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Enzyme Immunoassay forAlpha-AmylaseKryzysztof A. Sobiech a & Regina Slowinska ba Department of Biochemistry , AgriculturalAcademy , 50-375, Wroclaw, Polandb Department of Cardiology , Institute ofInternal Medicine Medical Academy , 50-367,WroclawPublished online: 05 Dec 2006.

To cite this article: Kryzysztof A. Sobiech & Regina Slowinska (1983) EnzymeImmunoassay for Alpha-Amylase, Journal of Immunoassay, 4:2, 127-134, DOI:10.1080/15321818308057005

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JOURNAL OF IMMLTNOASSAY, 4 ( 2 ) , 127-134 (1983)

ENZYME IMMUNOASSAY FOR ALPHA-AMYLASE

Kryzysz tof A . Sobiech Department o f Biochemis t ry , A g r i c u l t u r a l Academy

50-375 Wroclaw, Poland.

and

Regina Slowinska Department of Card io logy , I n s t i t u t e of I n t e r n a l Medicine

Medical Academy, 50-367 Wroclaw

ABSTRACT

A s e n s i t i v e sandwich enzyme immunoassay has been developed f o r q u a n t i t a t i o n o f a l p h a - a m y l a s e i n p i g p a n c r e a s . T h e alpha-amylase-antibody con juga te was prepared from h o r s e r a d i s h peroxidase coupled wi th a n t i b o d i e s t o hog pancreas alpha-amylase. With t h i s method t h e measuring range is 1-10 ng/ml and t h e d e t e c t i o n l i m i t i s 0.2 ng/ml. The amount o f p i g p a n c r e a t i c amylase was measured t o be 6.21 2.05 mg/g of tissue. The enzymatic a c t i v i t y de t e rmina t ion and immunoassay were compared i n t h e m a t e r i a l s t u d i e d .

KEY WORDS: alpha-amylase, p i g pancreas , enzyme immunoassay.

I" The de te rmina t ion of alpha-amylase a c t i v i t y i n t i s s u e homogenates

and b i o l o g i c a l f l u i d s is h indered by d i f f e r e n t f a c t o r s which i n t e r f e r e

wi th t h e a s say . Th i s f a c t , t o g e t h e r wi th problems i n s u b s t r a t e

s p e c i f i c i t y , has prompted u s t o deve lop an immunological t echn ique f o r

measurement o f amylase.

Th i s paper d e s c r i b e s a new s e n s i t i v e enzyme-linked immunosorbent

a s say f o r q u a n t i t a t i o n of t h e amylase i n p i g pancreas .

127

Copyright 0 1983 by Marcel Dekker, Inc. 0 197-1 522/83/0402-O 127$3.50/0

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128 SOBIECH AND SLOWINSKA

Antiserum. Antibodies. Enzvme and Chemicak

Antiserum t o p i g pancreas alpha-amylase (Koch L igh t L a b o r a t o r i e s ,

Colnbrook, Bucks, England) was ob ta ined by t h e immunization o f r a b b i t s

a s r epor t ed p rev ious ly ( 1 1 ) .

The I@ was i s o l a t e d from t h e an t i s e rum o r c o n t r o l serum by

ammonium s u l p h a t e p r e c i p i t a t i o n a n d DE-52 c e l l u l o s e c o l u m n

chromatography, a s desc r ibed by Liv ings ton ( 5 ) . S p e c i f i c an t i amylase

a n t i b o d i e s were obta ined by a f f i n i t y chromatography, u s i n g immobilized

alpha-amylase as an immunosorbent ( 1 2 ) . P u r i t y of t h e p r e p a r a t i o n s was

v e r i f i e d by d i s c e l e c t r o p h o r e s i s , i m m u n o d i f f u s i o n , a n d

immunoelectrophoresis. Goat a n t i r a b b i t IgG was ob ta ined from

Behringwerke A G , Marburg Lahn, West Germany. F resh ly i s o l a t e d pancreas

from a hea l thy a d u l t p i g (Sus domest ica) was homogenized wi th 4 v o l . of

0.15 M NaCl i n a Potter-Elvehjem g l a s s homogenizer cooled wi th i c e ;

t h e sediment was removed by c e n t r i f u g a t i o n a t 10.000 g f o r 10 min, and

t h e supe rna tan t used f o r t h e experiment. Horseradish pe rox idase HRP

( E C 1 .11 .1 .7) was purchased from Serva Feinbiochemica, Heide lberg , West

Germany.

Sephadex G-25 and Sepharose-Con A were t h e p roduc t s o f Pharmacia

F ine Chemicals, Sweden. Po lys ty rene p l a t e s MRC ware ob ta ined from

Linbro USA. P r o t e i n s were determined by t h e c o l o r i m e t r i c method o f

Lowry e t a 1 (161, o r , i n t h e case of low p r o t e i n c o n c e n t r a t i o n s , by t h e

method of Blakes ley and Boezi (13) w i th bovine serum albumin (Sigma

Chemical Co., USA) used a s t h e s t anda rd .

Alpha-amylase a c t i v i t y was determined by t h e method o f Pick and

Wober (9) u s i n g amylase from Koch L igh t L a b o r a t o r i e s as t h e s u b s t r a t e .

Horseradish peroxidase a c t i v i t y was assayed c o l o r i m e t r i c a l l y ( 1 4 ) .

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ENZYME 1MM"OASSAY FOR ALPHA-AMYLASE 129

PreDara t ion of Conluaate

Conjugate HRP-anti-alpha-amylase an t ibody was prepared u s i n g t h e

two s t e p method accord ing t o Avrameas and Ternynck (2). B r i e f l y , 4.95

mg HRP d i s so lved i n 100 ul phosphate b u f f e r , pH 6.8, 100 mmol/L was

reac t ed a t 22OC f o r 18 h wi th g lu t a ra ldehyde (Se rva ) a t a f i n a l

concen t r a t ion of 1.2%. A sample o f modified HRP was sepa ra t ed from

excess of g lu t a ra ldehyde by column f i l t r a t i o n on Sephadex G-25.

F r a c t i o n s c o n t a i n i n g HRP were concen t r a t ed and allowed t o react wi th

2.5 mg anti-alpha-amylase a n t i b o d i e s in ca rbona te b u f f e r , pH 9 .5 , 50

mmol/L a t 4OC f o r 24 h. Then 50 u l of 0.2 mol/L L- lys ine was added,

mixed and kep t 2 h a t 4OC. After d i a l y s i s a g a i n s t s a l i n e , t h e

con juga te was p r e c i p i t a t e d wi th s a t u r a t e d ammonium s u l p h a t e . The

con juga te was sepa ra t ed from f r e e a n t i b o d i e s on a Con A-Sepharose

column e q u i l i b r a t e d in a c e t a t e b u f f e r , pH 6.0, 100 mmol/L c o n t a i n i n g 1

mol/L NaC1, 1 mmol/L MgC12, and 1 mmol/L CaC12 (1). F r a c t i o n s

c o n t a i n i n g t h e con juga te were e l u t e d by 100 mmol/L alpha-methyl

D-glucosid- (Sigma), concen t r a t ed t o 0.2 m l , and kept a t -12OC f o r over

6 months. In a double inmunodiffusion t e s t , an t ibody-peroxidase

con juga te r e a c t e d wi th enzyme s i m i l a r l y t o a n t i b o d i e s a g a i n s t

alpha-amylase. A c t i v i t y o f t h e con juga te was unchanged d u r i n g t h i s

pe r iod and r e p r o d u c i b i l i t y o f t h e s t anda rd c a l i b r a t i o n curve was

s a t i s f a c t o r y . - The procedure is summarized in F i g u r e 1 . Polys ty rene p l a t e s were

coa ted wi th 20 ug o f an t ibody pe r bead in phosphate-buffered s a l i n e

(PBS) a t 25OC for 16 h. After incuba t ion wi th g e l a t i n e 5 g/L a t 37OC

f o r 2 h, t h e p l a t e s were washed f i v e times wi th g e l a t i n e 1 g/L in PBS.

S tandard s o l u t i o n s of alpha-amylase o r samples c o n t a i n i n g enzyme were

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130

polystyrene bead

SOBIECH AND SLOWINSKA

rabbit anti hog pancereas amylase

sample containing amylase

rabbit anti amylase

-HRP conjugate

peroxidase activity assay

FIGURE 1 : Sandwich ELISA procedure for amylase.

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ENZYME IMMUNOASSAY FOR ALPHA-AMYLASE 131

incubated wi th beads a t 25OC f o r 2 h and ove rn igh t a t 4OC. Plates were

washed wi th g e l a t i n e 1 g/L and f i n a l l y t h e beads were incubated wi th

t h e conjugate s o l u t i o n , 400 ng per bead, a t 4OC f o r 16 h. After

washing, HRP a c t i v i t y was determined by t h e c o l o r i m e t r i c method ( 1 4 ) .

RESULTS

e r i z a t i o n of Con i u

Goat a n t i - r a b b i t IgG was used t o s e p a r a t e f r e e and c o v a l e n t l y

bound enzyme from conjugate . Determina t ion o f enzymatic a c t i v i t y

showed 78% o f peroxidase i n t h e p r e c i p i t a t e . With t h e q u a n t i t y o f

enzyme cova len t ly bound i n t h e whole con juga te , and t h e q u a n t i t y o f

enzyme be fo re t h e con juga t ion , t h e y i e l d was c a l c u l a t e d t o be 65%. The

molar r a t i o o f peroxidase t o a n t i b o d i e s i n t h e con juga te was de termined

t o be 2 .8 . The l o s s o f an t ibody r e a c t i v i t y due t o con juga t ion was

about 20% of t h e o r i g i n a l .

i s o n o f qglylase bv -tic A c t l v i t v and bv

ELISB

The amount o f p i g pancreas amylase was measured both by ELISA and

by enzymatic a s say u s i n g amylase a s t h e s u b s t r a t e . A s t a n d a r d

c a l i b r a t i o n curve o f amylase is i l l u s t r a t e d i n F igu re 2 . For

comparison, t h e enzymatic a c t i v i t y was conver ted t o ug/L us ing t h e

p u r i f i e d amylase f o r s t anda rd curve c a l i b r a t i o n . The mean amount o f

p a n c r e a t i c amylase determined by ELISA i n t h e 6 p i g s was 6.21 2.05

mg/g o f t i s s u e (mean f S.D.). The con ten t o f amylase de te rmined by

enzymatic a s say was 9.96 f 3.88 mg/g o f t i s s u e (59.8 mg/g o f homogenate

p r o t e i n ) . The amylase r a t i o determined by enzymatic a s say and by ELISA

was es t ima ted t o be 1 .62 .

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132 SOBIECH AND SLOWINSKA

1 10 1 00 ng amylase

FIGURE 2: S tandard c a l i b r a t i o n curve of amylase by t h e ELISA method. Hor i zon ta l bar-working range o f t h e ELISA t e s t .

DISCUSSION

Since t h e f i r s t communication about enzyme imnunoassay was

publ i shed by Engval l and Perlmann ( 4 ) i n 1971, s e v e r a l a s s a y s f o r

q u a n t i t a t i o n of serum p r o t e i n s , a n t i g e n s ( i n c l u d i n g enzymes), hormones,

and drugs have been publ i shed . During t h e l a s t t e n y e a r s many

immunological s t u d i e s of p a n c r e a t i c and s a l i v a r y amylases have been

presented (7,8). Ryan e t a 1 (10) and Takatsuka e t a1 (13) r e c e n t l y

have developed s p e c i f i c radioimmunoassays f o r p ig and human p a n c r e a t i c

amylases. Previous s t u d i e s i n o u r l a b o r a t o r y on t h e immunological

p r o p e r t i e s of p a n c r e a t i c amylase and p u r i f i c a t i o n of s p e c i f i c

a n t i b o d i e s a g a i n s t amylase s t i m u l a t e d f u r t h e r i n v e s t i g a t i o n s (11 ,12 ) .

In t h e p re sen t paper , a s p e c i f i c and h igh ly s e n s i t i v e ELISA f o r

p i g panc rea t i c amylase is repor t ed f o r t h e f i r s t time. The r a t i o of

p a n c r e a t i c amylase determined by c o l o r i m e t r i c a s say and ELISA was 1 .62 .

These r e s u l t s may be expla ined by t h e e f f e c t o f t h e complex k i n e t i c s of

t h e ca t a lyzed r e a c t i o n and t h e l a c k o f s p e c i f i c s u b s t r a t e f o r amylase.

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ENZYME IMMUNOASSAY FOR ALPHA-AMYLASE 133

Moreover, i n t h e enzymatic a s s a y , amylase con ten t may be dependent on

t h e presence o f o t h e r p a n c r e a t i c enzymes a c t i n g on amylase o r i t s

degrada t ion products . It seems t h a t u se o f t h e ELISA t e s t has d i s t i n c t

advantages over t h e enzymatic a s say . The ELISA method desc r ibed he re

can be used even when alpha-amylase i s i n a c t i v a t e d o r i n h i b i t e d by low

molecular compounds i n b i o l o g i c a l m a t e r i a l . The p r e s e n t r e s u l t s a l low

u s t o recommend t h i s method f o r amylase as say .

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