of Western blot was confirmed by immunoprecipitation analysisin which CA 125 antigen precipitated by 10A6 and 7A3 mAbs showed diffusedbands in 3% separating gel and a band of about 200 KD when detected by OC125. Interestingly, the same pattern was observed when 10A6 or 7A3 itselfwas used as detecting antibody except that the 200 KD band was not detected.The reason for such differential behavior of our antibodies and OC 125 is notclear, but may be due to different concentrations of detection antibodies (10A610 μg/ml and OC 125 1:100) or recognition of different epitopes as confirmedby the antibody competitive ELISA. In conclusion, we report the generationand characterization of three novel anti-CA 125 mAbs by ELISA, dot blotting,immunoprecipitation, western blotting, intracellular and surface immunofluores-cence staining of OVCAR-3 cell line and IHC staining of ovarian cancer tissues.The anti-CA 125 mAbs produced in the study are invaluable tools in diagnosisand research fields for assessment of CA 125 expression in cancerous tissues.
ACKNOWLEDGMENTS
The authors thank to A. R. Mahmoodi, J. Ghasemi,. L. Balay-goli, M. Babaeiand M. Ostadkarampour for technical assistance. This work was supported bya grant from Iranian Nanotechnology Initiative.
Declaration of interest: The authors report no conflicts of interest. Theauthors alone are responsible for the content and writing of the paper.
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