An Immunoassayof Human Band 5 (“Tartrate-Resistant”)Acid ...

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prostatic cancer with metastasis to bone assaysbased on inhibitionby tartratecompared. The isoenzyme of human acid phosphatase (EC 3.1.3.2) that is normally ...
CLIN.

CHEM.

35/1,

86-89

(1989)

An Immunoassayof Human Band 5 (“Tartrate-Resistant”)Acid PhosphataseThat Involvesthe Use of Anti-PorcineUteroferrinAntibodies Katrlne B. Whltaker, Timothy M. Cox, and Donald W.

Moss1

We describe an immunoassay for human band-5 acid phosphatase in which antibodies to porcine uterofernn, immobi-

lized on Sepharose particles, are used. Band-5 acid phosphatase is the tartrate-resistant isoenzyme normally expressed in tissue macrophages such as osteoclasts and alveolar macrophages. The immunoassay is similar in reproducibility and sensitivity to assays based on inhibition by dtartrate. However, compared with the latter, the greater specificity of the immunoassay makes it markedly less susceptible to errors arising from the presence of non-band-5 acid phosphatases, e.g., from prostate. AddItIonal Keyphrases: isoenzymes teoclastic activity

.

inhibitionkinetics os prostatic cancer with metastasis to bone

assaysbased on inhibitionby tartratecompared

The isoenzyme of human acid phosphatase (EC 3.1.3.2) that is normally expressed in certain differentiated cells of the monohistiocytic system-notably in osteoclasts (1) and alveolar macrophages (2, 3) and possibly in Kupifer cells, but not in the precursor monocytes (3)-is of continuing interest to clinical chemists because of its potential value as a marker of osteoclastic activity, and because of its inappropriate expression in Gaucher’s disease (4) and in the hairy cells of leukemic reticuloendotheliosis (5). Recognition and measurement of the isoenzyme generally depend on its marked resistance to inhibition by dextrorotatory tartrate, in contrast to prostatic (6) or lysosomal (7) acid phosphatases.However, although qualitatively clear, this distinction is not quantitatively absolute: because tartrate is a competitive inhibitor of lysosomal and prostatic phosphatases (8), inhibition is necessarily incomplete under conditions in which

activity

is measurable,

whereas,

as reported

here, there is some inhibition of the “tartrate-resistant” isoenzyme. Furthermore, erythrocyte acid phosphatase also resists inhibition by tartrate (6). Thus, assays based on inhibition

by tartrate

lack absolute

specificity.

The human acid phosphatase derived from osteoclasts and other tissue macrophages can also be identified by its cathodal (“band 5”) mobility on electrophoresis at pH 4 (9). Because its electrophoretic mobility is a unique property of the acid phosphatase isoenzyme from these cells, unlike resistance to tartrate-inhibition, we designate the macrophage isoenayme “band-5 acid phosphatase” in this paper. Band-5 acid phosphatase is a member of a class of widelydistributed iron-containing proteins with acid phosphatase activity (10). We have reported that a rabbit antiserum raised against one such protein, porcine uteroferrin, crossreacts with human band-5 acid phosphatase and can be used as the basis of immunoassays of the human isoenzyme (11). Departments of Chemical Pathology and Haematology, Royal Postgraduate Medical School, Hammersmith Hospital, London W12

ONN, U.K. 1 Addresscorrespondence to this author. ReceivedAugust 12, 1988; accepted October

14,

86 CLINICALCHEMISTRY, Vol. 35, No. 1, 1989

1988.

We now describe such an immunoassay. In particular, we have investigated whether the greater isoenzyme specificity of the immunoassay offers a significant analytical advantage as compared with assays based on inhibition by tartrate.

MaterIals and Methods We measured

acid phosphatase

ed, continuous-monitoring

activity by the automatof Warren and Moss (12) as substrate at pH 5.6 and 37 #{176}C

method

with 1-naphthyl phosphate during the purification of acid phosphatase from prostate and spleen. Specific activities are quoted as micromoles of substrate hydrolyzed per minute (U) under these conditions. In experiments on the inhibition of band-5 acid phosphatase, and in all immunoassays, we measured activity by fixedtime assay at pH 5.0 in citrate buffer (100 mmoli’L) and 37 #{176}C, with 4-nitrophenyl phosphate (10 mmolJL) as substrate. This substrate is hydrolyzed more rapidly than 1naphthyl phosphate by the band-5 isoenzyme, while the use of a fixed-time assay allowed us to use longer incubation periods, further to increase sensitivity. Liberated 4-mtrophenol was measured at 405 nm after stopping the reaction with NaOH (500 mmol/L). Band-S acid phosphatase was extensively purified from the spleen from a patient with Gaucher’s disease by successive stages of CM-cellulose ion-exchange chromatography, gel-filtration through Sephadex G200 (Pharmacia, Uppsala, Sweden), and affinity-chromatography on concanavalin ASepharose 4B (10, 11). Our most nearly pure preparations had specific activities of 85 kU per gram of protein. We purified prostatic acid phosphatase from human prostate glands removed at autopsy by animonium sulfatefractionation, affinity-chromatography on d-tartrate-Sepharose-4B (13), and chromatography on concanavalin A-Sepharose, the specific activity of the product being 660 kU per gram of protein. Human alveolar macrophages were obtained by bronchoalveolar lavage, cultured,and separated from nonmacrophage cells as described by Fuller et al. (14). We extracted band-5 acid phosphatase from the resulting cell population (more than 95% macrophages) by homogenization in 300 mol/L KCI containing 1 g of Triton X-100 surfactant per liter. The supernate after high-speed centrifugation contained about 400 U of acid phosphatase activity per liter, as measured with 4-mtrophenyl phosphate as substrate. Lysed erythrocyteswere the source of erythrocyte acid phosphatase. Anti-porcine uteroferrin antibodies were raised in rabbits, purified by immunoaffinity chromatography on a uterofer. rin-Sepharose 4B column, and coupled to particles of CNBr. activated Sepharose-4B (Pharmacia) as previously described (11). Immunoassay procedure: Let anti-uteroferrin--Sepharose particles settle overnight, remove the supernatant fluid, and resuspend in four volumes of Ti-is HC1 (10 mmol/L; pH 8.2 containing NaC1 (300 mmol/L).

Set up two microfuge

tubes for each sample: Test

60 Control

mL

ResuspendedSepharose-antibodyparticles Tns buffer Sample

-J

50

D

40

C

0.15

0

0.15 0.15

0.3 0.15

Mix, and leave for 2 h in crushed ice, mix about every 30 mm. Centrifuge in a microfuge at -10 000 x g for 2 mm. Measure the acid phosphatase activity by adding 0.15 mL of each supernate to 0.3 mL of citrate buffer (150 mmol/L, pH 5.0) containing 4-nitrophenyl phosphate, 15 mmolJL, giving final concentrations in the assay mixture of 100 and 10 mmoIJL, respectively. Incubate this mixture for 15 mm at 37 #{176}C, then stop the reaction by adding 0.2 mL of 500 mmoll

L NaOH. Measure the absorbances at 405 nm in small-volume cuvettes. Calculate enzyme activities in control and test tubes,using the absorbance coefficient of 4-mtrophenol, 1.86 L mmol’ nim1. The difference (control test) corresponds to band-5 acid phosphatase activity that is antibodybound. -

Results and Discussion We validated the selectedimmunoassay conditions, using Gaucher’s disease spleen or human alveolar macrophages as the sources of band-5 acid phosphatase. At an activity concentration of 60 U/L [i.e., about sixfold the upper reference limit for serum from normal subjects as determined by a tartrate-inhibition assay under similar conditions of temperature, pH, and substrate concentration (15)], the same fraction of the band-5 isoenzyme activity in the sample was bound when either 0.15 mL or 0.10 mL of the resuspended Sepharose-bound antibody was present in the assay. Binding was only slightly less with 0.05 mL; however, we selected 0.15 mL to ensure an excess of binding capacity. Binding of acid phosphatase to the antibody was more than 80% complete within 1 h of mixing and was 92% complete after 2 h. Some increase in binding (-5%) was apparent between 2 and 5 h, when 97% of the specific isoenzyme became bound (Figure 1), but no more binding occurred after 5 h. We chose 2-h incubation of the sample with the antibody as a compromise between increased binding and the possibility of enzyme inactivation during longer incubations. Because total and unbound acid phosphatase activities are measured in a fixed-time assay, we investigated the linearity of response in this part of the procedure as well as in the complete imniunoassay. Measured activity was linearly related to added acid phosphatase up to at least 160 UI L. The complete immunoassay responded linearly to added band-5 acid phosphatase up to at least 60 U/L (Figure 2).

20

30 .20 10 10

20

30

40

Addedband 5 activity

50 (U/L)

60

Fig. 2. Relationship between added band-5 acid phosphatase activity

(from alveolar macrophages) and actMty bound to anti-uterofemnSepharose particles in the immunoassay

The within-run CVs (n = 8) for immunoassay were 9.0% at a mean concentration of 5 UIL, 5.4% at 14 UIL, and 4.2% at 34 U/L. These values are similar to those found with a typical tartrate-inhibition assay (12). We have shown previously (11) that the anti-porcine uteroferrin antibodies are highly specific for band-S acid phosphatase from human alveolar macrophages, bone, and spleen, and that they do not cross-react with acid phosphatase isoenzymes from erythrocytes or prostate. The effect of the specificity of the antibodies in analysis can be seen by comparing the immunoassay with a typicalassay in which

specificity depends on inhibition by tartrate. Dextrorotatory tartrateis a fully competitive inhibitor of lysosomal and prostatic acid phosphatases. The value of K for purified prostatic acid phosphatase is approximately 0.04 mmol/L, compared with aKm of 0.1 mmol/L for the substrate (8, 16). Thus, at typical inhibitor and substrate concentrations of 100 mmolJL and 10 mmol/L, respectively, calculation according to Michaelis-Menten kinetics indicates that inhibition of the prostatic isoen.zyme will be 96% complete. As a result, an increasing background of tartrate-inhibitable acid phosphatase (e.g., from prostate) in the sample will result in a progressive overestimation of the contribution of non-prostatic (or non-lysosomal) isoenzymes to the tartrateresistant fraction. In practice, using highly-purified prostatic acid phosphatase, we found 95% inhibition by 100 mmolJL tartrateat a substrate concentration of 10 mmol/L. Although band-5 acid phosphatase is essentially tartrate resistant, a small degree of inhibition, e.g., of the purified spleen isoenzyme, can be demonstrated. This is of uncompetitive type, with a K, of 1.6 mollL (Figure 3). We found the value of Km for band-S acid phosphatase to be -0.6 mniol/L with 4-nitrophenyl phosphate as substrate. Calculation on the basis of these values indicates that “tartrate-resistant” acid phosphatase will be inhibited to the extent of 6% by 100 3.0

-2.5-2.0-1.5-1.0-0.5

0.0 0.5 1.0 1.5

1/s (LimmoI) Time of incubation (h)

Fig.1. Percentageofband-5acidphosphatasepurifiedfrom Gaucher’s disease spleen (0.15 mLofa 60 U/Lsolution)bound to anti-uteroferrinSepharoseparticles(0.15 mL) afterdifferentincubationperiods

Fig. 3. Plots of reciprocals of v (absorbance change per mm) against reciprocal of substrate concentration (4-nitrophenyl phosphate, mmol/ L; pH 5.0) in the presence of increasingconcentrationsof d-tartrate (0, 0; S, 0.125 m0IIL; A, 0.375 mol/L; U, 0.5 mol/L) Band-5 acid phosphatase was purified fromGauchersdisease spleen

CLINICAL

CHEMISTRY,

Vol. 35, No. 1, 1989 87

d-tartrate at a substrate concentration of 10 mmoll L. We found purified spleen acid phosphatase (37-kDa fraction, i.e., free from 100-kDa lysosomal phosphatase) to be inhibited by 14%. The difference between the observed and calculated values probably reflects uncertainty in the estimate of K. Thus, even though d-tartrate is an inefficient inhibitor of non-prostatic and non-lysosomal acid phosphatases, partial inhibition of these “tartrate-resistant” isoenzymes results in their underestimation when the activity is measured in the presence of tartrate. The resulting effect of incomplete inhibition on the one hand and partial inhibition on the other on the accuracy of measurement of specific isoenzymes will depend on the relative proportions of tartrate-sensitive and tartrate-resistant isoenzymes present in the sample. As the background of tartrate-sensitive isoenzymes (e.g., prostatic acid phosphatase) increases, the effect of incomplete inhibition of these isoenzymes becomes predominant, leading to a significant overestimation of “tartrate-resistant” acid phosphatase by the inhibition assay (Figure 4). The presence of prostatic acid phosphatase does not interfere with binding of the band-5 isoenzyme by the antiuteroferrin antibody and, as a result, the presence of an increasing background of prostatic phosphatase shows a greatly decreased overestimate of the band-5 isoenzyme (e.g., from spleen) in the immunoassay (Figure 4). We further confirmed that acid phosphatases other than the band-5 isoenzyme do not interfere in the immunoassay by looking for an effect of adding increasing amounts of alveolar-macrophage acid phosphatase to a normal serum pool and to pools containing respectively added high activities of prostatic or erythrocytic acid phosphatases. Activities of the band-5 acid phosphatase measured in the pools containing added prostatic or erythrocytic acid phosphatases were identical to that found in the normal serum pool (Figure 5). Unlike the immunoassay, inhibition assays cannot distinguish between band-5 and erythrocyte acid phosphatases, both of which are essentially tartrate-resistant. Contamination of serum taken for the measurement of band-S acid phosphatase with the erythrocyte isoenzyme is rarely a problem with careful sampling. However, assessment of osteoclastic activityis often of interest in metastatic carcinoma of the prostate, when high concentrations of prostatic acid phosphatase are almost invariably present. The immunoassay is much less affected than the inhibition assay under these circumstances, as we have shown in the model experiment in Figure 4. The increased specificity of the immunoassay in the presence of other isoenzymes of acid phosphatase is also valuable when one is studying the mmolfL

30 20

10

0

20 40 60 80 100 120 Prostaticacid phosphataseactivity (U/LI

Fig. 4. Estimates of band-5 acid phosphatase activity (7 U/L; purified from Gauchers disease spleen) obtained by tartrate-inhibition assay (0) and by immunoassay (#{149}) in the presence of increasingactivities of

prostatic acid phosphatase 88 CLINICALCHEMISTRY,Vol. 35, No. 1, 1989

100 -

a -J

80 >-

>

a,

20 0 10 20 30 40 Total acid phosphatase added (U/L)

b >>

U (‘3

20

0 10 20 30 40 Band 5 acidphosphataseadded (U/L)

Fig. 5. (a) Immunoassay of increasing activities of band-5 acid phosphatase (from alveolarmacrophages) added to serum pools containing non-band-5 acid phosphatases

0, S. normal serum;

., A, hemolyzedserum; LI, #{149},serumwith high activityof prostaticacid phosphatase. Solid symbolstotalacidphosphatase activity (i.e., without addition of antibody);opensymbols,activitynot boundby antibody

(b) Relation between band-5 activity added to various serum pools and amount bound to antibody Data arederivedfrom a by subtracting the unbound activity from the corresponding total activity for each serum, andcorrecting for the small amount of band-5 activity initially present in each. Serum pools: 0 normal; , hemotyzed; LI, containing prostatic acid phosphatase. Where only one or two symbols are visible, other(s) are exactly coincident expression of band-5

acid phosphatase in cells such as in culture, because of the lack of interference by lysosomal acid phosphatase. Acid phosphatase assays whose specificity depends on inhibition by d-tartrate and the immunoassay that we describe here both depend on measurement of differences in activity before and after reaction with the inhibitor or with the antibodies, respectively. Thus they can be expected to display comparable sensitivities and precisions. The precision of the immunoassay is indeed similar to that of a typicalinhibition assay, and the sensitivity is also similar. One unit of band-S acid phosphatase gives the same difference in absorbance between the “control,” in which total activity is measured, and the “test,” in which residual activity is determined afterimmuno- or chemical inhibition, when the same sample volumes and dilutions are used in the two assays. Immunoassays in which the analyte is measured after immunoprecipitation or capture by immobilized antibodies offer the advantage of increased sensitivity through the ability to concentrate the analyte into a smaller volume than that of the original sample. However, we have previously reported that the band-5 acid phosphatase isoenzyme is partly inactivated when bound by the antibody and cannot be quantitatively eluted from it (11). We have been equally unsuccessful in developing a “sandwich”-type immunoassay with polyclonal rabbit anti-uteroferrin antiserum. The molecular mass of the major protein component of human band-5 acid phosphatase from spleen, present after electrophoresis in gels containing sodium dodecyl sulfate, is similar to that of the native enzyme (10, 11), with smallermolecular-mass components present in minor amounts that osteoclasts

probably result from proteolytic degradation (10). This implies that band-5 acid phosphatases are monomeric, and therefore are unlikely to exhibit repeated epitopes recognizable by a single species of antibody. However, a sandwichtype assay based on two different monoclonal antibodies may be feasible. In the meantime, we believe that the immunoassay described here offers significant advantages of specificity over tartrate inhibition in the measurement of band-S acid phosphatase. References 1. Minkin C. Bone acid phosphatase: tartrate-resistant acid phosphat.ase as a marker of osteoclast function. Calcif Tissue mt 1982;34:285-90. 2. Efstratiadis T, Moss DW. Tartrate-resistant acid phosphatase of human lung. Apparent identity with osteoclastic acid phosphatase. Enzyme 1985;33:34-40.

3. Efstratiadis T, Moss DW. Tartrate-resistant acid phosphatase in human alveolar macrophages. Enzyme 1985;34:140-3. 4. RobinsonDB, Glew RH. Acid phosphatase in Gaucher’s disease. Clin Chem 1980;26:371-82. 5. Yam LT, Li CY, Finkel HE. Leukemic reticuloendotheliosis. The role of tartrate-resistant acid phosphatase in diagnosisand splenectomy in treatment. Arch InternMed 1972;130:248-56. 6. Abul-Fadl MAM, King EJ. Properties of the acid pho8phatases of prostate gland. Biochem J 1949;45:51.-60. 7. PodhajcerOL, Filmus JE, Mordoh J. Characterization of lyso-

erythrocytesand of the human

somal acid phosphatase from normal and malignant mammary tissue. Clin Chem 1986;32:279.-82. 8. Jacobsson K. On the inhibition of prostatic phosphatase by tartrate. Scand J Clin Lab Invest 1958;11:358-60. 9. Ki CY, Yam LT, Lam KW. Studies of acid phosphatase isoenzymea in human

leukocytes.Demonstration

of isoenzyme cell

specificity. J Histochem Cytochem 1970;18:901-1O. 10. Ketcham CM, Baumbach GA, Bazer FW, RobertsEM. The type 5 acid phosphatase from spleen of humans with hairycell leukemia. Purification, properties, immunological characterization, and comparison with porcine uteroferrin. J Biol Chem 1985;260:5768-76. 11. Echetebu ZO, Cox TM, Moss DW. Antibodies to porcine uteroferrin used in measurement of human tartrate-resistant acid phosphatase.Clin Chem 1987;33:1832-6. 12. Warren RJ, Moss DW. An automated continuous-monitoring procedure for the determination of acid phosphatase activity in serum. Clin Chim Acta 1977;77:179-88. 13. Vihko P, KontturiM, Korhonen LK. Purification of human prostatic acid phosphatase by affinity chromatography and isoelectricfocusing. Part 1. Clin Chem 1978;24:466-70. 14. Fuller RW, Morris PK, Richmond R, et al. Immunoglobulin Bdependent stimulation of human alveolar macrophages: significance in type 1 hypersensitivity. Clin Exp hnmunol 1986;65:41626. 15. Moss DW, Efatratiadis T. Selective expression of human acid phosphataseisoenzymes.In:GoldbergDM, de Ia Morena E, Werner M, eds. Adv Clin Enzymol, Vol. 4., Carnitine,enzymes and isoenzymes in disease. Basel:Karger, 1986:1-11. 16. Campbell DM, Moss DW. Spectrofluorimetric determination of acid phosphataseactivity. Clin Chim Acta 1960;6:307-15.

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