[37] Use of antibodies and the primary enzyme immunoassay (PEIA) to study enzymes: The arylsulfatase A-anti-arylsulfatase A system

550

ANTIGEN

AND

ANTIBODY

DETECTION

BY IMMUNOASSAY

[37]

parable from one experiment to another and one laboratory to another. While it is preferable that the antigen be covalently attached, difficulties may be encountered in binding the antigen or hapten to a solid support, and a standard reference on the preparation of immunoadsorbents may have to be consulted. 29 The antigen or hapten against which the antibody was produced may not be available as a purified material; the method then would be useful only as a qualitative estimate (providing the antigen or hapten can be covalently attached to a solid support). Finally, the method described here has two other factors that have been addressed in detail, z° Of the two, one is sufficiently important to repeat: namely, when compared to results obtained by precipitin reactions, UNLIM yields a 10% larger value for antibody. This effect cannot be ignored if the method is adapted to clinical situations, as it may lead to undue concern if estimates of antibody levels against an infectious material are being sought. Although results obtained using this procedure may be viewed as "false positive," it is more likely that previous methodology may have resulted in "false negatives." Until this situation is resolved, care must be exercised in interpreting results. Nevertheless, U N L IM represents a method of general usefulness in a variety of research and clinical situations for the difficult task of antibody quantitation. 29O. Hoffman-Osteuhof,ed., "AffinityChromatography." Pergamon, Oxford, 1978.

[37] U s e o f A n t i b o d i e s a n d t h e P r i m a r y E n z y m e I m m u n o a s s a y (PEIA) to Study Enzymes: The Arylsulfatase A-Anti-Arylsulfatase

A System

By EDWARD A. NEUWELT* Production and Applications of Antibody to Arylsulfatase A Purpose Sulfatase A (arylsulfatase A, EC 3.1.6. I) (ASA-A) is a lysosomal acid hydrolase. The activity of this enzyme is deficient in metachromatic leukodystrophy, ~ a human neurologic disorder. An antibody to human sulfatase A was needed for two reasons: to determine whether the enzyme protein is absent in the disease or whether it is present in a defective form; * Present address: University of Oregon Health Science Center, Portland, Oregon. i j. Austin, D. Armstrong, and L. Shearer,Arch. Neurol. 13, 593 (1965).

METHODS IN ENZYMOLOGY, VOL. 73

Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181973-6

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551

and to enable purification of sufficient e n z y m e f r o m the diseased patients to study further its properties. The p u r p o s e s of this section are (a) to describe a method for preparing the h u m a n e n z y m e in relatively pure form; (b) to describe the preparation of an antibody to it; (c) to c o m m e n t on some novel, quite u n e x p e c t e d , kinetics of the reaction between this antibody and the normal enzyme; and (d) to describe a method for purification of the e n z y m e itself using its monospecific antibody. A s s a y o f Arylsulfatase Activity

Arylsulfatase A activity was determined colorimetrically with the substrate dipotassium 2-hydroxy-5-nitrophenyl sulfate (nitrocatechol sulfate). 2 The reagent system contained 20 m M nitrocatechol sulfate, 1 m M Na4P2OT, and 3.4 M NaCI in l M sodium a c e t a t e - a c e t i c acid, p H 5.2. E n z y m e (0.2 ml), reagent (0.1 ml), and w a t e r (0.1 ml) were incubated at 37 ° for 1 hr; 1 M N a O H (0.6 ml) terminated the reaction. F r e e nitrocatechol was measured with a B e c k m a n DB-G spectrophotometer (hmax = 515 nm, ~515 = 12,200). One unit = 1.0 micromole of nitrocatechol sulfate hydrolyzed per milliliter of enzyme per hour. Protein concentration was determined by the method of L o w r y et al. 3 To c o n s e r v e e n z y m e when sulfatase A was highly purified, protein was quantitated by absorption at 280 nm using albumin as a standard. Specific activities are e x p r e s s e d as units per milligram of protein. Steps in the Purification of Arylsulfatase A (see also T a b l e I) The method used was a modification of that e m p l o y e d to purify ox sulfatase. 4 All centrifugations w e r e at 10,000 g for 30 min. All steps were p e r f o r m e d at 4 °. Dialysis was against water overnight. The p H of Tris-HCl buffers was determined at r o o m t e m p e r a t u r e . Step 1. Two normal h u m a n livers were obtained flesh at autopsy. Vessels and encapsulating connective tissue were r e m o v e d . The tissue ( 1.7 kg) was diced and homogenized in three volumes (volume/wet weight) of w a t e r using a Waring CB-5 blender. L y s o s o m e s were disrupted by sonication at 50 W for I min ( H e a t Systems-Ultrasonic Sonicator). Insoluble cellular debris was r e m o v e d by centrifugation. Step 2. The supernatant was adjusted to p H 5.7, and solid (NH4)zSO4 2 H. Baum, J. Dodgson, and B. Spencer, Clin. Chim. Acta 4, 453 (1959). 3 O. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). 4 L. W. Nichol and A. B. Roy, J. Biol. Chem. 55, 643 (1964).

552

ANTIGEN AND ANTIBODY DETECTION BY IMMUNOASSAY

[37]

TABLE I PURIFICATION OF SULFATASE A FROM HUMAN LIVER

Step

Volume (ml)

Total units sulfatase A

Specific activity

Relative specific activity

1 2 3 4 5 6 7 8

7500 650 670 56 7.5 12.2 32 7.5

26,820 28,150 20,800 19,910 12,930 10,830 3,350 590

0.5 1.7 3.8 16.6 17.4 19.9 39.8 77.4

1.0 3.4 7.6 33.2 34.8 39.8 76.9 154.8

was added to a concentration of 2.47 molal. The precipitate obtained by centrifugation was resuspended in water and dialyzed. Step 3. Proteins precipitating between 20 and 55% acetone at 0° were recovered, washed twice with 55% acetone, resuspended in water, and dialyzed. Step 4. The sample was centrifuged, the pellet was discarded, and the solution was adjusted to pH 5.7. Proteins precipitating between 1.7 and 2.34 molal (NH4)2SO4 were recovered by centrifugation, resuspended in water, and dialyzed. Step 5. After standing for 6 hr at pH 4.3, the sample was centrifuged (40,000 g for 90 min). Enzyme proteins were precipitated with 50% acetone from the supernatant, resuspended, and dialyzed. Proteins precipitating between 1.35 and 2.25 molal (NH4)2SO4 were recovered by centrifugation, resuspended in a minimum volume of water, and dialyzed against 0.1 M Tris-HCl, pH 7.4. Step 6. The sample was applied to a Sephadex G-200 upward flow column (2.5 cm × 35 cm) previously equilibrated with 0.1 M Tris-HCl, pH 7.35, and eluated with the same buffer. Fractions containing sulfatase A activity were pooled, concentrated by precipitation with 2.70 molal (NH4)2SO4, resuspended, and dialyzed against water. The solution was then further dialyzed against 1% glycine in water. Step 7. Isoelectric fractionation was performed by the method of Vesterberg and Svensson, ~ with minor variations. An LKB 8101 electrofocusing system and ampholytes (LKB Ampholine, 8142, pH 3-6) were used. The anode was at the bottom of the column. An anodal solution of 1% O. Vesterberg and H. Svennson, Acta Chem. Scand. 20, 820 (1966).

[37]

USE OF ANTIBODIES TO STUDY ENZYMES AND THE P E I A

553

concentrated sulfuric acid and a cathodal solution of 2% 2-aminoethanol were used. Sulfatase A activity was found between pH 4.33 and 4.72. Step 8. Because large amounts of protein had been applied to the first column, resolution was not completely satisfactory. A second column was prepared with the enzyme as before. The peak of sulfatase A activity now focused at pH 4.83 (adjacent fractions were pH 4.70 and 4.98).

Preparation of Antibody Fraction 17 from step 8 (pH 4.83) and one adjacent fraction (fraction 15, pHi 4.58) were selected as antigen preparations because they had the highest specific activity. Fraction 15 contained 553 mg of protein per milliliter; fraction 17 contained 1012 mg of protein per milliliter. Preinnoculation sera were drawn from four 1-kg New Zealand white rabbits. E n z y m e (0.1 ml) was homogenized with 0.1 ml of complete Freund's adjuvant. The popliteal lymph nodes of each anesthetized rabbit (1.5 ml o f 50% pentobarbital) were exposed just rostral to the junction of the two heads of the biceps femoris muscle. The enzyme-adjuvant solution was then injected directly into the lymph node in each leg. 6 Two rabbits were inoculated with each fraction. The rabbits were challenged with 1:1 homogenate of adjuvant and enzyme 16 days after inoculation. Each rabbit received four intramuscular injections of 0.1 ml each (i.e., 0.4 ml total). The rabbits were again challenged on days 30, 76, and 94. Initially, sera were obtained from an ear vein; subsequently, cardiac puncture proved to be much more efficient. Sheep anti-rabbit serum was obtained commercially.

Biochemical Assay and Titration of the Antibody Antiserum and enzyme were dialyzed separately against 0.02 M TrisHCI, pH 7.35. The samples were then incubated together for 90 rain at 37° and again overnight at 4° . When it was found that antibody precipitated within 10 min at 37°, the enzyme incubations were reduced to 60 min. The immunoprecipitate was centrifuged at 30,000 g for 30-60 rain and resuspended in 0.02 M Tris-HCl, p H 7.35. Enzymic activity was then determined separately in both the resuspended pellet and the supernatant.

Immunological Techniques For Ouchterlony double diffusion studies, r microscope slides (1 inch × 3 inch) were covered with 3 ml of 1% agarose (SeaKem) in 0.03 M phos6 p. j. Polley and H. J. Miiller-Eberhard,J. Exp. Med. 128, 507 (1949). 70. Ouchterlony, Acta Pathol. Microbiol. Scand. 26, 507 (1949).

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ANTIGEN AND ANTIBODY DETECTION BY 1MMUNOASSAY

[37]

phate buffer, pH 8.0, containing 0.1 M NaCi, 0.01 M EDTA, and 0.01% sodium azide. Wells (0.2 mm in diameter) were made in these slides and filled with the appropriate samples. Precipitin lines developed after 24-48 hr at room temperature in a humidified atmosphere. Slides were then washed at room temperature for a total of 72 hr: first in 0.15 M NaC1 (24 hr), next in 0.075 M NaC1 (24 hr), and finally in water (24 hr). Washed slides were then analyzed first for the presence of protein and then for enzyme activity. To detect protein, slides were dried under filter paper and placed for 10 rain in a solution of 0.6% Amido Black in methyl alcohol-acetic acidwater (9 : 2 : 9, v/v/v). Slides were rinsed in three changes of the above solvent for a total of 30 min to remove the excess stain. To detect sulfatase A activity in the precipitin lines, washed (but not dried) slides were soaked for 60 min in the arylsulfatase A reagent at 37° and then developed by immersion in l M N a O H for about 10 sec. A narrow red band along a former precipitin line indicated sulfatase A activity. The red band was transient because the nitrocatechol chromogen diffused (in contrast to the sharp black protein lines produced by amido black). Microimmunoelectrophoresis slides were prepared 8 using 2% agar (Difco) in barbital buffer (pH 8.2) of ionic strength 0.04. Antibody was electrophoresed for 45 min and enzyme for 3 hr across a potential of 35-40 V per slide. Precipitin lines were developed for 24-48 hr at room temperature, then washed and stained for protein or enzyme activity as above.

Preparation of a Crude Liver Enzyme This crude preparation was used for routine testing of the antibody. Fresh-frozen human liver tissue obtained at autopsy was diced and homogenized in 5 ml of 0.02 M Tris-HCl, pH 7.35, per gram using a Polytron PTI0 homogenizer. After centrifugation for 60 rain at 30,000 g, supernatant proteins precipitating between 1.35 and 2.7 molal (NH4)2SO4 at pH 5.7 were resuspended and dialyzed overnight against the Tris-HC1 buffer. Thereafter, when necessary, the dialyzate was centrifuged to remove precipitated protein.

Purification of Enzyme Using Its Antibody The source of the crude liver e n z y m e preparation was a normal adult human male. Nonspecific precipitates in both antiserum and crude liver preparations were removed by centrifugation of each, separately, after J. J. Scheidegger, Int. Arch. Allergy 7, 103 (1955).

[37]

USE OF ANTIBODIES TO STUDY ENZYMES AND THE P E I A

555

preincubation for l hr at 37°. Sufficient antibody was then added to precipitate all enzymic activity. The immunoprecipitate was recovered by centrifugation and washed with 0.02 M Tris-HC1, pH 7.35. The pellet was then resuspended in 5 ml of 0.02 M citric acid-sodium citrate, pH 3.2, to dissociate the enzyme-antibody complex, sonicated at 40 W for 1 min at 0°, and incubated for 15 min at 37°. Insoluble protein trapped in the original immunoprecipitate was removed by centrifugation at 30,000 g for 30 rain. The supernatant was dialyzed overnight against 0.02 M Tris-HC1, pH 7.35, to reprecipitate the enzyme-antibody complex. The complex was recovered as a pellet and resuspended by sonication and incubation in a minimum volume of citrate buffer, as before. Undissolved precipitate which contained no enzymic activity, was removed by centrifugation. The supernatant was then applied to a refrigerated (4°) Sephadex G-200 column (previously equilibrated with the above citrate buffer) and eluted with the citrate buffer to separate enzyme from antibody. The eluted enzyme was dialyzed against 0.02 M Tris-HC1, pH 7.35, then stored at 4°.

Disc Gel Electrophoresis The method of Davis 9 was used. Results

Purification of Enzyme The specific activity of sulfatase A increased 160 times through steps 1-8. Actual purification of the enzymic protein may have been greater because there was a substantial loss of units (95%) during the final electrofocusing steps, presumably by inactivation. Mixing experiments did not confirm the possibility that sulfatase A was resolved into two less active fractions. The final preparation of sulfatase A contained three bands by disc gel electrophoresis (pH 8.9).

Antibody Titration One rabbit yielded a high antibody titer when immunized with fraction 17. Even when (after the second challenge) this antibody was diluted 20 times, it still precipitated 75% of the enzyme activity in a crude liver supernatant. After the third challenge, 50% of the activity was precipitated at a dilution of I : 40, and after the fourth challenge, 90% at a dilution of 1 : 160. Subsequently the titer remained at this high level for 2 months. 9 B. J. Davis, A n n . N. Y. A c a d . Sci. 121,404 (1964).

556

[37]

ANTIGEN A N D ANTIBODY DETECTION BY IMMUNOASSAY

s

~°'~

~ , ~

~m

. . . .

SE A PLUS ANTIBODY

.,. s s

RELATIVE VELOCITY

,Y

/

/

UL FATASE A

!

|

i

i

I0

20

30

SUBSTRATE

CONCENTRATION

FI~. 1. The effect of increasing substrate concentration on the relative velocity of the sulfatase A reaction, with and without antibody. Antibody inhibits hydrolysis at low substrate concentrations (increased K,0 and stimulates hydrolysis at high substrate concentrations (increased Vmx). The characteristic inhibition of the normal enzyme by excess substrate is not seen when antibody is present. Values are means - standard deviations; incubations were for 3 hr.

Kinetics of the Enzyme-Antibody Reaction Antibody alters the kinetic properties of sulfatase A. These effects are more evident if the usual assay conditions are modified (e.g., to a 3-hr incubation at greater than 5 m M substrate concentrations instead of a 1-hr incubation at a substrate concentration of 5 mM). Antibody slightly inhibits e n z y m e activity at low p H but stimulates hydrolysis at high pH. In interpreting the shape of this and subsequent kinetic curves, it is of note that normal sulfatase A, even in the absence o f antibody, typically displays anomalous kinetics. 10 The antibodyprecipitated enzyme differs from the enzyme exposed to buffer both in its K m and Vmaxand in the fact that it is not inhibited by excess substrate (Fig. 1). Antibody increases the Km11 from 1.74 -+ 0.19 to 4.11 -+ 0.22 m M (relative to the hydrolysis at 40 m M nitrocatechol sulfate). Figure 2 indi~0 H. Baum, K. Dodgson, and N. Spencer, J. Biochem. 69, 567 (1958). ~1 B. Wilkinson, J. Biochem. 80, 324 (1961).

w

40

[37]

USE OF ANTIBODIES TO STUDY ENZYMES AND THE P E I A

2.5 2.4 2.3 2.2 2: o 2.1 I-2.0 D ~E 1.9 I.',,0 1.8 (.j, 1.7 ~E >.a 1.6 Z W 1.5 c~ 1.4 N J 1.3 <~ 1.2 nO 1.1 Z 1.O 0.9 0.8 0.7

o.oi

557

40.0 m M / 20.0 mM

olos

o11

o12

RELATIVE ANTIBODYCONCENTRATION FIG. 2. Relationship between the concentration of antibody, the concentration of substrate, and the stimulation of enzyme activity. A log scale is indicated on the abscissa. Note that the stimulation of enzyme activity bears a linear logarithmic relationship to the antibody concentration at higher substrate concentrations. Enzymic activity in the presence of antibody is normalized tbr each point by relating it to the activity observed using normal rabbit serum. The concentrations of rabbit antiserum or of normal rabbit serum were varied. The amount of crude liver enzyme with which they were allowed to react was kept constant. The immunoprecipitate was not separated from the enzyme-serum reaction mixture. The enzyme-serum mixtures were allowed to react for 3 hr with an equal volume of substrate. Final substrate concentrations were varied from 5 to 40 mM.

c a t e s t h a t t h e e n z y m e is n o t i n h i b i t e d b y i t s a n t i b o d y . I t s a c t i v i t y , i n f a c t , is e n h a n c e d .

Effect of Antibody Concentration and of Substrate Concentration on Enzyme Stimulation (Fig. 2) E n z y m e a c t i v i t y is c o n s i s t e n t l y s t i m u l a t e d o n l y a t s u b s t r a t e c o n c e n t r a t i o n s o f 5 m M a n d a b o v e . T h e a c t i v i t y is a l s o s t i m u l a t e d in d i r e c t p r o p o r -

558

A N T I G E N A N D A N T I B O D Y D E T E C T I O N BY I M M U N O A S S A Y

[37]

tion to the logarithm of the antibody concentration at higher antibody concentrations.

Effect of Enzyme Concentration The hydrolysis of nitrocatechol sulfate by either unreacted or antibody-reacted enzyme is directly proportional to the amount of protein under routine assay conditions.

Effect of pH 5.2 on the Enzyme-Antibody Precipitate Routinely, the enzyme was precipitated at pH 7.35 and assayed at pH 5.2. This change in pH resolubilized variable amounts of the enzymeantibody complex. The enzyme was more stable in the presence of antibody at pH 5.2. For example, the enzyme-antibody complex retained five times more activity than did the enzyme alone after (pre-) incubation at 45° for 1 hr at pH 5.2.

Immunodiffusion and Immunoelectrophoresis Unabsorbed antiserum gave three precipitin lines, indicating at least three different antigen-antibody reactions. However, serum absorbed with fractions electrofocused on either side of the enzyme produced a single precipitin line (Figs. 3 and 4). The single line of identity between the purified electrofocused enzyme (fraction 17) and the crude liver enzyme indicated that the reacting proteins in each preparation were antigenically equivalent. When such a slide was stained for enzymic activity, a curved red band replaced the single line of identity. This finding indicated that the line of identity also contained sulfatase A, precipitated by antibody, yet still enzymically active. Although enzyme protein was present (Fig. 3A), metachromatic leukodystrophy patients showed no detectable enzyme activity (Fig. 3B). A single distinct line was also obtained when either the normal or metachromatic leukodystrophy crude liver enzyme was studied by immunoelectrophoresis (Fig. 4). The enzyme migrated 1.5 cm in 3 hr. Fraction 17 also gave a single line of the same appearance and mobility. The antibody was immunoelectrophoresed for a shorter period of time (45 min). The antiserum, previously fractionated with 33% (NH4)2SO4 and absorbed, gave a single precipitin line against normal sulfatase A. This line had the same mobility as did y-globulin. After reacting with enzyme solution, the precipitin line also yielded the expected band of sulfatase enzyme activity.

[37]

USE OF ANTIBODIES TO STUDY ENZYMES AND THE P E I A

559

Purification of Enzyme Using Its Antibody The enzyme-antibody complex could not be dissociated with 4 M NaC1, 5 M NaI, or 0.1% Triton X-100. However, citrate, pH 3.2, proved useful in dissociating the enzyme-antibody complex while preserving its activity. Although sulfatase activity is normally stable at 37°, and in citrate at 4 °, at higher temperatures and pH 3.2 it is much more labile. Thus at 45 °, the loss of activity is almost instantaneous.

F l f . 3. Immunodiffusion of normal and abnormal sulfatase A against monospecific antibody. The wells at the top and at the lower right and left sides contain normal (N) liver enzyme. This serves as the antigen. The other three wells (ML) contain liver enzyme from three different patients with metachromatic leukodystrophy. Top right: late infantile form; bottom: juvenile form; top left: variant form. The center well contains monospecific absorbed antibody (Ab). In (A) the arrow points to a single white, unstained precipitin line formed after 24 hr. This indicates that there is a reacting protein in each patient that is antigenically equivalent to that in the other patients. (B) A reverse negative of the exact same slide in which the precipitin lines were now stained to detect sulfatase A activity. Enzyme reaction product diffuses away in a band from the original precipitin lines. Therefore, what is seen here is a broad white area (dashed lines) curved around the wells (the original color was red). Note that only those precipitin lines that formed in relation to the normal enzyme contain detectable enzymic activity. No enzymic activity was seen in relation to the three ML wells. Taken together, the findings suggest that there is a sulfatase A enzyme protein both in normals and in metachromatic leukodystrophy, but that the sulfatase A activity is deficient in metachromatic leukodystrophy.

560

A N T I G E N A N D A N T I B O D Y DETECTION BY I M M U N O A S S A Y

FIG. 3 (continued).

[37]

FIG. 4. Immunoelectrophoresis of normal and abnormal liver enzymes. Both the normal and the abnormal livers contain a protein that reacts with the sulfatase A antibody. The top well contains normal enzyme (N). The next three wells (ML) contain enzyme from the same three patients with metachromatic leukodystrophy, respectively, as in Figs. 3A and 3B. Each white arrow points to a single precipitin line. Both troughs contain monospecific, absorbed antibody.

562

A N T I G E N A N D A N T I B O D Y D E T E C T I O N BY I M M U N O A S S A Y

[37]

Three conditions allowed separation of the enzyme from the antibody on Sephadex G-200: (a) The enzyme is stable in pH 3.20 citrate; (b) antigen-antibody complexes are dissociated at an acid pH; (c) there is a large difference between the molecular weight of the enzyme (approximately 400,000) 4"12 and its corresponding antibody (approximately 160,000). Sulfatase A activity started to emerge from the column (void volume 52 ml) at an elution volume of 55 ml, corresponding to a molecular weight of about 400,000. The specific activity of the eluted enzyme was as high as 65.9. The temperature sensitivity of the Sephadex-purified enzyme was studied to test how much the enzyme had been dissociated from its antibody. The crude enzyme quickly loses activity at 67° when no antibody is present. However, in the presence of antibody, the enzymic activity is very stable. Therefore, aliquots of the Sephadex-purified enzyme were treated with either antibody or buffer and incubated at 67°. Tubes to which antibody were added were quite stable at 67°, but tubes without antibody rapidly lost activity (82% after 120 min) (Figs. 5 and 6). Thus, most of the antibody appears to have been removed by the Sephadex. As another test for the presence of residual rabbit antibody, sheep anti-rabbit serum was added to the Sephadex-purified enzyme. After this treatment, 70% of the enzymic activity was precipitated into the pellet fraction (Table I). This experiment shows that some antibody does remain attached to the enzyme, although not in sufficient quantity to render the enzyme-antibody complex heat-stable. Thus, to make this purification process applicable, the antibody would have to be covalently bound to an insoluble carrier. We have done this with cyanogen bromide-activated Sepharose 4B to purify a human ribonuclease. TM Potential contamination of the enzyme preparation with sulfatase B was not an issue because the rabbit antibody to sulfatase A did not crossreact with sulfatase B. The Use of Antibody to ASA-A in the Primary Enzyme Immunoassay (PEIA)

Principle A primary enzyme-antibody binding test has been developed to study antigenic differences between normal and mutant enzymes that cannot be distinguished by routine immunoprecipitation techniques. The test de12L.

W. Nichol and A. B. Roy, Biochemistry 4, 386 (1965). 13 E. A. Neuwelt, J. Frank, et al. J. Biochem. 163, 419 (1977).

[37]

USE OF ANTIBODIES

TO STUDY

ENZYMES

AND THE PEIA

563

5.0

CRUDE LIVER ENZYME PLUS ANTIBODY

>i-

1.0 ~ I - - ' T 0.5

I--

M >N Z

nuJ 0.10

~

i

.

CRUDE LIVER

N

-i 0.05

IT 0 Z

0.01

,

,

,

,

,

,

,

,

,

,

,

,

0 10 20 3040 5 0 6 0 7 0 8090100110120 TIME (minutes)

FIG. 5. Incubation of crude liver enzyme at 67° with and without antibody. Note that the antibody confers stability on the enzyme at pH 7.35 in 0.02 M Tris-HCl buffer. The activity of the enzyme plus its antibody at 120 min is taken as 1.0, and all other enzymic activities were normalized to this value. The standard error of the mean is indicated. pends on enzyme substrate specificity and does not require monospecific antibody or pure antigen, one of which is needed for most other primary binding tests. Goat antiserum to normal human arylsulfatase A was coupled to Sepharose 4B using cyanogen bromide. The mutant enzyme protein from patients with metachromatic leukodystrophy or normal enzyme protein was assayed by its ability to saturate the Sepharose-bound antibody and thus block the subsequent binding of a standard normal enzyme preparation to the immunoabsorbent. Unbound normal enzyme activity in the supernatant was then measured by hydrolysis o f the substrate, nitrocatechol sulfate. Significant binding differences between normal and mutant enzymes were detected using this method. These antigenic differences were n o t detectable using immunodiffusion or immunoelectrophoresis. The method also made it possible to compare the binding of enzymes from different species. Monkey enzyme, and, to a lesser extent, dog enzyme, were found

564

A N T I G E N A N D A N T I B O D Y D E T E C T I O N BY I M M U N O A S S A Y

[37]

10.1

>-

F-

SEPHADEX PURIFIED ENZYME PLUS ANTIBODY >N Z LU a Ld U d

1.0

0.50

(3C

0 Z

0.10

II

0

•

!

•

!

!

I

•

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|

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10 20 30 40 50 60 70 80 90 100110120

TIME (minutes) FiG. 6. Incubation of Sephadex-purified e n z y m e at 67 ° with and without antibody. Note that the Sephadex-purified e n z y m e per se is not as stable as when it is allowed to react again with antibody. This suggests that the column has removed some antibody (but not necessarily all). In these experiments the e n z y m e was first precipitated with antibody and the e n z y m e activity was eluted from a Sephadex G-200 column using citrate buffer at pH 3.2. The e n z y m e w a s then dialyzed overnight against 0.02 M Tris-HCl, pH 7.35, before incubation at 67 ° . It was then studied exactly in the same manner as was the crude liver e n z y m e in Fig. 5.

to be related to the human enzyme. The primary binding test was also useful in quantitating normal enzyme protein during enzyme purification. The determination of enzyme specific activity based on enzyme protein rather than total protein concentration may now be possible in the carrier state. The primary enzyme immunoassay (PEIA) suitably modified may be extended to study qualitative and quantitative enzyme or inhibitor protein deficiencies in other diseases caused by inborn errors of metabolism. It has also been successfully adapted to compare several normal human ribonucleases. 13

Purpose Although biochemical methods have been developed to detect a deficiency of enzyme activity, a sensitive and simple immunologic method to

[37]

USE OF ANTIBODIES TO STUDY ENZYMES AND THE P E I A

565

differentiate active from inactive enzyme protein would be of diagnostic value, especially in the detection of the heterozygous carrier state. ~4 Moreover, an assay for inactive e n z y m e is essential if the abnormal protein is to be isolated and characterized. In previous studies, inactive ASA-A protein was detected immunologically but could not be distinguished from normal active enzyme. 1~,1GThe purpose of the present study is to describe a new procedure that (a) measures the primary immunological interaction between enzyme protein and antibody; (b) identifies and distinguishes between active and inactive e n z y m e immunologically; and (c) quantitates both functional and mutant inactive enzyme protein. The procedure has practical application because it obviates the need for monospecific antibody and purified antigen, one of which is usually required for primary binding tests. ~r This method has the potential to be extended to the study o f other inborn errors with relative ease.

Materials and Methods E n z y m e Preparation

Soluble crude e n z y m e samples were prepared by homogenizing fresh frozen liver obtained at autopsy or necropsy from normals and patients with metachromatic leukodystrophy (MLD). The crude liver supernatant was either used directly or concentrated by 0-60% ammonium sulfate fractionation in 0.1 M acetate buffer, pH 5.8.1~ A semipurified normal enzyme preparation was also prepared by ammonium sulfate fractionation, fi~llowed by acetone fractionation, column chromatography and isoelectric focusing and used for immunization as described above in the first part of this chapter covering the production and application of antib o d y to arylsulfatase A and in a previous communication. 15 All e n z y m e preparations were dialyzed against 0.2 M Tris-HCl, pH 7.35 before use. A pure ASA-A enzyme obtained from urine by affinity chromatography was kindly provided by Dr. J. L. Breslow, National Heart and Lung Institute. ~s

14

E. A. Neuwelt, D. Stumpf, P. Kohler, and J. Austin, Symp. Sphingolipids, Sphingolipidoses Allied Disorders, p. 421 (1971).

15E. A. Neuwelt, D. Stumpf, J. Austin, and P. Kohler, Biochim. Biophys. Acta 236, 333 (1971). 16D. Stumpf and J. Austin, Arch. Neurol. 24, 117 (1971). 17p. Minden and R. Farr, in "Immunological Diseases" (M. Samter, ed.), 2nd ed., p. 179. Little, Brown, Boston, Massachusetts. 18j. L. Breslow and H. R. Sloan, Bioehem. Biophys. Res. Commun. 46, 919 (1972).

566

A N T I G E N A N D A N T I B O D Y D E T E C T I O N BY I M M U N O A S S A Y

[37]

Enzyme Assays The presence of active ASA-A was measured spectrophotometrically by the hydrolysis of the artificial chromogenic substrate nitrocatechol sulfate as described in the first part of this chapter. ASA-B was assayed by the method of Baum et al. 2 Acid phosphatase was assayed using nitrophenyl phosphate. Specific activities are expressed as units of enzyme per milligram of total protein or as units of enzyme per relative units of enzyme protein (see section entitled: Blocking PEIA).

Preparation of Antibody Four-tenths milliliter of a 1 : 1 mixture of semipurified enzyme preparation from normal liver in complete Freund's adjuvant was injected into both popliteal lymph nodes of a 42-kg goat.'3 The specific activity of the enzyme was 554 units per milligram of protein, and the protein concentration was 1.96 mg/ml. Subsequent injections of the same dose were given intramuscularly 4, 6, and 10 weeks later. The experiments in this study were performed using serum obtained 12 weeks after the initial injection. When the goat antiserum was studied by double immunodiffusion and immunoelectrophoresis with crude human liver enzyme, 4-6 precipitin bands developed. Using a chromatographically pure urinary ASA-A preparation '~ both the multivalent goat and a monospecific rabbit antisera produced a line of identity between normal liver enzyme and the pure ASA-A preparation.

Conjugation of Goat Antiserum to Sepharose Sepharose 4B was activated with cyanogen bromide according to the method of Cuatrecasas.'9 The activated Sepharose was then allowed to react at 4° for 20 hr with the undiluted goat antiserum (Ab) or normal goat serum (NGS) after dialysis against 0.1 M NaHCO3. The conjugated Sepharose (control NGS Sepharose or Ab-Sepharose) was then sequentially washed with 10 volumes of each of the following buffers: (a) 0.5 M NaHCO3; (b) 0. I M acetate buffer, pH 4.0; (c) 0.1 M Tris-HC1, pH 7.35; (d) 0. I M Tris-HC1, pH 7.35 + 0.2% bovine serum albumin; (e) 0.1 M Tris-HCI, pH 7.35; (f) 0.02 M citrate buffer, pH 3.2; and (g) 0.02 M Tris-HC1, pH 7.35. Both control and Ab-Sepharose were stable when stored in the last buffer at 4° .

Direct Prima~ Enzyme Immunoassay (PEIA ) The binding of enzyme to Ab-Sepharose represents the primary interaction between antigen and antibody. After incubation of normal liver ,9 p. Cautrecasas, J. Biol. Chem. 245, 3059 (1970).

[37]

USE OF ANTIBODIES TO STUDY ENZYMES AND THE P E I A

567

enzyme with Ab-Sepharose, the immunoabsorbent was allowed to settle spontaneously. The supernatent fluid was then removed, and the immunoabsorbent was washed twice in 0.02 M Tris buffer. Enzyme activity was then measured in both the initial supernatant fluid and in an aliquot of resuspended Ab-Sepharose. Enzyme bound to Ab-Sepharose had the same activity as free enzyme.

Blocking PEIA In rhe blocking PEIA, an enzyme preparation is used either as blocking (test) enzyme or standard enzyme. A constant amount of unpurified norma~ human liver enzyme is used as the standard enzyme. A preparation that is being quantitated by its ability to block subsequent binding of the standard enzyme preparation to the Ab-Sepharose is the blocking enzyme. The source of blocking enzyme in this study was either purified or crude normal human liver enzyme.

Preparation of Standard Enzyme Conjugated Sepharose was allowed to react with different amounts of standard enzyme as follows: 0.1 ml of 1:2, 1:4, and 1:6 dilutions of Ab-Sepharose:buffer and control (unbound) Sepharose:buffer were reacted with serial twofold dilutions of crude normal human liver enzyme. The volume was brought to 1.5 ml with 0.02 M Tris-HC1, pH 7.35, and incubated at 37° for 30 min. To ensure adequate mixing, the mixtures were placed on an oscillating shaker (Lab Line Model 2095). The Sepharose enzyme complexes were then allowed to settle for 20 min at room temperature. Sepharose-frequent supernatent (0.3 ml) was assayed for ASA-A activity. Figure 7 shows several such experiments using different concentrations of enzyme and amounts of Sepharose. As illustrated, the fraction of enzyme bound to Ab-Sepharose (optical density units) increased with increasing amounts of Sepharose and with decreasing concentrations of enzyme (mg/ml). The optimal combination of standard enzyme and AbSepharose for the blocking PEIA was derived from this relationship. In Fig. 7, the optimal combination would be either 1 : 2 Ab-Sepharose and 2.48 mg of crude liver enzyme per milliliter or 1 : 4 Ab-Sepharose and 1.24 mg of crude liver enzyme per milliliter. Either of these combinations would result in a supernatant activity of 0.2-0,5 OD units using AbSepharose and 1 . 0 - 1 . 2 0 D units using NGS-Sepharose. This range of activity was chosen because it could be most accurately measured under the assay conditions used. These appropriate reagent quantities were employed to give the upper and lower base line values shown in Fig. 8, which illustrates the theoretical basis by which standard enzyme (crude normal

568

ANTIGEN AND ANTIBODY DETECTION BY IMMUNOASSAY

[37]

10.0

6,3 I'-Z D

o

1.0

O >.I-t.-(.9 ,< LLI >.N Z LLI I-Z

< I-.-

0,1

Z cr Ld (3._ 3 o3

I /// / 0.01 0.62

•- e -

Ul i ' ~ g o a ~ t ) l l u l u s e 1 : 6 Sephorose : Buffer 1 : 4 Sephorose : B u f f e r 1:2 Sephorose : Buffer

1124 2148 4197 9%

CRUDE

LIVER

ENZYME

(mg/ml)

FIG. 7. Arylsulfatase A binding to Ab-Sepharose with varying amounts of conjugated Sepharose and enzyme. Each line represents increasing amounts of enzyme (total crude liver protein) added to a constant amount of conjugated Sepharose. The distance between a point on any of the lines produced using Ab-Sepharose and the point on the line produced using no Sepharose or control-normal goat serum (NGS) Sepharose at the same total protein concentration, represents the amount of enzyme activity bound by the immunoabsorbent. h u m a n l i v e r e n z y m e ) is u s e d to a s s a y v a r i o u s test e n z y m e in the b l o c k i n g P E I A . T h e t e c h n i q u e u s e d is d e s c r i b e d b e l o w and o u t l i n e d in Fig. 9.

Blocking PEIA Procedure T w o f o l d serial dilutions o f e a c h b l o c k i n g (test) e n z y m e preparation w e r e p r e p a r e d to a dilution o f 1 : 1024 w i t h 0.02 M T r i s - H C l , p H 7.35.

[37] I

c

U S E OF A N T I B O D I E S TO S T U D Y E N Z Y M E S A N D T H E P E I A

NormalGoatSerumonSepharoseDIusNormalEnzyme

.

~

569

n

T.,.,,..o,,,.

"°°

In

,< z

tn

÷

O

BLOCKING(ENZYME)PROTEIN(Relat,veConcentrat,on) Anl~- Normal Non- CtossEntyme + Relctmg

[nlyme

FIG. 8. Theoretical basis of the different curves produced by using different arylsulfatase A (ASA-A) enzymes in the blocking PEIA. The distance between the upper plateau, produced using high concentrations of a given enzyme, and the upper base line, varies with the type of antigenic sites present on that enzyme. Enzymes, such as normal human enzyme, are able to react with all the antigen binding sites on the antibody to normal human enzyme. Therefore, high concentrations of the normal enzyme result in a supernatant value equal to the upper base line. In contrast, metachromatic leukodystrophy and animal enzyme share only some of the antigen binding sites present on the normal enzyme. Thus, the upper plateau using tihese enzymes is somewhere below the upper base line. Finally, a nonrelated enzyme such as arylsulfatase B gives a flat line equal to the lower base line since it shares no antigenic sites with normal ASA-A.

Buffer w a s added to duplicate tubes containing either 0.5 or 1.0 ml o f test e n z y m e to give a v o l u m e o f 1.5 ml and mixed with 0. I ml o f the predetermined concentration of control Sepharose or Ab-Sepharose. The mixtures were incubated on the oscillating shaker for 60 min at 37 °. The AbSepharose w a s then washed to r e m o v e unbound e n z y m e with 9.0 ml o f 0.02 M Tris-HCl, pH 7.35 and allowed to settle spontaneously for 20 min; the added buffer (9 ml) was r e m o v e d . When this was done carefully the Sepharose remained in the tube, all the unbound e n z y m e was r e m o v e d , and the total v o l u m e in each tube before and after each w a s h was unchanged. One-half milliliter o f the previously determined concentration o f standard e n z y m e (see section: Preparation o f Standard E n z y m e ) w a s then added to each tube, and this mixture w a s incubated for 30 min at 37 ° on the oscillating shaker. The e n z y m e in the Ab-Sepharose-free supernatant w a s r e m o v e d from each tube and assayed for e n z y m e activity. The relative amount o f A S A - A protein in a given test sample w a s

570

A N T I G E N A N D A N T I B O D Y D E T E C T I O N BY I M M U N O A S S A Y CONTROL TUBES

EXPERIMENTALTUBES

DILUTIONS OF

DILUTIONS OF

BLOCKING ENZYME

BLOCKING ENZYME

+

CONTROLTUBES

BUFFER

+

+

NGS- SEPHAROSE

AB-SEPHAROSE

AB-SEPHAROSE

60' ot 3 7 ° C

60' at 3 7 ° C

60' ot 3 7 ° C

WASH

STANDARD

AWAY

UNBOUND

AMOUNT

[37]

OF

ENZYME

NORMAL

ENZYME

30' ot 3 7 ° C

ASSAY

RESULT ~ ALL ACTIVITY IN SUPERNATANT

SUPERNATANT

ACTIVITY

RESULT :

RESULT

VARIABLE A C T I V I T Y

MINIMUM

IN SUpICRNATANT DEPENDING ON

ACTIVITY IN SUPERNATANT

OILUTION OF BLOCKING

ENZYME

FIG. 9. Schematic diagram of the blocking PEIA assay. The upper and lower base line values in Figs. 10-12 and 13B were obtained from the tubes in the left-hand and right-hand columns, respectively. calculated from these blocking studies. The blocking PEIA curves in Fig. 10 were derived using e n z y m e s from the normal livers of R. L. and G. W. Supernatant optical density determinations, which are a measure of enz y m e activity, were plotted against the amount of blocking (test) e n z y m e protein added. Undiluted e n z y m e was assigned a relative blocking enz y m e protein concentration of 1.0. The upper base line represents those tubes in which buffer was added to control Sepharose, followed by standard enzyme. The lower base line differs from the upper base line in that Ab-Sepharose was used instead of control Sepharose. Using R. L. as a reference curve, the relative amount of e n z y m e protein from G. W. was calculated. Since the midpoint between the baselines represents the amount o f blocking e n z y m e that occupies 50% o f the antigen binding sites,

[37]

USE OF A N T I B O D I E S TO S T U D Y E N Z Y M E S A N D T H E P E I A

571

1.2! 1.1

\

"

No

1,0

a Goat Serum on Sepharose plus Normal Enzyme

"~O.9 ~

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0.8

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O.3 0.2 0.1 O

I

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BLOCKING (ENZYME) PROTEIN (Relotive Concentrotion) FIG. 10. Use of the blocking PEIA to assay normal human liver enzyme. Note that at high concentrations of normal human enzyme (blocking enzyme), the supernatant values equal the upper base line, and that at low concentrations of enzyme the supernatant values equal the lower base line. The dashed lines were used to calculate the relative arylsulfatase A protein concentrations in G. W. as compared to R. L. as described in the text. The line labeled: "Goat Antibody to Enzyme on Sepharose plus Normal Enzyme" represents the lower base line, and "Normal Goat Serum on Sepharose plus Normal Enzyme" the upper base line. The undiluted blocking (test) enzyme is assigned a relative protein concentration of 1.0. a p e r p e n d i c u l a r line was d r a w n f r o m this p o i n t o n e a c h c u r v e to the axis l a b e l e d b l o c k i n g e n z y m e as s h o w n in Fig. 10. T h e v a l u e so o b t a i n e d f r o m a test c u r v e (i.e., G. W.) was t h e n d i v i d e d into the c o r r e s p o n d i n g v a l u e o b t a i n e d f r o m a r e f e r e n c e c u r v e (i.e., R. L.). This c a l c u l a t e d v a l u e gives the c o n c e n t r a t i o n o f e n z y m e p r o t e i n in the test p r e p a r a t i o n (G. W.) relative to the r e f e r e n c e p r e p a r a t i o n (R. L.).

Specificity of PEIA To d e m o n s t r a t e the specificity o f the a s s a y for A S A - A , e x p e r i m e n t s w e r e c a r r i e d out to see w h e t h e r acid p h o s p h a t a s e a n d A S A - B , w h i c h w e r e also p r e s e n t in the u n f r a c t i o n a t e d c r u d e liver e n z y m e p r e p a r a t i o n , w e r e b o u n d to the A b - S e p h a r o s e . It was f o u n d that the a m o u n t o f u n b o u n d acid

572

A N T I G E N A N D A N T I B O D Y D E T E C T I O N BY I M M U N O A S S A Y

[37]

phosphatase and ASA-B activity in the supernatant was unchanged by the addition of Ab-Sepharose.

Reaction of Animal Enzyme with Goat-Anti-Human ASA-A The reaction of antibody with animal enzymes was studied by measuring the binding of enzyme to Ab-Sepharose in the direct and blocking PEIA as described above. Monkey and dog liver ASA-A were studied. E n z y m e was not concentrated with (NH4)zSO4 in any of these studies because each animal enzyme has a different solubility in this salt. Results

Blocking PEIA In the blocking PEIA, the normal liver ASA-A enzyme occupied all the antigen binding sites on the Sepharose-bound antibody, giving supernatant value equal to the upper base line (Fig. 10). In contrast, with the addition of saturating amounts of mutant (MLD) enzyme, the maximum blocking was considerably below the upper base line (Fig. 1 l). As both normal and mutant enzyme were diluted, as expected proportionately fewer antigen binding sites were blocked, resulting in less standard enzyme activity in the supernatant. With further dilutions of blocking enzyme (either normal or MLD), none of the binding sites were occupied, giving a minimum supernatant activity equal to the lower base line value. The shape of these curves can be further explained by reexamining the theoretical curves illustrated in Fig. 8. The distance between the upper plateau produced using high concentrations of a given enzyme and the upper base line varies with the antigenic sites present on that enzyme. A reaction o f identity such as that between normal human enzyme and its antibody gives an upper plateau equal to the upper base line, since all the antigen binding sites are filled at high enzyme concentrations. In contrast, M L D enzymes share only some of the antigenic sites present on the normal human enzyme. Thus, there is a reaction of partial identity giving an upper plateau below the upper base line. Finally, a nonrelated enzyme, such as ASA-B, results in a flat line equal to the lower base line because it shares no antigenic sites with normal human ASA-A. Using the same technique, binding differences were found between enzyme from human and animal sources. Even though enzyme concentrations in Fig. 12 were not high enough to produce upper plateaus, antigenic differences can be inferred from the different slopes o f the middle portions of each of the curves. If the concentration of ASA-A was the only difference between the different preparations, the middle portion of each curve

[37]

USE OF ANTIBODIES TO STUDY ENZYMES AND THE P E I A

573

1.2 1.1

~Normol

Go~, Serum on Sephorose plu~ Normal Enzyme

1.0 0.9 ~0.8 tr)

• Juvenile MLD Enzyme .it Infontile MLD Enzyme

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o

0.4 0.3 0.2

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O.1 .O

1.O

o's

0,0078 0.0039 o 6 o z . . 0.063 . .0.031 . 0.016 . 0.125 BLOCKING (ENZYME) PROTEIN (Relolive Concentrotion) o'.2s

0.001 '

FiG. I 1. Use of the blocking PEIA to assay metachromatic leukodystrophy (MLD) liver enzyme. This figure is analogous to Fig. 10; except that the blocking enzymes were derived from patients with MLD rather than from normal individuals. Note that the upper plateau values obtained with high concentrations of enzyme are well below the upper base line. The base lines are different in this figure from those in Fig. 10 because a different standard enzyme preparation was used. Nonetheless, the form of the curves using normal enzyme in this experiment remained the same as those in Fig. 10. w o u l d h a v e the s a m e slope b u t w o u l d b e shifted to the right or left. T h u s , the slope o f the m i d s e c t i o n o f e a c h c u r v e also reflects a n t i g e n i c differences. As i l l u s t r a t e d in Fig. 13A, m o n k e y e n z y m e a n d , to a lesser e x t e n t , dog e n z y m e w e r e o n l y partially able to b l o c k the a n t i g e n b i n d i n g sites on the i m m u n o a b s o r b e n t . T h e s e b i n d i n g differences were c o r r o b o r a t e d by the results o f the direct P E I A in Fig. 13B.

Specific Activity of ASA-A A c o m p a r i s o n was m a d e b e t w e e n the specific a c t i v i t y of A S A - A as c a l c u h t t e d b o t h f r o m the relative a m o u n t o f e n z y m e p r o t e i n a n d from the total a m o u n t of p r o t e i n p r e s e n t in different e n z y m e p r e p a r a t i o n s (Table II). W h e n the total a m o u n t o f p r o t e i n is u s e d to c a l c u l a t e the specific

ANTIGENAND ANTIBODY DETECTION BY IMMUNOASSAY

574

[37]

1.3 I 1.2 1.1 ,, "~

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x

,,.) 0,9'

,~_~

--

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~.

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a~5

ai2s

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BLOCKING (ENZYME) PROTEIN (Relative Concentration) FIG. 12. Comparison of human and animal enzymes using the blocking PEIA. Note the differences in the slope of each line. Enzyme concentrations were not high enough in these preparations to produce plateaus at the highest enzyme concentrations used.

activities of the purified and crude liver enzymes, the values vary between 15 and 554 units per milligram of total protein. In contrast, the values vary only between 1087 and 1419 units per relative unit enzyme protein when the blocking PEIA curves are used to calculate specific activities. Thus, crude human enzyme and purified human enzyme have similar specific activities when calculated on the basis of relative enzyme protein units. In contrast, they have markedly different specific activities when calculated on the basis of total liver protein. Direct PEIA

This test, a measure of the direct binding between test enzyme and Ab-Sepharose, was used to confirm the results of the blocking PEIA. Since MLD enzyme is inactive, it could not be studied by this procedure, which directly measures enzyme activity bound to Ab-Sepharose as the experimental parameter. As is shown in Figs. 13A and B, the amount of a given test enzyme bound directly to Ab-Sepharose correlated well with its blocking activity.

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, 0.031

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~ .... 0.016 0.0078

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B

13'

\.0

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E ¢ 0.9"

----(>--- Monkey

~, o.s pz 0.7

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0.4, a

0

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,.o

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F~G, 13. Comparison of monkey and dog ASA-A using both direct (A) and blocking (B) PEIA. As can be seen by comparing A and B the amount of each animal enzyme that binds to the Ab-Sepharose in the direct PEIA correlates with that enzyme's blocking ability in the blocking PEIA.

576

ANTIGEN A N D ANTIBODY DETECTION BY IMMUNOASSAY

~~

o

~

~ c5~

Z Z 0

Z

L~

o~ oz

o..

rr

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u~

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m

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7 <

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[37]

[37]

USE OF ANTIBODIES TO STUDY ENZYMES AND THE PEIA

577

Potential Significance of the PEIA The unique ability of ASA-A to hydrolyze nitrocatechol sulfate under the above-specified conditions has been carefully established. 2,16a° this enzyme-substrate specificity has been used in the present work to develop a sensitive and potentially widely applicable primary binding test. The advantages of the test are its specificity and the elimination of the necessity for monospecific antibody and pure antigen, one of which is required for most immunoassays.17 The sensitivity of a primary binding test can be utilized in this assay by employing an antiserum containing antibody populations to multiple unrelated proteins, and an enzyme (antigen) preparation that has not undergone purification. Indeed, a single antiserum containing antibodies to several enzymes potentially could be used sensitively and specifically to assay the amount of each of these enzymes present in a single crude preparation. The only requirement for such a system is that an enzyme or inhibitor assay be used that is specific for each activity to be quantitated. Since the blocking PEIA does not require active enzyme and is unaffected by antibody-mediated enzyme modification, it is more generally applicable than the direct assay.

Sensitivity of the PEIA The blocking PEIA reveals definite antigenic differences between mutant and normal human enzymes (Figs. 10 and 11). Using rabbit antiserum and goat antiserum, these differences could not be detected in immunodiffusion and immunoelectrophoresis studies. TM As expected, the studies shown in Fig. 13 suggest that monkey ASA-A is antigenically more closely related to human enzyme than is dog enzyme.

Use of the PEIA during Enzyme Purification As reported previously, '5 normal liver enzyme may be partially purified biochemically to the point where it contains only four protein bands on acrylamide gel electrophoresis. As shown in Table II, normal human crude liver enzyme has a specific activity of 15.3-24 units per milligram of protein, whereas purified enzyme has a specific activity of 292-554 units per milligram of protein. In the purification process, 97.5% of the enzyme activity was lost. The specific activities of crude and purified enzyme, however, are quite similar when the values are calculated on the basis of relative enzyme protein units. Therefore, it seems that not only was enzyme activity lost during purification, but enzyme protein, or at least antigenicity, was lost also. 2oj. Au,;tin, D. Armstrong, L. Shearer, and D. McAfee,Arch. Neurol. 14, 259 (1966).

578

A N T I G E N A N D A N T I B O D Y D E T E C T I O N BY I M M U N O A S S A Y

[38[

Enzyme Purification Using an Immunoabsorbent Column In earlier attempts to purify enzyme immunologically, ASA-A was precipitated with antibody and subsequently antigen and antibody were separated on Sephadex G-200 using pH 3.2 citrate as the eluent. Unfortunately, ASA-A-antibody complexes persisted.l~ The attachment of the antibody to an insoluble carrier has eliminated this problem. Breslow and Sloan is have recently prepared pure urinary ASA-A chromatographically. A monospecific antibody to such a preparation, when conjugated to Sepharose, could result in a much more rapid purification of ASA-A. It would be important to use the PEIA during such purification to measure enzyme protein because, as was shown above, it is not sufficient to monitor enzyme activity alone. Acknowledgments This m a n u s c r i p t was derived in large part from Biochim. Biophys. Acta 236, 333-346 (1971) and Immunochemistry 10, 767-773 (1973) with the permission of the copyright holders. The assistance o f Mr. A n d r e w Michael on the preparation o f this m a n u s c r i p t and the typographical expertise of Ms. Mary A n n M e a n s was m u c h appreciated.

[38] I m m u n o n e p h e l o m e t r i c A s s a y for I m m u n o g l o b u l i n s Released by Cultured Lymphocytes*

By JOSE Mur~oz, t GABRIEL VIRELLA, and H. HUGH FUDENBER~ The human immune system, like those of many other mammals and vertebrates, is a complex conglomerate of specific and nonspecific components, among which immunologists have paid particular attention to two groups of immunocompetent cells: T lymphocytes (thymus-derived) and B lymphocytes (bursa-derived in birds, of unknown origin in humans). Both types of lymphocytes can be studied by measuring their mitogenic responses after stimulation with lectins or bacterial products. In the case of B lymphocytes, the most commonly used mitogens are * Publication No. 418 from the D e p a r t m e n t of Basic and Clinical I m m u n o l o g y and Microbiology. R e s e a r c h supported in part by South Carolina State R e s e a r c h Appropriation 1979/80 and by U S P H S Grants HD-09938 and CA-25746. Present address: Unidad de Immunologia Clinica Facultad de Medicina, Universidad de L o s Andes, Merida, Venezuela.

METHODS IN ENZYMOLOGY, VOL. 73

Copyright ~) 1981by AcademicPress, Inc. All fights of reproduction in any form reserved. ISBN 0-12-181973-6