Clinica Chimicu Acta, 186 (1989) 315-320 Elsevier
315
CCA 04612
Short Communication
The preparation of monoclonal antibodies which react preferentially with human bone alkaline phosphatase and not liver alkaline phosphatase Craig S. Hill and Robert L. Wolfert Hybritech Inc., San Diego, CA (USA) (Received 2 March 1989; revision received 7 September 1989; accepted 11 September 1989)
Key words: Alkaline phosphatase; Enzyme immunoassay; Radioimmunoassay; Monoclonal antibody
Four different genes code for the main groups of alkaline phosphatase (ALP) isoenzymes: the intestinal, placental, placental-like, and the hepatic/renal/skeletal isoenzyme groups [l-3]. Although the isoenzymes derived from the liver, kidney and bone are the same gene product, they differ from each other on the basis of electrophoretic mobility and, heat and urea stability. These differences are thought to be due to posttranslational modifications such as glycosylation. Identification and quantitation of alkaline phosphatases in human serum is commonly performed for disease diagnosis. However, it is difficult to distinguish between the liver and bone isoenzymes which are often the most clinically relevant [4-61 using procedures such as electrophoresis and/or urea and heat denaturation. Most previous attempts by other laboratories to produce antibodies specific for bone alkaline phosphatase (BAP) over liver alkaline phosphatase (LAP) have resulted in cross-reactive antibodies [l]. Two recent studies have reported monoclonal antibodies which partially distinguish between BAP and LAP [7,8]. However, the antibodies are only 2-5 times more specific for one isoenzyme over the other. We have immunized mice with crude extracts of SAOS-2 cells. This cell line is derived from a human osteosarcoma and produces high levels of BAP which has previously been shown to be biochemically identical to that found in human serum [9-111. We have obtained five different antibodies which preferentially react with BAP in a competitive RIA. In the case of one of the antibodies, we have observed c 3% cross-reactivity with LAP.
Correspondence to: Dr. C.S. Hill, Hybritech Inc., 11085 Torreyana Road, San Diego, CA 92126, USA.
0009-8981/89/$03.50
0 1989 Ekevier Science Publishers B.V. (Biomedical Division)
316
Materials and methods Purification of bone and liver alkaline phosphatase
BAP was extracted from the SAOS-2 human osteosarcoma cell line (ATCC no. HTB 85) which was grown in complete MEM supplemented with 8% horse serum and 2% fetal calf serum. The cells were scraped from the culture flask and washed twice in PBS by centrifugation followed by incubation in an extraction buffer containing 1% NP-40 in a 0.1 mol/l Tris-HCl buffer, pH 8.0. The cells were extracted for one hour at 25” C with gentle stirring followed by centrifugation at 10000 X g for 15 min to remove the cellular debris. The BAP preparation was further purified by running the extract through an anti-ALP immunoaffinity column consisting of p%rified monoclonal antibody (which binds both BAP and LAP) coupled to Affigel-10 (Bio-Rad Laboratories, Richmond, CA). The column was washed with extraction buffer followed by elution with buffer containing 125 mmol/l KCl, 10 mmol/l lysine, pH 11.0. Fractions were collected and monitored for ALP activity. Purification was analyzed by SDS gel electrophoresis. SAOS-2 cells were also extracted with T&on-X 100 for immunization purposes. This was performed by repeated freeze thawing of the cells in PBS followed by centrifugation and resuspension in a T&on-X buffer consisting of 0.1% Triton X-100,1 mmol/l MgC12, 20 mmol/l ZnC12, and 10 mmol/l Tris-HCl, pH 7.4. The cells were sonicated for 30 s at the maximal setting (Heat Systems Ultrasonics Inc., Plainview, NY) and then centrifuged at 10000 x g and resuspended in PBS followed by preparation with Freunds adjuvant for injection into mice. LAP was extracted from human liver by suspending diced, washed liver in an extraction buffer consisting of 30% butanol in a buffer containing 2 mmol/l MgC12, 0.025 mmol/l ZnC12, 10 mmol/l Tris-HCl, pH 7.5, and homogenizing with a Polytron homogenizer (Brinkman Instruments, Westbury, NY). The homogenate was incubated at 25 o C for 16 h followed by 8 h at 4” C with gentle stirring. Centrifugation was performed at 9000 X g for 30 min and the aqueotis phase was separated from the butanol phase and pellet. The aqueous phase was clarified by centrifuging at 20000 X g for 20 min. ALP activity assay
ALP activity was measured by combining 50 ~1 of appropriately diluted sample with 100 ~1 of PNPP solution in microtit:r plates and measuring the rate of PNPP turnover at 405 nm in a V,, microtiter @late reader (Molecular Devices, Palo Alto, CA) in the kinetic reading mode at 25 o C. One unit (U) of activity was defined as the quantity of epzyme that catalyzes the hydrolysis of 1 pm01 of substrate per minute under these conditions. One unit of activity was considered to equal 1 pg of pure BAP or LAP. Monoclonal antibody production BALB/c and A/J mice were immunized
with SAOS-2 cells which had been extracted with a T&on-X containing buffer. Mice were immunized using complete
317
Freunds adjuvant for the initial injection followed by incomplete Freunds adjuvant at day 14. Boosts were repeated at 14day intervals with PBS. Fusion of P3 myeloma and spleen cells from immunized animals was done by routine techniques [12]. Serum titers and initial screening of clones were done by the immuno-assisted enzyme assay or RIA procedures described below. Hybridomas which were identified to be secreting ALP reactive antibodies were grown in mouse ascites. Antibody
assays
An RIA for the detection of BAP or LAP specific antibody was performed by adding 25 ~1 of hybridoma supematant sample to microtiter plate wells followed by 50 1.11of ‘251-labeled, purified BAP. Sepharose beads (Pharmacia, Uppsala, Sweden) coupled with sheep anti-mouse IgG were added and the plates were incubated overnight at 25’ C with gentle shaking. The Sepharose beads from each plate were washed and collected with a cell harvester on paper discs and the discs were counted in a gamma counter. Saturation analysis was performed by: (1) determining the 50% titer point of the antibody sample by the above RIA procedure, and (2) displacing the tracer antigen with increasing concentrations of crude BAP or LAP added to the appropriate dilution of antibody sample along with a fixed amount of 1251purified BAP. The rest of the procedure was as described above for the RIA. An immuno-assisted enzyme assay was performed similarly to Bailyes et al. [8]. Monoclonal antibodies were partially purified from ascites by salt cut and dried down at 1 pg/well on microtiter plates overnight at 37 o C. The plates were rinsed with distilled water followed by a 2-min incubation in a 0.1% Tween-20, PBS solution and then rinsed again with distilled water. Plates were blocked with a 1% BSA, 0.1% Tween-20, PBS solution for 30 min at 37 o C and washed as before. The BAP or LAP extracts were diluted to appropriate concentrations in a solution consisting of 5% Alba and 0.1% thimerosal in PBS. Antigen was added to the plates at 50 @well and incubated 4 h at 37 o C. The plates were washed and incubated with 100 pi/well PNPP substrate and read on a V,, kinetic microtiter plate reader at 405 mn. Results and conclusions
BAP for use as an ‘251-labeled antigen in the RIA was purified from SAOS-2 cells using an NP-40 extraction buffer followed by immunoaffinity purification on a column containing monoclonal antibody against alkaline phosphatase. The final purification resulted in a preparation that was estimated by SDS-gel electrophoresis to be greater than 95% pure using either Coomassie blue or silver staining techniques (data not shown). The final yield was typically approximately 50% from the crude detergent extract based on ALP activity. Antibodies were assayed using an immuno-assisted enzyme assay (IAE assay). When a cross-reactive antibody is tested in this way, using crude BAP or LAP extracts as the antigen, no difference in response is observed between the two antigens over a range of 0 to 1.0 IU/ml (Fig. 1A). A total of 20441 clones were screened from the 19 fusions that were performed. Of these, 354 were positive for
318
A. Cross-reactive
180--
0.00
B.Specific
Ab,
Ab,
BAlB
BAlB
067
[ALPJ
IU/ml
419
0.20 [ALP]
0.40 IU/ml
0.60
0.80
Fig. 1. Plot of an immune-assisted enzyme assay. The x-axis represents the W/ml of the alkaline phosphatase in the added samples as measured enzymatically. The y-axis represents the turnover of PNPP substrate by the alkaline pbosphatase captured by the antibodies in the assay. Antibodies used were: (A) crossreactive antibody, BAlB 067; and (B) specific antibody, BAlF 419. The antigens are SAOS extract(o), or live extract (0).
BAP. Five antibodies were found that had a differential response with BAP and LAP in the IAE assay. Figure 1B is a plot of one of these antibodies. BAlF 419. This antibody shows a 2- to 4-fold preference for BAP over LAP.
319
I
1.0 Alkaline
phosphatase
10.0 concentration
(ng/25
100.0 uL)
1000.0
Fig. 2. Plot of au inhibition RIA using the specific antibody, BAIG 121. Badickdinated BAP was used as the tracer (purified SAOS-2 extract) and crude SAOS-2 extract (0), or Iiver extract (O), were the i~bit~g antigens. Each data point represents the average of duplicate dete~ations.
TABLE I Summary of ra~o~uno~say Antibody
data
Ascites titer WA)
Isotope.
BAP
LAP
l/6,400 l/124,000 l/129,000 l/256,000 l/2,000
IgGl IgG2a IgG2a IgG2a IgG2a
17 5.0 6.0 8.8 16
2200 >lOO 2200 ,200 >200
1,‘256,000 l/282,000 l/84,008 1/830,000 1/106,000
IgG2a IgGl IgGl IgM IgGl
4.0 7.6 13 3.0 21
7.0 6.6 16 1.1 9.0
Spefific
BAIF BAlG BAlG BAlG BAlG
419 017 121 151 339
Cone of unlabeled antigen for 50% inhibition (ng/25 al)
Crvss-Reuctive
BAlB BAlH BAlH BAlH BAlH
067 415 359 1463 1424
% Cross-reactivity with LAP
> > > > >
8.5 5.0 3.0 4.4 8.0
320
Antibodies were further characterized in the RIA format. Figure 2 is a plot of an inhibition RIA using a radio-iodinated BAP tracer (purified from SAOS-2 cells), and either BAP (crude SAOS-2 cell extract), or LAP (liver extract) as the competing antigen. An antibody (BAIG 121) which was previously shown to react preferentially with BAP in the IAE assay was used in the RIA. When unlabeled BAP was used as the competing antigen, a concentration of 6 ng/25 ~1 was required for 50% tracer antigen displacement. In contrast, greater than 200 ng/25 ~1 of the LAP antigen was required to displace 50% of the tracer antigen. This indicates that the BAlG 121 antibody is less than 3% cross-reactive with LAP as compared to BAP. Table I is a summary of the RIA results for the five specific antibodies and representative data for five of the 354 cross-reactive antibodies that we have obtained. The five antibodies that bound BAP preferentially had -C9% cross-reactivity with LAP. References 1 McComb 2 3
4 5 6
7
8
9
10
11
12
RB, Bowen GN, Posen S. Alkaline phosphatase. New York and London: Plenum Press, 1979;313-524. Seargeant LE, Stinson RA. Evidence that three structural genes code for human alkaline phosphatases. Nature 1979;281:152-154. Weiss MJ, Henthom PS, Lafferty MA, Slaughter C, Taducha M, Harris H. Isolation and characterization of a cDNA encoding a human liver/bone/kidney-type alkaline phosphatase. Proc Nat1 Acad Sci USA 1986;83:7182-7186. Moss DW, Edwards RK. Improved electrophoretic resolution of bone ,and liver alkaline phosphatases resulting from partial digestion with neuraminidase. Clin Chim Acta 1984;143:177-182. Onica D, Sundblad L, Waldenlind LL. Affinity electrophoresis of human serum alkaline phosphatase isoenzymes in agarose gel containing lectin. Clin Chim Acta 1986;155:285-294. Sinha PK, Bianchi-Bosisio A, Meyer-Sabellek W, Righetti PG. Resolution of alkaline phosphatase isoenzymes in serum by isoelectric focusing in immobilized pH gradients. Clin Chem 1986;32:1264-1268. Lawson GM, Katzmann JA, Kimlinger TK, O’Brien JF. Isolation and preliminary characterization of a monoclonal antibody that interacts preferentially with the liver isoenzyme of human alkaline phosphatase. Chn Chem 1985;31:381-385. Bailyes EM, Seabrook RN, Calvin J, Maquire GA, et al. The preparation of monoclonal antibodies to human bone and liver alkaline phosphatase and their use in immunoaffinity purification and in studying these enzymes when present in serum. Biochem J 1987;244:725-733. Farley JR, Kyeyune-Nyombi E, Tarbaux NM, Hall SL, Strong DD. Alkaline phosphatase activity from human osteosarcoma cell line SAOS-2: an isoenzyme standard for quantifying skeletal alkaline phosphatase activity in serum. Chn Chem 1989;35:223-229. Fedde KN, Lane CC, Whyte MP. Alkaline phosphatase is an ectoenzyme that acts on micromolar concentrations of natural substrates at physiologic pH in human osteosarcoma (SAOS-2) cells. Arch Biochem Biophys 1988;264:400-409. Murray E, Prowedini D, Curran D, Catherwood B, Sussman H, Manolagas S. Characterization of a human osteoblastic osteosarcoma cell line (SAOS-2) with high bone alkaline phosphatase activity. J Bone Min Res 1987;2:231-238. Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of pre-defined specificity. Nature (London) 1975;256:495-497.
315
CCA 04612
Short Communication
The preparation of monoclonal antibodies which react preferentially with human bone alkaline phosphatase and not liver alkaline phosphatase Craig S. Hill and Robert L. Wolfert Hybritech Inc., San Diego, CA (USA) (Received 2 March 1989; revision received 7 September 1989; accepted 11 September 1989)
Key words: Alkaline phosphatase; Enzyme immunoassay; Radioimmunoassay; Monoclonal antibody
Four different genes code for the main groups of alkaline phosphatase (ALP) isoenzymes: the intestinal, placental, placental-like, and the hepatic/renal/skeletal isoenzyme groups [l-3]. Although the isoenzymes derived from the liver, kidney and bone are the same gene product, they differ from each other on the basis of electrophoretic mobility and, heat and urea stability. These differences are thought to be due to posttranslational modifications such as glycosylation. Identification and quantitation of alkaline phosphatases in human serum is commonly performed for disease diagnosis. However, it is difficult to distinguish between the liver and bone isoenzymes which are often the most clinically relevant [4-61 using procedures such as electrophoresis and/or urea and heat denaturation. Most previous attempts by other laboratories to produce antibodies specific for bone alkaline phosphatase (BAP) over liver alkaline phosphatase (LAP) have resulted in cross-reactive antibodies [l]. Two recent studies have reported monoclonal antibodies which partially distinguish between BAP and LAP [7,8]. However, the antibodies are only 2-5 times more specific for one isoenzyme over the other. We have immunized mice with crude extracts of SAOS-2 cells. This cell line is derived from a human osteosarcoma and produces high levels of BAP which has previously been shown to be biochemically identical to that found in human serum [9-111. We have obtained five different antibodies which preferentially react with BAP in a competitive RIA. In the case of one of the antibodies, we have observed c 3% cross-reactivity with LAP.
Correspondence to: Dr. C.S. Hill, Hybritech Inc., 11085 Torreyana Road, San Diego, CA 92126, USA.
0009-8981/89/$03.50
0 1989 Ekevier Science Publishers B.V. (Biomedical Division)
316
Materials and methods Purification of bone and liver alkaline phosphatase
BAP was extracted from the SAOS-2 human osteosarcoma cell line (ATCC no. HTB 85) which was grown in complete MEM supplemented with 8% horse serum and 2% fetal calf serum. The cells were scraped from the culture flask and washed twice in PBS by centrifugation followed by incubation in an extraction buffer containing 1% NP-40 in a 0.1 mol/l Tris-HCl buffer, pH 8.0. The cells were extracted for one hour at 25” C with gentle stirring followed by centrifugation at 10000 X g for 15 min to remove the cellular debris. The BAP preparation was further purified by running the extract through an anti-ALP immunoaffinity column consisting of p%rified monoclonal antibody (which binds both BAP and LAP) coupled to Affigel-10 (Bio-Rad Laboratories, Richmond, CA). The column was washed with extraction buffer followed by elution with buffer containing 125 mmol/l KCl, 10 mmol/l lysine, pH 11.0. Fractions were collected and monitored for ALP activity. Purification was analyzed by SDS gel electrophoresis. SAOS-2 cells were also extracted with T&on-X 100 for immunization purposes. This was performed by repeated freeze thawing of the cells in PBS followed by centrifugation and resuspension in a T&on-X buffer consisting of 0.1% Triton X-100,1 mmol/l MgC12, 20 mmol/l ZnC12, and 10 mmol/l Tris-HCl, pH 7.4. The cells were sonicated for 30 s at the maximal setting (Heat Systems Ultrasonics Inc., Plainview, NY) and then centrifuged at 10000 x g and resuspended in PBS followed by preparation with Freunds adjuvant for injection into mice. LAP was extracted from human liver by suspending diced, washed liver in an extraction buffer consisting of 30% butanol in a buffer containing 2 mmol/l MgC12, 0.025 mmol/l ZnC12, 10 mmol/l Tris-HCl, pH 7.5, and homogenizing with a Polytron homogenizer (Brinkman Instruments, Westbury, NY). The homogenate was incubated at 25 o C for 16 h followed by 8 h at 4” C with gentle stirring. Centrifugation was performed at 9000 X g for 30 min and the aqueotis phase was separated from the butanol phase and pellet. The aqueous phase was clarified by centrifuging at 20000 X g for 20 min. ALP activity assay
ALP activity was measured by combining 50 ~1 of appropriately diluted sample with 100 ~1 of PNPP solution in microtit:r plates and measuring the rate of PNPP turnover at 405 nm in a V,, microtiter @late reader (Molecular Devices, Palo Alto, CA) in the kinetic reading mode at 25 o C. One unit (U) of activity was defined as the quantity of epzyme that catalyzes the hydrolysis of 1 pm01 of substrate per minute under these conditions. One unit of activity was considered to equal 1 pg of pure BAP or LAP. Monoclonal antibody production BALB/c and A/J mice were immunized
with SAOS-2 cells which had been extracted with a T&on-X containing buffer. Mice were immunized using complete
317
Freunds adjuvant for the initial injection followed by incomplete Freunds adjuvant at day 14. Boosts were repeated at 14day intervals with PBS. Fusion of P3 myeloma and spleen cells from immunized animals was done by routine techniques [12]. Serum titers and initial screening of clones were done by the immuno-assisted enzyme assay or RIA procedures described below. Hybridomas which were identified to be secreting ALP reactive antibodies were grown in mouse ascites. Antibody
assays
An RIA for the detection of BAP or LAP specific antibody was performed by adding 25 ~1 of hybridoma supematant sample to microtiter plate wells followed by 50 1.11of ‘251-labeled, purified BAP. Sepharose beads (Pharmacia, Uppsala, Sweden) coupled with sheep anti-mouse IgG were added and the plates were incubated overnight at 25’ C with gentle shaking. The Sepharose beads from each plate were washed and collected with a cell harvester on paper discs and the discs were counted in a gamma counter. Saturation analysis was performed by: (1) determining the 50% titer point of the antibody sample by the above RIA procedure, and (2) displacing the tracer antigen with increasing concentrations of crude BAP or LAP added to the appropriate dilution of antibody sample along with a fixed amount of 1251purified BAP. The rest of the procedure was as described above for the RIA. An immuno-assisted enzyme assay was performed similarly to Bailyes et al. [8]. Monoclonal antibodies were partially purified from ascites by salt cut and dried down at 1 pg/well on microtiter plates overnight at 37 o C. The plates were rinsed with distilled water followed by a 2-min incubation in a 0.1% Tween-20, PBS solution and then rinsed again with distilled water. Plates were blocked with a 1% BSA, 0.1% Tween-20, PBS solution for 30 min at 37 o C and washed as before. The BAP or LAP extracts were diluted to appropriate concentrations in a solution consisting of 5% Alba and 0.1% thimerosal in PBS. Antigen was added to the plates at 50 @well and incubated 4 h at 37 o C. The plates were washed and incubated with 100 pi/well PNPP substrate and read on a V,, kinetic microtiter plate reader at 405 mn. Results and conclusions
BAP for use as an ‘251-labeled antigen in the RIA was purified from SAOS-2 cells using an NP-40 extraction buffer followed by immunoaffinity purification on a column containing monoclonal antibody against alkaline phosphatase. The final purification resulted in a preparation that was estimated by SDS-gel electrophoresis to be greater than 95% pure using either Coomassie blue or silver staining techniques (data not shown). The final yield was typically approximately 50% from the crude detergent extract based on ALP activity. Antibodies were assayed using an immuno-assisted enzyme assay (IAE assay). When a cross-reactive antibody is tested in this way, using crude BAP or LAP extracts as the antigen, no difference in response is observed between the two antigens over a range of 0 to 1.0 IU/ml (Fig. 1A). A total of 20441 clones were screened from the 19 fusions that were performed. Of these, 354 were positive for
318
A. Cross-reactive
180--
0.00
B.Specific
Ab,
Ab,
BAlB
BAlB
067
[ALPJ
IU/ml
419
0.20 [ALP]
0.40 IU/ml
0.60
0.80
Fig. 1. Plot of an immune-assisted enzyme assay. The x-axis represents the W/ml of the alkaline phosphatase in the added samples as measured enzymatically. The y-axis represents the turnover of PNPP substrate by the alkaline pbosphatase captured by the antibodies in the assay. Antibodies used were: (A) crossreactive antibody, BAlB 067; and (B) specific antibody, BAlF 419. The antigens are SAOS extract(o), or live extract (0).
BAP. Five antibodies were found that had a differential response with BAP and LAP in the IAE assay. Figure 1B is a plot of one of these antibodies. BAlF 419. This antibody shows a 2- to 4-fold preference for BAP over LAP.
319
I
1.0 Alkaline
phosphatase
10.0 concentration
(ng/25
100.0 uL)
1000.0
Fig. 2. Plot of au inhibition RIA using the specific antibody, BAIG 121. Badickdinated BAP was used as the tracer (purified SAOS-2 extract) and crude SAOS-2 extract (0), or Iiver extract (O), were the i~bit~g antigens. Each data point represents the average of duplicate dete~ations.
TABLE I Summary of ra~o~uno~say Antibody
data
Ascites titer WA)
Isotope.
BAP
LAP
l/6,400 l/124,000 l/129,000 l/256,000 l/2,000
IgGl IgG2a IgG2a IgG2a IgG2a
17 5.0 6.0 8.8 16
2200 >lOO 2200 ,200 >200
1,‘256,000 l/282,000 l/84,008 1/830,000 1/106,000
IgG2a IgGl IgGl IgM IgGl
4.0 7.6 13 3.0 21
7.0 6.6 16 1.1 9.0
Spefific
BAIF BAlG BAlG BAlG BAlG
419 017 121 151 339
Cone of unlabeled antigen for 50% inhibition (ng/25 al)
Crvss-Reuctive
BAlB BAlH BAlH BAlH BAlH
067 415 359 1463 1424
% Cross-reactivity with LAP
> > > > >
8.5 5.0 3.0 4.4 8.0
320
Antibodies were further characterized in the RIA format. Figure 2 is a plot of an inhibition RIA using a radio-iodinated BAP tracer (purified from SAOS-2 cells), and either BAP (crude SAOS-2 cell extract), or LAP (liver extract) as the competing antigen. An antibody (BAIG 121) which was previously shown to react preferentially with BAP in the IAE assay was used in the RIA. When unlabeled BAP was used as the competing antigen, a concentration of 6 ng/25 ~1 was required for 50% tracer antigen displacement. In contrast, greater than 200 ng/25 ~1 of the LAP antigen was required to displace 50% of the tracer antigen. This indicates that the BAlG 121 antibody is less than 3% cross-reactive with LAP as compared to BAP. Table I is a summary of the RIA results for the five specific antibodies and representative data for five of the 354 cross-reactive antibodies that we have obtained. The five antibodies that bound BAP preferentially had -C9% cross-reactivity with LAP. References 1 McComb 2 3
4 5 6
7
8
9
10
11
12
RB, Bowen GN, Posen S. Alkaline phosphatase. New York and London: Plenum Press, 1979;313-524. Seargeant LE, Stinson RA. Evidence that three structural genes code for human alkaline phosphatases. Nature 1979;281:152-154. Weiss MJ, Henthom PS, Lafferty MA, Slaughter C, Taducha M, Harris H. Isolation and characterization of a cDNA encoding a human liver/bone/kidney-type alkaline phosphatase. Proc Nat1 Acad Sci USA 1986;83:7182-7186. Moss DW, Edwards RK. Improved electrophoretic resolution of bone ,and liver alkaline phosphatases resulting from partial digestion with neuraminidase. Clin Chim Acta 1984;143:177-182. Onica D, Sundblad L, Waldenlind LL. Affinity electrophoresis of human serum alkaline phosphatase isoenzymes in agarose gel containing lectin. Clin Chim Acta 1986;155:285-294. Sinha PK, Bianchi-Bosisio A, Meyer-Sabellek W, Righetti PG. Resolution of alkaline phosphatase isoenzymes in serum by isoelectric focusing in immobilized pH gradients. Clin Chem 1986;32:1264-1268. Lawson GM, Katzmann JA, Kimlinger TK, O’Brien JF. Isolation and preliminary characterization of a monoclonal antibody that interacts preferentially with the liver isoenzyme of human alkaline phosphatase. Chn Chem 1985;31:381-385. Bailyes EM, Seabrook RN, Calvin J, Maquire GA, et al. The preparation of monoclonal antibodies to human bone and liver alkaline phosphatase and their use in immunoaffinity purification and in studying these enzymes when present in serum. Biochem J 1987;244:725-733. Farley JR, Kyeyune-Nyombi E, Tarbaux NM, Hall SL, Strong DD. Alkaline phosphatase activity from human osteosarcoma cell line SAOS-2: an isoenzyme standard for quantifying skeletal alkaline phosphatase activity in serum. Chn Chem 1989;35:223-229. Fedde KN, Lane CC, Whyte MP. Alkaline phosphatase is an ectoenzyme that acts on micromolar concentrations of natural substrates at physiologic pH in human osteosarcoma (SAOS-2) cells. Arch Biochem Biophys 1988;264:400-409. Murray E, Prowedini D, Curran D, Catherwood B, Sussman H, Manolagas S. Characterization of a human osteoblastic osteosarcoma cell line (SAOS-2) with high bone alkaline phosphatase activity. J Bone Min Res 1987;2:231-238. Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of pre-defined specificity. Nature (London) 1975;256:495-497.
