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Quantitation of peroxidase-antibody binding to membrane fragments using column chromatography
ANALYTICAL
98, 53-59
RIOCHEMISTRY
Quantitation
of Peroxidase-Antibody Using Column
A method specifi~:ally The technique taining
(1979)
bound
which bound
can
be
used
to
Binding to Membrane Chromatography
measure
the
to placental uses Sepharose
alkaline phosphatase 4B chromatography
peroxidase-antibody
from
demonstrated by nonbinding tween membrane-associated ventin;< binding using pure
amount
unbound
to rabbit placental phosphatase and enzyme. The general
of peroxidase
in membrane to separate
antibody
fragments membrane
peroxidase-antibody.
Fragments
which
is
is described. fragments conSpecificity
is
membranes, by a strict correlation bebound peroxidase-antibody. and by preutility of this method for membrane antigens
is discussed.
Immunohistochemistry is accepted as a valid technique for localizirig antigens on the cell surface. The most popular reagents for this purpose are enzyme conjugates to specific antibodies, either covalent ( 1,2) or by use of enzyme-antibody complexes and “bridging” antiimmunoglobulins (3). The specificity of reaction is generally confirmed by finding an “appropriate” cellular localization of the antigen of interest and by competition of purified soluble antigen for binding of labeled antibody. If this approach for localization of specific antigen is valid, then it should be possible to confirm thle specificity of the technique by measuring the amount of membraneassociated antigen. by quantitating the amount of peroxidase-antibody which binds to it. and by demonstrating the competition of soluble antigen for this binding. This would have the advantage of rigorously validating the use of the immunohistochemical technique and might be useful in “probing” the relative exposure of a given antigen in several different cell types. Technical difficulties associated with this approach include the establishment of an independent measure of antigen concentra-
tion, and consideration of conditions for preparation of tissues so that the membrane containing antigen is maximally exposed for reaction with the labeled antibody. We were particularly interested in developing such an approach for human placental alkaline phosphatase. This enzyme is known to be associated with the microvillus membrane (4,5) and is found not only in human placentae, but also in neoplastic tissues (6), especially cancer of the ovary (7), and in cultured human cells (8- 10). Immunochemical (11) and chemical (12) evidence support an identity of the purified enzyme in tumor and placental tissue. It remains to be established whether the enzymes in these different tissues are in fact identical in their cellular localization, function, or microenvironment in the membrane. The specificity of the immunoperoxidase method for histochemical localization of this enzyme was previously demonstrated (13). The enzyme can be measured by enzyme assay when it is membrane bound, and this appears to be a reasonable measure of enzyme protein concentration. since (i) there is no loss of enzyme activity after 53
0003.2697179/130053-07$0?.00/0 Copynghl All right\
11 1979 by Academc Prer,. Inc. 01 reproductwn in any term rcxrved
54
TAYLOR
AND
butanol extraction of membrane preparations (14), (ii) immunoquantitation by radial immunodiffusion ( 15) and complement fixation (16) show no change in immunoreactive enzyme upon purification, and (iii) measurement of enzyme protein by radial immunodiffusion in Triton-containing gels showed that the membrane-associated enzyme did not differ from the soluble enzyme in the amount of enzyme protein per unit activity (15). The ability to measure the enzyme concentration in the membrane independently, and the availability of pure enzyme (14) made this a well-defined system for testing the specific interaction of peroxidase-antibody with membranes containing this antigen. The maximal exposure of membrane containing antigen was achieved by preparing membrane fragments in rabbit serum. The separation of bound from unbound peroxidase-antibody was achieved by using gel filtration on agarose media (Sepharose 4B). Using this approach, we have been able to reproducibly measure the amount of peroxidase-antibody which can bind to membraneassociated placental phosphatase. By testing the competition of soluble and membraneassociated phosphatase for peroxidaseantibody, it was found that the soluble enzyme binds the antibody more effectively. MATERIALS
AND METHODS
Rabbit serum (select hemolyzed sterile) was obtained from Pel-Freeze Biologicals, Rogers, Arkansas. Sephadex G-25, Sephadex G-200, and Sepharose 4B were purchased from Pharmacia Fine Chemicals, Uppsala. Sweden. Peroxidase, RZ 2.65, was obtained from Sigma Chemical Company, St. Louis, Missouri. All other chemicals were reagent grade from Fisher Chemical Company. Human term placentae were obtained from the obstetric service of Forsyth Memorial Hospital. Preterm placentae were
DOELLGAST
obtained from the gynecology service of North Carolina Baptist Hospital. Rabbit placentae were removed from a female San Juan rabbit 38 days after mating.
PrepLlration’c!f‘HM W-PAP.’ Tissues were homogenized for 60 s in a Waring blender with 2 ml Tris-acetate-buffered saline, pH 7.6, per gram of tissue. This was further homogenized for 30 s with a Polytron homogenizer. The homogenate was centrifuged for 30 min at 27,OOOg,,,. The pellet was discarded and the supernatant was centrifuged at 150,000gmax for 60 min. The pellet was washed twice, resuspended in rabbit serum, sonicated for 60 s in 15-s bursts, and centrifuged at lSO,OOOg,,, for 60 min. This supernatant was passed through a Sepharose 4B column (1.5 x 26 cm) equilibrated with rabbit serum. The excluded fractions (determined by assaying for PAP) were pooled and this pool was termed the “high-molecular-weight placental alkaline phosphatase” (HMW-PAP). Figure 1 gives an outline of this protocol. Prepcrration
of prrrijied atztihody to PAP.
Homogeneous human PAP was prepared as previously described (14). Rabbits were sensitized by injection of purified PAP and monospecific antiserum was obtained (14). An immunoabsorbent column was prepared by covalently coupling immunoabsorbentpurified PAP to cyanogen bromide-activated Sepharose. Monospecific antiserum was applied to the PAP-immunoabsorbent column; the column was exhaustively washed with 10 mM Tris-acetate-buffered saline, pH 7.6, and the antibodies were eluted using 5 mM sodium phosphate buffer, pH 12. The anti-PAP antibodies were further chromatoI Abbreviations used: HMW-PAP, high-molecularweight placental alkaline phosphatase (membranebound) excluded from Sepharose 4B gel: IgG. immunoglobulin G: PO-Ab. peroxidase-labeled antibody to human placental alkaline phosphatase: BSA. bovine serum albumin: DMA. ovarian cancer patient.
PEROXIDASE-ANTIBODY
Tissue
BINDING
+ 2 ml
10 mM Tris-buffered
Homogenize and then at 27,000
Pellet (save)
.\
TO MEMBRANE
saline,
FRAGMENTS
55
pH 7.6/gram
in a Waring blender 30 set in a Polytron. xgmax for 30 min.
for 60 set Centrifuge
supernatant Centrifuge
at
150,000
xgmax
for
60 min.
I
Resuspend in and centrifuge 60 min.
Supernatant
FIG.
I. Scheme
rabbit at
(HMW fragments
for the preparations
graphed on a Sephadex G-200 column (2.5 x 75 cm) to obtain anti-PAP IgG. Pcroxidrtsr lrrhrlittg. The purified IgG fraction was peroxidase labeled by a modification of the method of Nakane and Kawaoi (2) in that the “activated” peroxidase is separated from the reagents by chromatography on Sephadex G-25. The solution of PO-Ab was diluted to 3.4 x lo-’ mg peroxidase/ml in rabbit serum before use. Proteitl c’otz(.etttrvttiotts. These were determined by the method of Lowry et trl. (17) using a BSA protein standard and expressed as milligrams per milliliter. AIktrlitre pirosphtrtnsr ~~ssny. Alkaline phosphatase was assayed at pH 9.8 as described previously (14). All samples were heated at 65°C for 10 min and assayed in the presence of 0.1 mM bromotetramisole (18). The amount of PAP was determined as prn801.min’ . ml-’ and expressed as ngiml by dividing by the specific activity for PAP (450 /dmol.rnin~ ’ .mg-‘) x 10” (14). Po.o.rititrsr trsstr.~. Assays for peroxidase were performed by a variation of a previously published method (19). A stock peroxide solution containing 0.3% peroxide
of HMW
serum, 150,000
in
sonicate, zigmax for
rabbit
fragments
serum)
from
tissues.
(in distilled water) was diluted 1:50 with 10 mM phosphate buffer, pH 6.0 (fresh daily) to produce the working solution. The working solution (0.5 ml) was placed into each assay tube, in addition to 10 ~1 of 1% orthodianisidine, in isopropanoliwater ( 1: 1). The sample was added to each assay tube and the mixture was incubated at 24°C for 10 min. After 10 min. the reaction was stopped by the addition of 100 ~1 of 2% sodium azide followed by 1 ml of methanol. This was mixed thoroughly and the absorbance was measured at 460 nm. Peroxidase standards were prepared from a stock peroxidase solution (5 x IO--’ mg peroxidasei ml). The amount of peroxidase present in samples was calculated as ngiml, using a peroxidase standard assayed simultaneously as a reference. Mrrrstr~ettlrttt of’PO-Ah hitltlittg to HM WPAP. A quantity of HMW-PAP (in rabbit serum) was placed in a test tube and PO-Ab was added. This reaction volume was brought to 1 ml with rabbit serum and incubated for 2 h at 37°C. After 2 h, the sample was applied to a Sepharose 4B column (0.7 x 48 cm) and fractions were
56
TAYLOR
AND
DOELLGAST
collected. The fractions were assayed for protein, placental phosphatase. and peroxidase. RESULTS
The linearity of the modified peroxidase assay was tested over the range of 0 to 10 ng peroxidase. In Fig. 2, it is apparent that the linear range of the assay is 0 to 7 ng peroxidase (correlation coefficient equal to 0.997). All assays were therefore performed within this range. A typical Sepharose 4B elution profile for HMW-PAP with PO-Ab bound is shown in Fig. 3. A clear separation of the bound and unbound peroxidase antibody is obtained. To measure the amount of binding of POAb to HMW-PAP, increasing quantities of PO-Ab (O-40 pg protein) were added to a constant amount of placental HMW-PAP (containing 120 ng PAP). The amount of POAb binding to the HMW-PAP was plotted against the amount of PO-Ab added (Fig. 4). The relationship appears to be linear up to 20 pg PO-Ab, after which there is an apparent saturation of the antigenic binding sites. Using a concentration of 6.7 pg of peroxidase-antibody, we tested the amount of peroxidase antibody bound to varying amounts of HMW-PAP. In Fig. 5, it is seen that there is a linear correlation over the range of O-240 ng of HMW-PAP. To determine whether the PO-Ab were specifically binding to the HMW-PAP, three
q
of
Peroxidose
added
FIG. 2. Standard curve forperoxidase assay (straight line given by the least-squares method of linear regression).
FRACTION
NUMBER
FIG. 3. Elution profile of tumor (DMA) HMW-PAP, plus PO-Ab from a Sepharose 4B column assayed for protein in mgiml (A -A ); PAP. absorbance at 505 nm (0 ---0 ): and peroxidase. absorbance at 460 nm (O---O).
experiments were performed. First, HMWPAP of rabbit placentae was examined. No detectable PO-Ab bound to HMW-PAP of rabbit placentae. Second, pure soluble PAP was added to 120 ng of human placental HMW-PAP and increasing quantities of PO-Ab (O-10 pg protein) in an effort to competitively bind PO-Ab. Figure 6 shows the effects of adding 0- 120 ng soluble PAP. Eleven nanograms soluble PAP inhibits PO-Ab binding to the HMW-PAP by 16%, 58 ng by 55%. and 120 ng by 8470. Since there were equal amounts of HMW-PAP and pure PAP in the 120 ng experiment, it appears that the pure PAP is better able to bind the PO-Ab than the HMW-PAP, suggesting steric hindrance in the mem-
PO-Ab
added
IN
protein)
FIG. 4. Quantitation of Sepharose 4B-excluded POAb upon addition of increasing increments of PO-Ab to placental HMW-PAP (containing 120 ng PAP).
PEROXIDASE-ANTIBODY
200
100
present
P&P
BINDING
TO MEMBRANE
PO-Ab
300
trig PAP)
TABLE TP.TION
OF SEPHAROSE
4%EXCLUDED
CONTAINING
Sample Term
WITH
LEVELS
DIFFERENT
PI ACENTAL
SAMPLES
or- PAP”
Protein (mg)
PAP
(ng)
PO-Ab trig)
2.73
129.6 62.6
7.6 3.7
2.78 1.39
0.059 0.059
46.8 245.2
2.5 14.1
1.00
0.053
5.26
189.0
10.9 13.6
0.057 0.058
ng PO-Abimg
protein
ng PO-Abing
placentae
4546 4554
2.66 2.50
4560 4574 4581
2.68 2.71
4583 4587 4589
2.73 2.72 2.70
256.8 168.0 273.8
1.91 1.84
2.7 240.0
Early
PO-Ah binding to HMW ng PAP) upon addition of PAP, (b) I I ng soluble PAP. I?0 ng soluble PAP.
I
PO-Ab VARIOLIS
(Kg proteInI
early placentae. The 12-week placenta contained low levels of PAP (2.7 ng) and no PO-Ab could be detected in the excluded portion. The 22-week placenta contains appreciable levels of PAP (240 ng) and is capable of binding PO-Ab: however, the ratio of excluded PO-Ab to HMW-PAP is significantly different from that for the term placenta. We attempted to use the simpler approach of incubating labeled antibody with suspensions of membrane in buffer. then removing bound antibody by ultracentrif-
brane. Finally, we tested the binding of PO-Ab to different placental HMW-PAP preparations, containing varying amounts of PAP, but thl: same amount of membrane protein. Table 1 shows the results for this experiment in (eight normal-term placentae, differing significantly in HM W-PAP activity. Examining the ratios of PO-Ab to protein and PO-Ab to PAP, the amount of PO-Ab bound correlat.es best with the amount of HMW-PAP. Table I also shows the results of a similar study on the HMW-PAP of two
QUANTI
added
FIG. 6. Inhibition of fragments (containing 120 soluble PAP. (a) No soluble Cc) 58 ng soluble PAP, Cdl
FIG. 5. Quantitation of Sepharose 4B-excluded POAb upon addition of increasing increments of PAP containing HMW fragments to a constant amount of PO-Ab (6.7 pg).
57
FRAGMENTS
10.7 16.0
4.02 4.98 3.93 5.93
0.053 0.064 0.058
5.82
0.045
placentae
12 week 22 week
” 6.7 Fg PO-Ab ’ Not detectable.
added
to each sample.
-
ND” 10.7
PAP
58
TAYLOR AND DC)EI.LGAST
ugation (20). The amount of measurable peroxidase-antibody bound to the pellets was neither a linear function of the amount of peroxidase-antibody added at constant membrane concentration, nor of the amount of membrane added at constant peroxidaseantibody concentration. This was true whether or not the pellets were extracted with 1% Triton X-100 prior to peroxidase assay.
divalent antibody. Since we did not use this technique at saturating antibody concentrations, and since we have an independent measure of membrane-associated ligand by enzyme assay (see introduction), it is interesting to compare our results with Reynolds’s model. In her analysis, formation of predominantly divalent complexes and monovalent complexes of divalent antibody with cell surface ligand are compared. In the case of predominantly DISCUSSION divalent complexes, the fraction of antiThis study has demonstrated that per- body bound is proportional to the square oxidase-labeled antibody to human placental of the cell-bound ligand concentration In the alkaline phosphatase binds specifically to divided by the cell concentration. placental membrane fragments containing case of only a single “arm” of the antithis antigen. Specificity for the placenta1 body being bound to the cell membrane phosphatase was demonstrated by (i) non- ligand, the amount bound is directly proreactivity with rabbit placental phosphatase, portional to the cell membrane ligand con(ii) competition of pure placental phos- centration. In Table I, we compare the amount of bound peroxidase-antibody with phatase for binding to placenta1 membranes, and (iii) a strict correlation between the the amount of enzyme present on eight difamount of placental phosphatase present ferent placental membrane preparations on membrane fragments and the amount of with a sixfold range of enzyme specific peroxidase-antibody bound. activity. The amount bound was directly Placental alkaline phosphatase is a com- proportional to enzyme concentration over plex protein with numerous antigenic deter- this range, and so it would appear that the using this minants. We are using this assay in a con- binding of peroxidase-antibody by attachment centration range which results in only a assay occurs predominantly small fraction of the total labeled antibody of only one arm of the divalent antibody. binding to enzyme. Since we have found According to Reynolds (21), this result that 72% of this labeled antibody preparation suggests that orientation factors and recan bind to a large excess of placental mem- striction of motion of both surface ligand branes (Taylor, Homesley, and Doellgast, in and bound antibody had restricted the preparation), the small amount of bound binding of the second arm of the bound antibody would appear to be due to the peroxidase-antibody. It would be interesting to use this system to test other predictions specific binding of high avidity antibodies under these assay conditions, or to partial of Reynolds’s model. In this work. we did not attempt such a shielding of the antigenic sites in the membrane. The latter explanation is in part detailed analysis, but have used the measure supported by the very effective competition of membrane-bound enzyme activity as a of the soluble enzyme for binding of the measure of enzyme protein concentration. We thus are measuring the “availability” peroxidase-antibody (Fig. 6). As has been forcefully argued by of antigenic sites for peroxidase-antibody Reynolds (21), the complex equilibria binding. In the one early (22-week) placental between divalent antibody and membrane sample we tested, we found significantly less antigens precludes measurement of the labeled antibody bound. This result suggests number of antigenic sites per cell using the application of this technique as a
PEROXIDASE-ANTIBODY
BINDING
of the exposure of antigenic “probe” determinants of membrane-bound enzyme from different. sources. We are currently using the technique in this way for comparison of cancer tissue and ascites fluid (22) membrane fragments (Taylor, Homesley, and Doellgast ( in preparation). In conclusion. we note that the technique described above (i) rigorously confirmed the specificity of our peroxidase-labeled antiplacental phosphatase as a histochemical reagent, (ii) shows some potential for antigen quantitation on membrane preparations, and (iii) could be useful in comparing the relative exposure of antigenic sites on different membrane preparations if an independent measure of antigen concentration is available. ACKNOWLEDGMENTS This work was supported by National Cancer Institute, National Institutes of Health Grant CA2353301, and by the Gaston County Cancer Society and Forsyth Cancer Service. We are grateful to the obstetrical nursing staff of Forsyth Memorial Hospital for placental tissue and to Dr. Howard Homesley for prelerm placentae.
REFERENCES I. Nakane. P. K.. and H;.,tochem. Cytrdwrn. 2. Nakane, clror~.
Pierce. G. 14, 929.
B.
P. K.. and Kawaoi, A. (1974) Cy,ochm. 22. 1084- 1091.
(1966)
.1.
.I. Hi.\tw
3. Steinberger. L. A., Hardy, P. H . . Cuculis. J. J.. and Meyer. H. G. (1970) ./. Hi.rr~x.ltr~i. c,vroc~ltrrtl. 18. 315-333. 4. Zijlstra, J. B., Torringa. J. M. W. (1970) @rteco/.
J. L.. Ini.e.sf.
and Bowma. 1. 160- 170.
TO MEMBRANE
FRAGMENTS
59
5. Carlson. R. W.. Wada. H. G.. and Sussman. H. H. (1976) .I. Bid. Clwtt1. 251, 4139-4146. 6. Fishman, W. H., Inglis. N. R.. Stolbach, L. L., and Krant. M. J. (~~~~)CU)I(.PTR~S. 28. l50- 154. 7. Inglis. N. R.. Kirley. S., Stolbach. L. L.. and Fishman, W. H. (1973) Cnrrccr Rcr. 33. 1657-1661. 8. Fishman. W. H.. lnglis. N. R.. Anstiss. C. L.. Ghosh. N. K.. Reif. A. E., Rustigian, R.. Krant, M. J.. and Stolbach. L. L. (1968) Ntrrrlrr (Lorrclrw~~ 219, 697-700. 9. Elson. N. A., and Cox. R. P. (1969) Eiochc,rtt. Genct. 3. 549. IO. Singer. R. M.. and Fishman. W. H. (1975) irk Isozymes; Developmental Biology (Markert, C. L., ed.), Vol. 3. pp. 753-774, Academic Press. New York. 1 I. Fishman. W. H.. Inglis. N. R.. and Green, S. t 1971) Conc,er Res. 31. 1054- 1057. 12. Greene. P. J.. and Sussman, H. H. (1973) Pr-oc. Nor. Acud. SC.;. USA 70, 2936-2940. 13. Miyayama. H.. Doellgast, G. J.. Memoli, V. A.. Gandbhir. L., and Fishman, W. H. (1976) Cancer 38, l?37- 1250. 14. Doellgast, G. J., Spiegel, J.. Guenther, R. A.. and Fishman. W. H. t 1977) Bioclrinl. Biophy.r Acts 484, S9- 78. 15. Doellgast. G. J. (1977)A,rrrl. Birx,hem. 82,?78-285. 16. Rule. A. H.. DiNapoli. A., Green, S.. Fishman, W. H.. and Doellgast. G. J. (1979) 1. Imr~~nol. .Met/r.. in press. 17. Lowry, 0. H.. Rosebrough, N. J., Farr. A. L., and Randall. R. J. ( 1951) .I. Biol. Che177. 193, 265-275. 18. VanBelle. H., DeBroe, M. E.. and Wieme. R. J. (1977) Clirlictrl Chrm. 23, 454-459. 19. Wcwthirrgtm En;?rne M~nrrcrl (1972) Worthington Biochemical Corp., Freehold. N. J. 20. Louvard. D.. Vannier, C.. Maroux. S.. Pages. J.-M., and Lazdunski, C. (1976) Anal. Biwltam. 76. X3-94. 21. Reynolds, J. A. (1979) Bioc~hemisrr~ 18, 264-269. 22. Doellgast. G. J., and Fishman, W. H. (1974) iii lsozymes: Molecular Structure (Markert. C. L.. ed.). Vol. I. pp. 293-314, Academic Press. New York.
98, 53-59
RIOCHEMISTRY
Quantitation
of Peroxidase-Antibody Using Column
A method specifi~:ally The technique taining
(1979)
bound
which bound
can
be
used
to
Binding to Membrane Chromatography
measure
the
to placental uses Sepharose
alkaline phosphatase 4B chromatography
peroxidase-antibody
from
demonstrated by nonbinding tween membrane-associated ventin;< binding using pure
amount
unbound
to rabbit placental phosphatase and enzyme. The general
of peroxidase
in membrane to separate
antibody
fragments membrane
peroxidase-antibody.
Fragments
which
is
is described. fragments conSpecificity
is
membranes, by a strict correlation bebound peroxidase-antibody. and by preutility of this method for membrane antigens
is discussed.
Immunohistochemistry is accepted as a valid technique for localizirig antigens on the cell surface. The most popular reagents for this purpose are enzyme conjugates to specific antibodies, either covalent ( 1,2) or by use of enzyme-antibody complexes and “bridging” antiimmunoglobulins (3). The specificity of reaction is generally confirmed by finding an “appropriate” cellular localization of the antigen of interest and by competition of purified soluble antigen for binding of labeled antibody. If this approach for localization of specific antigen is valid, then it should be possible to confirm thle specificity of the technique by measuring the amount of membraneassociated antigen. by quantitating the amount of peroxidase-antibody which binds to it. and by demonstrating the competition of soluble antigen for this binding. This would have the advantage of rigorously validating the use of the immunohistochemical technique and might be useful in “probing” the relative exposure of a given antigen in several different cell types. Technical difficulties associated with this approach include the establishment of an independent measure of antigen concentra-
tion, and consideration of conditions for preparation of tissues so that the membrane containing antigen is maximally exposed for reaction with the labeled antibody. We were particularly interested in developing such an approach for human placental alkaline phosphatase. This enzyme is known to be associated with the microvillus membrane (4,5) and is found not only in human placentae, but also in neoplastic tissues (6), especially cancer of the ovary (7), and in cultured human cells (8- 10). Immunochemical (11) and chemical (12) evidence support an identity of the purified enzyme in tumor and placental tissue. It remains to be established whether the enzymes in these different tissues are in fact identical in their cellular localization, function, or microenvironment in the membrane. The specificity of the immunoperoxidase method for histochemical localization of this enzyme was previously demonstrated (13). The enzyme can be measured by enzyme assay when it is membrane bound, and this appears to be a reasonable measure of enzyme protein concentration. since (i) there is no loss of enzyme activity after 53
0003.2697179/130053-07$0?.00/0 Copynghl All right\
11 1979 by Academc Prer,. Inc. 01 reproductwn in any term rcxrved
54
TAYLOR
AND
butanol extraction of membrane preparations (14), (ii) immunoquantitation by radial immunodiffusion ( 15) and complement fixation (16) show no change in immunoreactive enzyme upon purification, and (iii) measurement of enzyme protein by radial immunodiffusion in Triton-containing gels showed that the membrane-associated enzyme did not differ from the soluble enzyme in the amount of enzyme protein per unit activity (15). The ability to measure the enzyme concentration in the membrane independently, and the availability of pure enzyme (14) made this a well-defined system for testing the specific interaction of peroxidase-antibody with membranes containing this antigen. The maximal exposure of membrane containing antigen was achieved by preparing membrane fragments in rabbit serum. The separation of bound from unbound peroxidase-antibody was achieved by using gel filtration on agarose media (Sepharose 4B). Using this approach, we have been able to reproducibly measure the amount of peroxidase-antibody which can bind to membraneassociated placental phosphatase. By testing the competition of soluble and membraneassociated phosphatase for peroxidaseantibody, it was found that the soluble enzyme binds the antibody more effectively. MATERIALS
AND METHODS
Rabbit serum (select hemolyzed sterile) was obtained from Pel-Freeze Biologicals, Rogers, Arkansas. Sephadex G-25, Sephadex G-200, and Sepharose 4B were purchased from Pharmacia Fine Chemicals, Uppsala. Sweden. Peroxidase, RZ 2.65, was obtained from Sigma Chemical Company, St. Louis, Missouri. All other chemicals were reagent grade from Fisher Chemical Company. Human term placentae were obtained from the obstetric service of Forsyth Memorial Hospital. Preterm placentae were
DOELLGAST
obtained from the gynecology service of North Carolina Baptist Hospital. Rabbit placentae were removed from a female San Juan rabbit 38 days after mating.
PrepLlration’c!f‘HM W-PAP.’ Tissues were homogenized for 60 s in a Waring blender with 2 ml Tris-acetate-buffered saline, pH 7.6, per gram of tissue. This was further homogenized for 30 s with a Polytron homogenizer. The homogenate was centrifuged for 30 min at 27,OOOg,,,. The pellet was discarded and the supernatant was centrifuged at 150,000gmax for 60 min. The pellet was washed twice, resuspended in rabbit serum, sonicated for 60 s in 15-s bursts, and centrifuged at lSO,OOOg,,, for 60 min. This supernatant was passed through a Sepharose 4B column (1.5 x 26 cm) equilibrated with rabbit serum. The excluded fractions (determined by assaying for PAP) were pooled and this pool was termed the “high-molecular-weight placental alkaline phosphatase” (HMW-PAP). Figure 1 gives an outline of this protocol. Prepcrration
of prrrijied atztihody to PAP.
Homogeneous human PAP was prepared as previously described (14). Rabbits were sensitized by injection of purified PAP and monospecific antiserum was obtained (14). An immunoabsorbent column was prepared by covalently coupling immunoabsorbentpurified PAP to cyanogen bromide-activated Sepharose. Monospecific antiserum was applied to the PAP-immunoabsorbent column; the column was exhaustively washed with 10 mM Tris-acetate-buffered saline, pH 7.6, and the antibodies were eluted using 5 mM sodium phosphate buffer, pH 12. The anti-PAP antibodies were further chromatoI Abbreviations used: HMW-PAP, high-molecularweight placental alkaline phosphatase (membranebound) excluded from Sepharose 4B gel: IgG. immunoglobulin G: PO-Ab. peroxidase-labeled antibody to human placental alkaline phosphatase: BSA. bovine serum albumin: DMA. ovarian cancer patient.
PEROXIDASE-ANTIBODY
Tissue
BINDING
+ 2 ml
10 mM Tris-buffered
Homogenize and then at 27,000
Pellet (save)
.\
TO MEMBRANE
saline,
FRAGMENTS
55
pH 7.6/gram
in a Waring blender 30 set in a Polytron. xgmax for 30 min.
for 60 set Centrifuge
supernatant Centrifuge
at
150,000
xgmax
for
60 min.
I
Resuspend in and centrifuge 60 min.
Supernatant
FIG.
I. Scheme
rabbit at
(HMW fragments
for the preparations
graphed on a Sephadex G-200 column (2.5 x 75 cm) to obtain anti-PAP IgG. Pcroxidrtsr lrrhrlittg. The purified IgG fraction was peroxidase labeled by a modification of the method of Nakane and Kawaoi (2) in that the “activated” peroxidase is separated from the reagents by chromatography on Sephadex G-25. The solution of PO-Ab was diluted to 3.4 x lo-’ mg peroxidase/ml in rabbit serum before use. Proteitl c’otz(.etttrvttiotts. These were determined by the method of Lowry et trl. (17) using a BSA protein standard and expressed as milligrams per milliliter. AIktrlitre pirosphtrtnsr ~~ssny. Alkaline phosphatase was assayed at pH 9.8 as described previously (14). All samples were heated at 65°C for 10 min and assayed in the presence of 0.1 mM bromotetramisole (18). The amount of PAP was determined as prn801.min’ . ml-’ and expressed as ngiml by dividing by the specific activity for PAP (450 /dmol.rnin~ ’ .mg-‘) x 10” (14). Po.o.rititrsr trsstr.~. Assays for peroxidase were performed by a variation of a previously published method (19). A stock peroxide solution containing 0.3% peroxide
of HMW
serum, 150,000
in
sonicate, zigmax for
rabbit
fragments
serum)
from
tissues.
(in distilled water) was diluted 1:50 with 10 mM phosphate buffer, pH 6.0 (fresh daily) to produce the working solution. The working solution (0.5 ml) was placed into each assay tube, in addition to 10 ~1 of 1% orthodianisidine, in isopropanoliwater ( 1: 1). The sample was added to each assay tube and the mixture was incubated at 24°C for 10 min. After 10 min. the reaction was stopped by the addition of 100 ~1 of 2% sodium azide followed by 1 ml of methanol. This was mixed thoroughly and the absorbance was measured at 460 nm. Peroxidase standards were prepared from a stock peroxidase solution (5 x IO--’ mg peroxidasei ml). The amount of peroxidase present in samples was calculated as ngiml, using a peroxidase standard assayed simultaneously as a reference. Mrrrstr~ettlrttt of’PO-Ah hitltlittg to HM WPAP. A quantity of HMW-PAP (in rabbit serum) was placed in a test tube and PO-Ab was added. This reaction volume was brought to 1 ml with rabbit serum and incubated for 2 h at 37°C. After 2 h, the sample was applied to a Sepharose 4B column (0.7 x 48 cm) and fractions were
56
TAYLOR
AND
DOELLGAST
collected. The fractions were assayed for protein, placental phosphatase. and peroxidase. RESULTS
The linearity of the modified peroxidase assay was tested over the range of 0 to 10 ng peroxidase. In Fig. 2, it is apparent that the linear range of the assay is 0 to 7 ng peroxidase (correlation coefficient equal to 0.997). All assays were therefore performed within this range. A typical Sepharose 4B elution profile for HMW-PAP with PO-Ab bound is shown in Fig. 3. A clear separation of the bound and unbound peroxidase antibody is obtained. To measure the amount of binding of POAb to HMW-PAP, increasing quantities of PO-Ab (O-40 pg protein) were added to a constant amount of placental HMW-PAP (containing 120 ng PAP). The amount of POAb binding to the HMW-PAP was plotted against the amount of PO-Ab added (Fig. 4). The relationship appears to be linear up to 20 pg PO-Ab, after which there is an apparent saturation of the antigenic binding sites. Using a concentration of 6.7 pg of peroxidase-antibody, we tested the amount of peroxidase antibody bound to varying amounts of HMW-PAP. In Fig. 5, it is seen that there is a linear correlation over the range of O-240 ng of HMW-PAP. To determine whether the PO-Ab were specifically binding to the HMW-PAP, three
q
of
Peroxidose
added
FIG. 2. Standard curve forperoxidase assay (straight line given by the least-squares method of linear regression).
FRACTION
NUMBER
FIG. 3. Elution profile of tumor (DMA) HMW-PAP, plus PO-Ab from a Sepharose 4B column assayed for protein in mgiml (A -A ); PAP. absorbance at 505 nm (0 ---0 ): and peroxidase. absorbance at 460 nm (O---O).
experiments were performed. First, HMWPAP of rabbit placentae was examined. No detectable PO-Ab bound to HMW-PAP of rabbit placentae. Second, pure soluble PAP was added to 120 ng of human placental HMW-PAP and increasing quantities of PO-Ab (O-10 pg protein) in an effort to competitively bind PO-Ab. Figure 6 shows the effects of adding 0- 120 ng soluble PAP. Eleven nanograms soluble PAP inhibits PO-Ab binding to the HMW-PAP by 16%, 58 ng by 55%. and 120 ng by 8470. Since there were equal amounts of HMW-PAP and pure PAP in the 120 ng experiment, it appears that the pure PAP is better able to bind the PO-Ab than the HMW-PAP, suggesting steric hindrance in the mem-
PO-Ab
added
IN
protein)
FIG. 4. Quantitation of Sepharose 4B-excluded POAb upon addition of increasing increments of PO-Ab to placental HMW-PAP (containing 120 ng PAP).
PEROXIDASE-ANTIBODY
200
100
present
P&P
BINDING
TO MEMBRANE
PO-Ab
300
trig PAP)
TABLE TP.TION
OF SEPHAROSE
4%EXCLUDED
CONTAINING
Sample Term
WITH
LEVELS
DIFFERENT
PI ACENTAL
SAMPLES
or- PAP”
Protein (mg)
PAP
(ng)
PO-Ab trig)
2.73
129.6 62.6
7.6 3.7
2.78 1.39
0.059 0.059
46.8 245.2
2.5 14.1
1.00
0.053
5.26
189.0
10.9 13.6
0.057 0.058
ng PO-Abimg
protein
ng PO-Abing
placentae
4546 4554
2.66 2.50
4560 4574 4581
2.68 2.71
4583 4587 4589
2.73 2.72 2.70
256.8 168.0 273.8
1.91 1.84
2.7 240.0
Early
PO-Ah binding to HMW ng PAP) upon addition of PAP, (b) I I ng soluble PAP. I?0 ng soluble PAP.
I
PO-Ab VARIOLIS
(Kg proteInI
early placentae. The 12-week placenta contained low levels of PAP (2.7 ng) and no PO-Ab could be detected in the excluded portion. The 22-week placenta contains appreciable levels of PAP (240 ng) and is capable of binding PO-Ab: however, the ratio of excluded PO-Ab to HMW-PAP is significantly different from that for the term placenta. We attempted to use the simpler approach of incubating labeled antibody with suspensions of membrane in buffer. then removing bound antibody by ultracentrif-
brane. Finally, we tested the binding of PO-Ab to different placental HMW-PAP preparations, containing varying amounts of PAP, but thl: same amount of membrane protein. Table 1 shows the results for this experiment in (eight normal-term placentae, differing significantly in HM W-PAP activity. Examining the ratios of PO-Ab to protein and PO-Ab to PAP, the amount of PO-Ab bound correlat.es best with the amount of HMW-PAP. Table I also shows the results of a similar study on the HMW-PAP of two
QUANTI
added
FIG. 6. Inhibition of fragments (containing 120 soluble PAP. (a) No soluble Cc) 58 ng soluble PAP, Cdl
FIG. 5. Quantitation of Sepharose 4B-excluded POAb upon addition of increasing increments of PAP containing HMW fragments to a constant amount of PO-Ab (6.7 pg).
57
FRAGMENTS
10.7 16.0
4.02 4.98 3.93 5.93
0.053 0.064 0.058
5.82
0.045
placentae
12 week 22 week
” 6.7 Fg PO-Ab ’ Not detectable.
added
to each sample.
-
ND” 10.7
PAP
58
TAYLOR AND DC)EI.LGAST
ugation (20). The amount of measurable peroxidase-antibody bound to the pellets was neither a linear function of the amount of peroxidase-antibody added at constant membrane concentration, nor of the amount of membrane added at constant peroxidaseantibody concentration. This was true whether or not the pellets were extracted with 1% Triton X-100 prior to peroxidase assay.
divalent antibody. Since we did not use this technique at saturating antibody concentrations, and since we have an independent measure of membrane-associated ligand by enzyme assay (see introduction), it is interesting to compare our results with Reynolds’s model. In her analysis, formation of predominantly divalent complexes and monovalent complexes of divalent antibody with cell surface ligand are compared. In the case of predominantly DISCUSSION divalent complexes, the fraction of antiThis study has demonstrated that per- body bound is proportional to the square oxidase-labeled antibody to human placental of the cell-bound ligand concentration In the alkaline phosphatase binds specifically to divided by the cell concentration. placental membrane fragments containing case of only a single “arm” of the antithis antigen. Specificity for the placenta1 body being bound to the cell membrane phosphatase was demonstrated by (i) non- ligand, the amount bound is directly proreactivity with rabbit placental phosphatase, portional to the cell membrane ligand con(ii) competition of pure placental phos- centration. In Table I, we compare the amount of bound peroxidase-antibody with phatase for binding to placenta1 membranes, and (iii) a strict correlation between the the amount of enzyme present on eight difamount of placental phosphatase present ferent placental membrane preparations on membrane fragments and the amount of with a sixfold range of enzyme specific peroxidase-antibody bound. activity. The amount bound was directly Placental alkaline phosphatase is a com- proportional to enzyme concentration over plex protein with numerous antigenic deter- this range, and so it would appear that the using this minants. We are using this assay in a con- binding of peroxidase-antibody by attachment centration range which results in only a assay occurs predominantly small fraction of the total labeled antibody of only one arm of the divalent antibody. binding to enzyme. Since we have found According to Reynolds (21), this result that 72% of this labeled antibody preparation suggests that orientation factors and recan bind to a large excess of placental mem- striction of motion of both surface ligand branes (Taylor, Homesley, and Doellgast, in and bound antibody had restricted the preparation), the small amount of bound binding of the second arm of the bound antibody would appear to be due to the peroxidase-antibody. It would be interesting to use this system to test other predictions specific binding of high avidity antibodies under these assay conditions, or to partial of Reynolds’s model. In this work. we did not attempt such a shielding of the antigenic sites in the membrane. The latter explanation is in part detailed analysis, but have used the measure supported by the very effective competition of membrane-bound enzyme activity as a of the soluble enzyme for binding of the measure of enzyme protein concentration. We thus are measuring the “availability” peroxidase-antibody (Fig. 6). As has been forcefully argued by of antigenic sites for peroxidase-antibody Reynolds (21), the complex equilibria binding. In the one early (22-week) placental between divalent antibody and membrane sample we tested, we found significantly less antigens precludes measurement of the labeled antibody bound. This result suggests number of antigenic sites per cell using the application of this technique as a
PEROXIDASE-ANTIBODY
BINDING
of the exposure of antigenic “probe” determinants of membrane-bound enzyme from different. sources. We are currently using the technique in this way for comparison of cancer tissue and ascites fluid (22) membrane fragments (Taylor, Homesley, and Doellgast ( in preparation). In conclusion. we note that the technique described above (i) rigorously confirmed the specificity of our peroxidase-labeled antiplacental phosphatase as a histochemical reagent, (ii) shows some potential for antigen quantitation on membrane preparations, and (iii) could be useful in comparing the relative exposure of antigenic sites on different membrane preparations if an independent measure of antigen concentration is available. ACKNOWLEDGMENTS This work was supported by National Cancer Institute, National Institutes of Health Grant CA2353301, and by the Gaston County Cancer Society and Forsyth Cancer Service. We are grateful to the obstetrical nursing staff of Forsyth Memorial Hospital for placental tissue and to Dr. Howard Homesley for prelerm placentae.
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TO MEMBRANE
FRAGMENTS
59
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