HYBRIDOMA Volume 11, Number 6, 1992 Mary Ann Liebert, Inc., Publishers
Generation of Murine Triomas Secreting Bi-specific Monoclonal Antibodies That Recognize HBsAG ad and ay Subtypes G.
FALERO,1 I. RODRIGUEZ,2 J. SARRACENT,3 A.J. OTERO,4 B.L. RODRIGUEZ,2 A. ROJAS,2 and E. OCHOA5
1FINLAY Institute, Monoclonal Antibodies Group, 2National Center for Scientific Research, Hybridoma Lab., Chemistry Faculty, Havana University, Institute of Tropical Medicine "Pedro Kouri, Immunoassay Center, Havana City, Cuba "
ABSTRACT We report the generation of murine triomas by fusing splenocytes from mice previously immunized with HBsAg ay-subtype and a hybridoma, secreting anti-HBsAg acï-subtype monoclonal antibody, which was rendered HGPRT by induced mutagenesis with N-methyl-N* ni tro-N-ni trosoguani di ne. The fusion yielded a 83.8% of hybrids showing the antigen specificity of the parental hybridoma and a 16.1% of bi-specific monoclonal antibodies. One of them, coded as 1C8A5, showing a heavy chain isotype CI gGi/T gGzbD was used as capture reagent in an of both HBsAg ad- and ul tramicro-ELISA. As little as O. 78 I U. ay-subtypes could be realiably detected. .
INTRODUCTION or more are derived two fusing Hybrid hybridomas by The resulting cells secrete hybrid antibody-producing cells. immunoglobulins exhibiting characteristics of the two individual parental hybridomas in a single molecule C1.25. These bi—specific are but antibodies CbsMAbD monoclonal structurally bivalent functionally univalent for each combining site. noncovalent The possible applications of these heterofunctional cross-linking agents are numerous in biology and medicine and offer considerable advantages over chemical cross-linking methods. In fact, bsMAbs have been shown to be useful in immunochemistry C3D immunoenzymoassay C4.5D and immunotherapy C6.7D. surface antigen CHBsAg} is a complex macromolecular Hepatitis viral DNA. structure involving several proteins coded by hepatitis Several studies have shown four major antigenic subtypes C83. Among them, a, d and y specificities exist on each of the constituent components of HBsAg, and, therefore, the corresponding antigenic determinants are specific for the S proteins. In the present work we report the generation of a trioma producing ,
815
bsMAb which recognizes both ad- and ay-subtypes of HBsAg. This monoclonal antibody could be very useful as a capture reagent in the desing of an enzyme-linked immunosorbent assay CELI SA) for the detection of HBsAg positive human sera. MATERIALS AND METHODS Immuni zati
ons
Six Balb/c mice were immunized by intraperitoneal injections with a dose of 5µg of HBsAg Cay-subtype}, hereafter mentioned as ay, emulsified in Freund's adjuvant CDifco; Detroit, MI). Each animal three such all administered at 2-week received injections, intervals. Five to IO days after the final injection, the mice were bled by their tail veins and the serum collected was tested for the presence of anti-HBsAg antibodies in a conventional indirect ELISA Three to four days before cell fusion, Csee hybri doma screening). the appropriate host mouse received a final intravenous injection of The dose of the the antigen in phosphate-buffered saline CPBS) antigen administered intravenously was the same as in the previous i mmuni zati ons. .
Hybr i doma production Several anti-HBsAg C ad-subtype)-produci ng hybridoma lines were established from polyethylene glycol-facili tate fusion of P3-X63Ag8.653 cells and splenocytes obtained from Balb/c mice immunized hereafter mentioned as ad C9). with purified HBsAg Cad-subtype), Resistant cells to 6-thioguanine C6-TG) CSigma Chemical Co.,St Louis, MO), 3.34g/L, were derived from the anti-ad-secreting clones by induced mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine CMNG), a generous gift from Dr Walderico M. Generoso COak Ridge National Labs). Briefly, cells/mL in RPMI 1640 medi urn CGibco; Grand Island, NY) adjusted to pH 6.8 with HEPES were treated with an appropriated dilution of mutagen, previously selected from survival The mutagen-containing medium is then curves, for 3 hours at 37 C. bovine serum CFBS) removed and fresh RPMI 1640 with 10% fetal CGibco) is added. Cell viability is then checked by trypan blue-exclusion. Cells are growing for further 48hrs to allow cell recovery after the mutagenic challenge, and then seeded in 96-well cell/well in selective medium CRPMI 1640, 10% FBS microplates at Resistant colonies were then tested for and 6-TG at 3. 34mg/mL). anti-ad and for to hypoxantine activity sensitivity CHAT) in C1 µ CO. 4pmol/L)-thymidine C16pmol/L) /L)-aminopteri The HAT-sensiti ve and RPMI-1640 supplemented with 20% FBS. anti-ad-producing hybridoma lines were then cloned by limiting dilution. One clone CHGPRT1) was eventually isolated and used for cell fusion CÍO).
SXIO5
IO5
Hybridoma-Splenocyte cell fusion and anti body production Fusions were carried out essentially as described by Campbell Cll), the aminopterin-sensitive and with the following modifications: anti-ad-producing hybridoma CHGPRT1) replaced the conventional myeloma cells for the production of bi-specific antibodies. Mouse splenocytes were mixed with hybridoma cells at a ratio of 2:1, and fusion was performed using polyethylene glycol 4000 CPEG 4000, Merck). Fusion wells containing hybrid-hybri domas secreting the desired bi-specific antibody were cloned twice by limiting dilution, and
816
injected into screening for
mice for production of ascites fluid Cll). Antibody clones was performed essentially as described for One clone was isolated and used for the final fusion wells. characterization. This clone was coded 1C8A5.
Hybr i doma screening We used an ELISA to screen for anti-ad/ay antibodies. Briefly, as follow: microassay plates Cpolyvinylchloride, Titertek) were coated 4 C at with of goat overnight lOO/L/L/well affinity-purified anti-HBsAg polyclonal antibody at a concentration of lOpg/mL in carbonate buffer, C34mmol/L, pH 9.6). The wells were subsequently filled with a 10% sheep serum solution in PBS and incubated for Ihr to reduce non-specific binding. The plate was divided in three sections and 100µL/well of human normal serum, ad-positive human serum and ay-positive human serum, all diluted 1:4 in PBS-Tween 20 were at 0.05%, respectively added and incubated for 2hr. Then, hybrid culture supernatants were added to each section and allowed to incubate for 2hr. To each well, lOO/jL/well of horseradish peroxidase goat anti-mouse IgG conjugate CSigma) were added and incubate for Ihr. The bound conjugate was detected by incubating the plate at 24 C during 5 minutes with a substrate solution containing o-phenylenediamine CO.4mg/mL) and O. 4% H2O2 in citrate buffer, C100mmol/L, pH 5.5). The reaction was stopped by adding 2.5N HzSO* and optical densities at 492nm were read with a Titertek Multiskan microplate reader CFlow Laboratories). All incubation times were performed at 37 C and all microassay wells were washed extensively with PBS-tween 20 between all the aforementioned incubation steps.
Class and subclass determinati
on
Class and subclass determination for the bsMAb secreted by the clone 1C8A5 was performed using the ImmunoType Kit CSigma) and following manufacturer's instructions. Purification of bi-specific antibodies Purification equipment, column and gel Pharmacia-LKB CUppsala, Sweden). 1C8A5 ascites fluid was filtered through
previously
glicin,
equilibrated
protein
medium
were
obtained
from
glass wool and applied to a A-Sepharose column with 1 5mol/L buffer CpH 8.9). The sample was
3mol/L sodium chloride loaded at a flow rate of 15mL/hr at 4 C. Elution of bound antibodies was carried out using pH-gradient of O.lmol/L sodium citrate/citric acid buffer from pH 8 to pH 3. Fractionation was monitored by optical density at 280nm. The eluates were neutralized to pH 7.4 immediately, dialyzed against PBS, pH 7.2, and concentrated by Ultrafiltration using an Amicon YM-lOO membrane. .
Checking specificity
primary screening the clone 1C8A5 was tested, by mean of a inhibition to that competitive enzyme immunoassay, verify specificity really corresponds to ad-ay combination and not to ad-a. the ad Briefly, moiety was labeled with peroxidase by the After
conventional two-step method for glutaraldehyde. Immunopiates were coated with purified bsMAb C10µg/mL in carbonate buffer, C34mmol/L, pH 9.6)). After reducing the non-specific binding with a 10% sheep serum solution in PBS, we then added lOOpL of a mixture consisting in 50/jL of increasing amounts of ay moiety Cfrom O. 312 to lO/jg/mL) and 50pL of an appropriated dilution of the ay-enzyme conjugate.
817
Incubation was performed at 37°C for Ihr. After color development was performed as mentioned above.
several
washes,
Chain composi ti on of bsMAb
Eluates obtained by affinity chromatography were analyzed using a Sodium Dodecyl Sulphate-polyacrylamide C12. 5%) gel electrophoresis of reduced chains CSDS-PAGE) C12). The protein bands were visualized by Coomassie brilliant blue R250, in order to know the different molecular
species
Use of bsMAb
as
secreted and the association
pattern.
capture reagent
Purified bsMAb was used as capture antibody in an ultramicro-ELISA C13). follow: as Briefly, polystyrene ul trami croassay plates CGreiner) were coated overnight at 4°C with lO/uL/well of bsMAb 1C8A5 at a concentration of lO/Lig/mL in tris 50mmol/L, pH 8. The wells were subsequently filled with a 10% sheep serum solution in PBS and incubated for Ihr to reduce non-specific binding. After, the wells were filled with lOpL of increasing concentrations of HBsAg ad- and ay-subtype standards CPaul Ehrlich Institute, Germany), during IHr at We added of 37 C. then IO/l/L polyclonal antibody goat anti-HBsAg-alkali ne phosphatase conjugate and the plate incubate for further 30 minutes at 37°C. After washing, the bound conjugate was detected by incubating the plate during 30 minutes with lOpL of 0.5mmol/L solution of methylumbelliferyl phosphate CSigma) at 24°C. The plate was then read in a microplate fluorometer. Adjusment as follow: O, water; 100, methylumbelliferone CO. 5mmol/L). the assay was determined by means The sensibility of of the procedure described by Rodbard C14).
RESULTS We fused the anti-ad-producing hybridoma with spleen cells of animals immunized with ay, and hybrids selection was performed in HAT medium. As Figure 1 shows, the proportion of growth-positive wells that contained anti-ad antibodies were 83.8%. However, when culture supernatants were assayed for the presence of bi-specific anti-ad/ay antibodies, only 16.1% wells exhibited bsMAb secretion.
100
%
hybrid aecretlng antibody
Figure 1: Antibody specificities of fusion supernatants anti -ad, CB) anti-ay and CO anti-ad/ay activities.
818
CA)
The fusion produced no detectable anti-ay antibodies. One well which was found to be positive for the presence of bsMAb The final clone, coded 1C8A5 was was cloned by limiting dilution. isolated and used for further characterization. Figure 2 showed that the antibodies secreted by hybrid-hybridoma The parental hybridoma HGPRT1 were of the IgGi and IgGzb subclasses. subclass antibodies of the monoclonal secretes CÍO), IgGi consequently, the IgGzb portion of this bsMAb has been inherited from its parental lymphocyte.
Figure
2:
Results of the class and subclass
immunotyping.
Antibodies from 1C8A5 ascites were purified by passage through a were retained column. and Protein A-sepharose species IgG subsequently eluted with a pH-continuous gradient. Three fractions The obtained were yield of the three fractions CFigure 3). II and III) Cmentioned as peaks I, correspond to l.O, 1.9 and l.lmg/mL of asci ti s fluid, respectively. Analysis of the light CD and heavy CH) chains composition of the immunoglobulins present in the three fractions showed that peaks I and III represent the detectable with respectively, parentals anti-ad and anti-ay, cross-contamination of L chains. The middle peak showed the H and L chains expected for a hybrid molecule.
PH
1,2
0,8
,ß
0.4 0,2 —
30 # fraotlons
20
60
Figure 3: Resolution pattern of hybrid hybridoma products on protein A-Shepharose column, and the corresponding SDS-PAGE pattern for each peak CHI and LI correspond to the heavy and light chains from parental anti-ay, H2 and L2 correspond to the heavy
and
light
chains from
parental
anti-ad).
As shown in Figure 4, the specificity of the bsMAb 1C8A5 corresponds to both ad- and ay-subtypes as checked by sandwich-ELISA using ad— as and ay-positive human sera as well by a competitive enzyme inhibition immunoassay. The Figure 5 shows the standard curves for the quantification of HBsAg ad- and ay-subtypes by ultramicro-ELISA. The working range was
819
OD. (482nm) BSS9 HQPflll
ESS 1.5- CU
1C8A6
supernatant
Bupernatant
no related
MAb
MM»H*^WlW-:·:·:·:-:·:-:! HNa> hwu aarnl «ata
QD. (4Q2nm)
ug
oompetltor/mL
4: Results of specificity analysis performed by mean of sandwish ELISA using ad- and ay-positive human sera C A) and by a competitive enzyme inhibition immunoassay CB).
Figure a
* FLUORESCENCE
Generation of Murine Triomas Secreting Bi-specific Monoclonal Antibodies That Recognize HBsAG ad and ay Subtypes G.
FALERO,1 I. RODRIGUEZ,2 J. SARRACENT,3 A.J. OTERO,4 B.L. RODRIGUEZ,2 A. ROJAS,2 and E. OCHOA5
1FINLAY Institute, Monoclonal Antibodies Group, 2National Center for Scientific Research, Hybridoma Lab., Chemistry Faculty, Havana University, Institute of Tropical Medicine "Pedro Kouri, Immunoassay Center, Havana City, Cuba "
ABSTRACT We report the generation of murine triomas by fusing splenocytes from mice previously immunized with HBsAg ay-subtype and a hybridoma, secreting anti-HBsAg acï-subtype monoclonal antibody, which was rendered HGPRT by induced mutagenesis with N-methyl-N* ni tro-N-ni trosoguani di ne. The fusion yielded a 83.8% of hybrids showing the antigen specificity of the parental hybridoma and a 16.1% of bi-specific monoclonal antibodies. One of them, coded as 1C8A5, showing a heavy chain isotype CI gGi/T gGzbD was used as capture reagent in an of both HBsAg ad- and ul tramicro-ELISA. As little as O. 78 I U. ay-subtypes could be realiably detected. .
INTRODUCTION or more are derived two fusing Hybrid hybridomas by The resulting cells secrete hybrid antibody-producing cells. immunoglobulins exhibiting characteristics of the two individual parental hybridomas in a single molecule C1.25. These bi—specific are but antibodies CbsMAbD monoclonal structurally bivalent functionally univalent for each combining site. noncovalent The possible applications of these heterofunctional cross-linking agents are numerous in biology and medicine and offer considerable advantages over chemical cross-linking methods. In fact, bsMAbs have been shown to be useful in immunochemistry C3D immunoenzymoassay C4.5D and immunotherapy C6.7D. surface antigen CHBsAg} is a complex macromolecular Hepatitis viral DNA. structure involving several proteins coded by hepatitis Several studies have shown four major antigenic subtypes C83. Among them, a, d and y specificities exist on each of the constituent components of HBsAg, and, therefore, the corresponding antigenic determinants are specific for the S proteins. In the present work we report the generation of a trioma producing ,
815
bsMAb which recognizes both ad- and ay-subtypes of HBsAg. This monoclonal antibody could be very useful as a capture reagent in the desing of an enzyme-linked immunosorbent assay CELI SA) for the detection of HBsAg positive human sera. MATERIALS AND METHODS Immuni zati
ons
Six Balb/c mice were immunized by intraperitoneal injections with a dose of 5µg of HBsAg Cay-subtype}, hereafter mentioned as ay, emulsified in Freund's adjuvant CDifco; Detroit, MI). Each animal three such all administered at 2-week received injections, intervals. Five to IO days after the final injection, the mice were bled by their tail veins and the serum collected was tested for the presence of anti-HBsAg antibodies in a conventional indirect ELISA Three to four days before cell fusion, Csee hybri doma screening). the appropriate host mouse received a final intravenous injection of The dose of the the antigen in phosphate-buffered saline CPBS) antigen administered intravenously was the same as in the previous i mmuni zati ons. .
Hybr i doma production Several anti-HBsAg C ad-subtype)-produci ng hybridoma lines were established from polyethylene glycol-facili tate fusion of P3-X63Ag8.653 cells and splenocytes obtained from Balb/c mice immunized hereafter mentioned as ad C9). with purified HBsAg Cad-subtype), Resistant cells to 6-thioguanine C6-TG) CSigma Chemical Co.,St Louis, MO), 3.34g/L, were derived from the anti-ad-secreting clones by induced mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine CMNG), a generous gift from Dr Walderico M. Generoso COak Ridge National Labs). Briefly, cells/mL in RPMI 1640 medi urn CGibco; Grand Island, NY) adjusted to pH 6.8 with HEPES were treated with an appropriated dilution of mutagen, previously selected from survival The mutagen-containing medium is then curves, for 3 hours at 37 C. bovine serum CFBS) removed and fresh RPMI 1640 with 10% fetal CGibco) is added. Cell viability is then checked by trypan blue-exclusion. Cells are growing for further 48hrs to allow cell recovery after the mutagenic challenge, and then seeded in 96-well cell/well in selective medium CRPMI 1640, 10% FBS microplates at Resistant colonies were then tested for and 6-TG at 3. 34mg/mL). anti-ad and for to hypoxantine activity sensitivity CHAT) in C1 µ CO. 4pmol/L)-thymidine C16pmol/L) /L)-aminopteri The HAT-sensiti ve and RPMI-1640 supplemented with 20% FBS. anti-ad-producing hybridoma lines were then cloned by limiting dilution. One clone CHGPRT1) was eventually isolated and used for cell fusion CÍO).
SXIO5
IO5
Hybridoma-Splenocyte cell fusion and anti body production Fusions were carried out essentially as described by Campbell Cll), the aminopterin-sensitive and with the following modifications: anti-ad-producing hybridoma CHGPRT1) replaced the conventional myeloma cells for the production of bi-specific antibodies. Mouse splenocytes were mixed with hybridoma cells at a ratio of 2:1, and fusion was performed using polyethylene glycol 4000 CPEG 4000, Merck). Fusion wells containing hybrid-hybri domas secreting the desired bi-specific antibody were cloned twice by limiting dilution, and
816
injected into screening for
mice for production of ascites fluid Cll). Antibody clones was performed essentially as described for One clone was isolated and used for the final fusion wells. characterization. This clone was coded 1C8A5.
Hybr i doma screening We used an ELISA to screen for anti-ad/ay antibodies. Briefly, as follow: microassay plates Cpolyvinylchloride, Titertek) were coated 4 C at with of goat overnight lOO/L/L/well affinity-purified anti-HBsAg polyclonal antibody at a concentration of lOpg/mL in carbonate buffer, C34mmol/L, pH 9.6). The wells were subsequently filled with a 10% sheep serum solution in PBS and incubated for Ihr to reduce non-specific binding. The plate was divided in three sections and 100µL/well of human normal serum, ad-positive human serum and ay-positive human serum, all diluted 1:4 in PBS-Tween 20 were at 0.05%, respectively added and incubated for 2hr. Then, hybrid culture supernatants were added to each section and allowed to incubate for 2hr. To each well, lOO/jL/well of horseradish peroxidase goat anti-mouse IgG conjugate CSigma) were added and incubate for Ihr. The bound conjugate was detected by incubating the plate at 24 C during 5 minutes with a substrate solution containing o-phenylenediamine CO.4mg/mL) and O. 4% H2O2 in citrate buffer, C100mmol/L, pH 5.5). The reaction was stopped by adding 2.5N HzSO* and optical densities at 492nm were read with a Titertek Multiskan microplate reader CFlow Laboratories). All incubation times were performed at 37 C and all microassay wells were washed extensively with PBS-tween 20 between all the aforementioned incubation steps.
Class and subclass determinati
on
Class and subclass determination for the bsMAb secreted by the clone 1C8A5 was performed using the ImmunoType Kit CSigma) and following manufacturer's instructions. Purification of bi-specific antibodies Purification equipment, column and gel Pharmacia-LKB CUppsala, Sweden). 1C8A5 ascites fluid was filtered through
previously
glicin,
equilibrated
protein
medium
were
obtained
from
glass wool and applied to a A-Sepharose column with 1 5mol/L buffer CpH 8.9). The sample was
3mol/L sodium chloride loaded at a flow rate of 15mL/hr at 4 C. Elution of bound antibodies was carried out using pH-gradient of O.lmol/L sodium citrate/citric acid buffer from pH 8 to pH 3. Fractionation was monitored by optical density at 280nm. The eluates were neutralized to pH 7.4 immediately, dialyzed against PBS, pH 7.2, and concentrated by Ultrafiltration using an Amicon YM-lOO membrane. .
Checking specificity
primary screening the clone 1C8A5 was tested, by mean of a inhibition to that competitive enzyme immunoassay, verify specificity really corresponds to ad-ay combination and not to ad-a. the ad Briefly, moiety was labeled with peroxidase by the After
conventional two-step method for glutaraldehyde. Immunopiates were coated with purified bsMAb C10µg/mL in carbonate buffer, C34mmol/L, pH 9.6)). After reducing the non-specific binding with a 10% sheep serum solution in PBS, we then added lOOpL of a mixture consisting in 50/jL of increasing amounts of ay moiety Cfrom O. 312 to lO/jg/mL) and 50pL of an appropriated dilution of the ay-enzyme conjugate.
817
Incubation was performed at 37°C for Ihr. After color development was performed as mentioned above.
several
washes,
Chain composi ti on of bsMAb
Eluates obtained by affinity chromatography were analyzed using a Sodium Dodecyl Sulphate-polyacrylamide C12. 5%) gel electrophoresis of reduced chains CSDS-PAGE) C12). The protein bands were visualized by Coomassie brilliant blue R250, in order to know the different molecular
species
Use of bsMAb
as
secreted and the association
pattern.
capture reagent
Purified bsMAb was used as capture antibody in an ultramicro-ELISA C13). follow: as Briefly, polystyrene ul trami croassay plates CGreiner) were coated overnight at 4°C with lO/uL/well of bsMAb 1C8A5 at a concentration of lO/Lig/mL in tris 50mmol/L, pH 8. The wells were subsequently filled with a 10% sheep serum solution in PBS and incubated for Ihr to reduce non-specific binding. After, the wells were filled with lOpL of increasing concentrations of HBsAg ad- and ay-subtype standards CPaul Ehrlich Institute, Germany), during IHr at We added of 37 C. then IO/l/L polyclonal antibody goat anti-HBsAg-alkali ne phosphatase conjugate and the plate incubate for further 30 minutes at 37°C. After washing, the bound conjugate was detected by incubating the plate during 30 minutes with lOpL of 0.5mmol/L solution of methylumbelliferyl phosphate CSigma) at 24°C. The plate was then read in a microplate fluorometer. Adjusment as follow: O, water; 100, methylumbelliferone CO. 5mmol/L). the assay was determined by means The sensibility of of the procedure described by Rodbard C14).
RESULTS We fused the anti-ad-producing hybridoma with spleen cells of animals immunized with ay, and hybrids selection was performed in HAT medium. As Figure 1 shows, the proportion of growth-positive wells that contained anti-ad antibodies were 83.8%. However, when culture supernatants were assayed for the presence of bi-specific anti-ad/ay antibodies, only 16.1% wells exhibited bsMAb secretion.
100
%
hybrid aecretlng antibody
Figure 1: Antibody specificities of fusion supernatants anti -ad, CB) anti-ay and CO anti-ad/ay activities.
818
CA)
The fusion produced no detectable anti-ay antibodies. One well which was found to be positive for the presence of bsMAb The final clone, coded 1C8A5 was was cloned by limiting dilution. isolated and used for further characterization. Figure 2 showed that the antibodies secreted by hybrid-hybridoma The parental hybridoma HGPRT1 were of the IgGi and IgGzb subclasses. subclass antibodies of the monoclonal secretes CÍO), IgGi consequently, the IgGzb portion of this bsMAb has been inherited from its parental lymphocyte.
Figure
2:
Results of the class and subclass
immunotyping.
Antibodies from 1C8A5 ascites were purified by passage through a were retained column. and Protein A-sepharose species IgG subsequently eluted with a pH-continuous gradient. Three fractions The obtained were yield of the three fractions CFigure 3). II and III) Cmentioned as peaks I, correspond to l.O, 1.9 and l.lmg/mL of asci ti s fluid, respectively. Analysis of the light CD and heavy CH) chains composition of the immunoglobulins present in the three fractions showed that peaks I and III represent the detectable with respectively, parentals anti-ad and anti-ay, cross-contamination of L chains. The middle peak showed the H and L chains expected for a hybrid molecule.
PH
1,2
0,8
,ß
0.4 0,2 —
30 # fraotlons
20
60
Figure 3: Resolution pattern of hybrid hybridoma products on protein A-Shepharose column, and the corresponding SDS-PAGE pattern for each peak CHI and LI correspond to the heavy and light chains from parental anti-ay, H2 and L2 correspond to the heavy
and
light
chains from
parental
anti-ad).
As shown in Figure 4, the specificity of the bsMAb 1C8A5 corresponds to both ad- and ay-subtypes as checked by sandwich-ELISA using ad— as and ay-positive human sera as well by a competitive enzyme inhibition immunoassay. The Figure 5 shows the standard curves for the quantification of HBsAg ad- and ay-subtypes by ultramicro-ELISA. The working range was
819
OD. (482nm) BSS9 HQPflll
ESS 1.5- CU
1C8A6
supernatant
Bupernatant
no related
MAb
MM»H*^WlW-:·:·:·:-:·:-:! HNa> hwu aarnl «ata
QD. (4Q2nm)
ug
oompetltor/mL
4: Results of specificity analysis performed by mean of sandwish ELISA using ad- and ay-positive human sera C A) and by a competitive enzyme inhibition immunoassay CB).
Figure a
* FLUORESCENCE
