Biochimie t 1991 ) 73, 1417-1425

1417

© Socift6 franqaise de biochimie el biologie mol6culaire / Elsevier, Paris

Antigen specificity and cross-species reactivity of a monoclonal antibody (mAb 72.11) against porcine pancreatic pr

colipase

C Dezan, N Rugani, L de la FourniSre, L Sarda*, B Bellon institut de chirnte biologique, facultd des sciences St Charles. place Victor Hugo, !333; Marseille Cedex 03. France

(Received 12 July 1991" accepted 17 October 1991)

Summary - - We have studied the antigen specificity and cross-reactivity of a monoclonal antibody (mAb 72.11) of subclass lgG~, raised against the precursor form of porcine colipase (procolipase), whose epitope lies near the amino terminal region of the polypeptide, mAb 72.11 cross-reacts with native porcine, equine and human procolipase, as shown by immuno-inactivation and ELISA titration studies carried out on pure proteins, pancreatic tissue homogenate or pancreatic juice. The epitope site recognized by mAb 72.11 was further characterized by studying antibody binding to denatured procolipase. Reduced carboxymethylated procolipase reacted with mAb 72.11 in ELISA. Heat inactivated or reduced carboxymethylated porcine procolipase displaced antigen from the complex formed between antibody and native ~procolipase. The lack of sensitivity of epitope recognized by mAb 72.11 on procolipase to heat denaturation or reduction of the disulfide bridges is indicative that antigen specificity of mAb 72. ! ! is not dependent on the conformation of the antigenic site. Cross-reactivity of mAb 72.11 with procolipase from the three species demonstrates that substitution of amino acid at positions 1 and 3 causes no loss of antigenicity. Finally, mAb 72. !1 was coupled to sepharose to isolate human procolipase from human pancreatic juice and to separate the precursor form from activated colipase non-adsorbed on the column. colipase / pancreas / pig / horse / man / monoclonal antibody crosn-reactivity / immunoaffinity chromatography / ELISA

Introduction

Pancreatic lipase activhy on emulsified triacylglycerol . . . . . . . . u,,,.,, ,_,.,,,, ~a~t wm~,~l prevents e n z y m e binding at the lipid-water interface. Inhibition is specifica!ly reversed by colipase, a protein found with lipase in the pancreatic secretion of mammals, birds and fish. Colipase binds to triacylglycerol particles in the presence of bile salt and anchors lipase to its substrate by forming a protein-protein complex in a one to one molar ratio [ 1]. The sequences of porcine, equine (isoforms A and B) and human colipases have been established using protein chemistry methods [2-4] and those of rat [5], man [6, 7] and dog [8] have been deduced from the nucleotide sequence of cDNA. Colipases contain 9 5 - 9 6 ami:m acid residues (55% homology) and five disulfide bridges located at invariant positions [9]. Colipase is a tightly packed molecule with short free ends of 16 residues (N-terminal) and 8 - 9 residues (Cterminal). *Correspondence and reprints Abbreviations: DTr, dithiothreitol; RCM-colipase, reduced

carboxymethylated colipase; mAb, monoclonal antibody.

Colipase is secreted by the pancreas as a precursor, procolipase [1t3]. Procolipase activates bile salt-inhibited pancreatic lipase in lipolysis systems cnntaining no phospholipid. Procolipase is c o n v e a e d to an activated form by trypsin that specifically cleaves the Args-Gly 6 peptide bond and removes the N-terminal pentapeptide. The newly formed N-terminal region is hydrophobic in all colipases (Gly6-11eT(Val, Leu) llea(Phe) - lleg). In contrast to procolipase, trypsintreated colipase activates pancreatic lipase in vitro, in lipolysis systems containing triacylglycerol, phospholipid and bile salt. It is generally considered that activation of procolipase by trypsin is essential for fat digestion in vivo. Actually, evidence has been given that the N-terminal activation pentapeptide of procolipase is biologically active and decreases food intake with a selective effect on fat intake l I t, 12], The specific activation of pancreatic lipase by colipase has been interpreted assuming that the cofactor molecule has two functionally important domains, a lipid and a lipase binding site. Identification of residues essential Ior colipase activity was made by studying kinetic and binding properties of chemically modified protein and by using physical techniques including spectrophotometry [ 13, 14], spectroflucrometry [15-17], proton N M R [18, 19] and photo-

i 41 S

C Dezan et al

C I D N P 1201. It was concluded, on the one hand, that hydrophobic regions 6 - 9 and 5 3 - 5 9 , h i g h l y consera, ed in all colipases, are i r v o l v e d in lipid binding. The ,-,mtral region 5 3 - 5 0 contains a cluster o f tyrosine ~esidues (Tyr:~. Tyros and Tyr~o). On the other hand, the carboxyl group o f Glut: or Asp72, present in all proteins, might play an essential role in c c l i p a s e lipase interaction by f o r m i n g an ionic b o n d with a lysine residue located in the C - t e r m i n a l d o m a i n o f lipase [2 I, 22 I. In recent years, we have prepared m o n o c l o n a l antibodies ( m A b s ) directed against porcine colipase (procolipase form) and studied their reactivity with antigen 123, 24]. Io a series o f eight a n t i b o d i e s characterized so far, six were found to inhibit procolipase activity by preventing the b i n d i n g o f the cofactor to triacylglycerol particles. Cotitrations in E L I S A have s h o w n that inhibitory a n t i b o d i e s react with two independent antigenic regions. T h e antigenic region recognized b y the first group o f a n t i b o d i e s contains residues o l the N-terminal part. o f the polypeptide. Antibodies o f the second group b i n d to a region located in or at the vicinity o f the cluster o f ~ r o s i n e residues (region 5 3 - 5 9 ) . Prediction studies have c o n c l u d e d that possible antigenic regions are located at positions 4 - 7 and 11-17 in the N - t e r m i n a l end. and at positions 4 0 - 5 0 and 6 1 - 7 6 , on both sides o f the tyrosine region [24]. C o m p a r a t i v e studies o f the reactivity o f m A b s with the precursor and trypsin activate~ forms o f the porcine cofactor h a v e s h o w n that they all reacted with the two forms o f antigen, except .rnAb 72.11 w h i c h reacted only with procolipase, m A b 7 2 . ! ! has been used for detection and differential determirtation o f porcine lzrocolipase in E L I S A . In this c o m m u n i c a t i o n , we report further studies.on the cross-species reactivity o f m A b 72.11 carried out with pure and crude s a m p l e s o f porcine, e q u i n e and h u m a n colipases. Results indicate that m A b 72.1 ! reacts with equine and h u m a n procolipases and can be used for the specific identification o f the precursor f o r m o f the h u m a n cofactor in crude s a m p l e s inc l u d i n g pancreatic j u i c e and for its purification by i m m u n o a f f i n i t y chromatography.

Materials and methods Materials Colipase samples Porcine and equine coiJpases (prccolipase forms) were prepared at the laboratory froia pancreatic tissue according to Frocedures already described [25, 26]. Trypsin-activated porcine colipase was obtained as reported previously except that the activation pentapeptide was separated from activated colipase by gel filtration on a Sephadex G-25 column (2.5 x 45 cm) equilibrated in distilled water [271. The activation peptide (Vai-Pro-Asp-Pro-Arg) was identified in fractions by

absorbance at 230 nM. These fractions were pooled and lyophilized. Samples of porcine and human pancreatic juice (lyophilized powder) were kindly provided by Dr Corring I INRA. Jouy-en-Josas) and Dr Dagorn (Inserm, Marseille). To obtain crude extracts of porcine and equine pancreas, defatted tissue (1 g) was homogenized in 3 ml of Tris 10 mM, ! mM benzamidine and 4 mM CaCI_,, pH 8.5 and insoluble material of the homogenate was removed by centrifugation (150 rain at I00 000 g). These preparations contained approximately equal molar amounts of lipase and colipase. To obtain samples ol inactive colipase, the protein was heated or treated with DTI'. A solution of pure porcine procolipase in distilled water (I mg/ml) was heated at 90°C. Under these conditions, activity was fully abolished after 150 mtn. Pure pore.me cofactol (procolipase or trypsin-activated form) was also inactivated by trealment with DTT (40 mM final concentration) for 5 min at 96°C, followed by addition of iodoacetate (100 mM final concentration) for 30 min at 37°C. Preparations of heat inactivated or RCM-colipase showed one single band with a molecular weight of 10 000 Da in gel electrophoresis in the presence of sodium dodecylsulfate. Determination of colipase activity Colipase activity was determined potentiometrically with the pH-stat method using triolein emulsified in gum arabic as substrate in the presence of 8 mM sodium deoxycholate and pancreatic lipase in excess to colipase ie under conditions where lipolytic activity was directly proportional to the amount of colipase in the assay syst,~m [281. Colipase dependent iipase activity was measured at pH 9 m-~d 25°C and expressed as colipase units. One colipase unit corresponds to the liberation of one microequivalent fatty acid per minute. Under the standard assay conditions, p~ocolipase and its trypsin-activated derivative showed the same activity (specific activity 20 000 units mg-t ). Monoclonal antibody (mAb 72.11) Antiporcine procolipase monoclonal antibody mAb 72.11 was produced and purified on a column of pure porcine procolipase coupled to sepharose 4B as reported earlier [23]. Stock solutions of antibody in 0. ! M sodium bicarbonate (1.5 mg Fer ml) were kept frozen at - 80°C. Methods lmmuno-inactivation studies Assays were carried out as described previously with minor modifications [29]. Pure procolipase, crude extracts of pancreatic tissue and samples of pancreatic juice containing about 2.5-3 lag of colipase corre'.;ponding to 50--60 cofactor units were mixed with increasing amounts of mAb 72.11 ranging from 5 to 40 tag, in a final volume of 500 lal of 0.1 M sodium bicarbonate with 0.5% bovine serum albumin and 1 mM benzamidine. The mixture was kept at 37°C for 1 h. Residual colipase activity was determined on an aliquot fraction (200 lai) of the colipase-antibody mixture. Inactivation curves were obtained by plotting the fraction of residual colipase activity against the concentration of aatibody in the incubation mixture. The value of the microscopic dissociation constant of the antibody-antigen complex formed between mAb 72.11 and procoiipase was obtained by comparing experimental and calculated curves obtained by assuming that the immunoglobulin (IgG) has two independant binding sites of equal affinity for colipase (C) and that the residual activity is representative of the fraction of antigen not bound to antibody.

Cross-reactivity of antiprocolipase monoclonal antibodv Then, the dissociation of the inactive antibody-ant!,,,.r, complexes, IgGC and IgGC., is described by equilibria ( 1) and (2). IgGC 2 !gGC + C (K 2)

(!)

IgGC

(2)

formed between native and inactive (denatured) pn,,colipa~: with m A b 72. I l antibody, respectively end c.. the concentration of free inactive cofactor. Then, it becomes: -

~IgG

+ C ( K I)

K~ and K, are the macroscopic dissociation equilibrium constants. The fi'action of antigen bound to antibody, z,

i s d L n ~ = c d_0 dLn c ~}dc

with Ln #, the binding potential [301, ~ the partition function [31] and c the concentration of free antigen (procolipase). Assuming that the immunoglobulin has two identical independent binding sites of microscopic dissociation constant equal to kd, 0 can be expressed as:

I. 419

-"

-,,.

2 k,c. k,c. + k,c i + k,k,

{5

Assuming that, under experimental conditions used. the two sites of the antibody are bound to either one of the two forms (native or denatured) of the aJitigen, z, + z, = 2 (z, = fraction of inactive colipase bound to antibody). C,T--C, +C,v--Ci _ 2 a n d c , = c l v _ 2 1 g G T + c , r _ % . with igG-r IgGlc,x and c~$, the total concentration of native and inactive procoiipase, and IgG v, the total concentration of antibody. If r represents the fraction of free native colipase r =

. equation

(5) can be written as follows: (~ = (c + kd)2 = c 2 + K2c + KIK-, (kd = K--z-2= 2 KO 2 dLnq_ Therefore z - - dLn c andz =

c , r ~ kr +i c , v ( l - r ) - 2 1 g G l + k ,

c 6~ _ 2 c ~ dc c + ka

IgGC ,- 2 IgGCC _ C v - C lgGr IgGT

2C (3) C + kd with CT and IgGr, the total

cor~centration of the colipase and antibody, respectively. C If r = ~---, equation (3) can be written t.-r

Co•

lgG r = - ( i - r ) +

kd-A---( ! -- r) 2 r

(4)

and theoretical inactivation curves are obta,~ned by plotting r versus IgGv, the total conce,:tradon of mAi~ 72.11 in the incubation mixture, for given values of kd.

Displacemem o f native procolipase front inactive antig~'n-antibody complex by denatured antigen Displacement of native colipase from inactive antigen-antibody complex by denatured antigen was studied to assess the importance of the conformation of procolipase for its association with m A b 72.11. Porcine procolipase (5 10-7 M) was mtxed with m A b 72.1 ! (1.25 10--7 M) under conditions described above. Denatured colipase was added to the incubation medium, at concentrations ranging from 0 to 10-5 M and the incubation mixture was kept at 37°C for 1 h. Colipase activity was determined on an aliquot fraction (200 lad of the incubation mixture under standard conditions. Reactivation curves were obtained by plotting the fraction of colipase activity as a function o f the logaf'thm o f the total concentration of denatured cofactor added to the complex. To estimate the dissociation constant o f the complex formed between inactive colipase and m A b 72. ! 1, the experimental curves were compared to theoretical curves calculated as follows: the fraction of native colipase bound to antibody, z.. is z. =

--~%. with c.. concentra-

tion o f free native colipase and • = (k~c. + k.c~ + k~k.)-', k.., and k: are the microscopic dissociation constants o f the complex

k, k, 2l -rr

lgG T = - c , r

(6)

From equation (6), theoretical reactivation curves are obiained by plotting r versus logc,v, with value of c,v equal to 5 10- 7 M. value o f IgG v equal to 1.25 10-7 M and k. equal to l0 -9 M. In our experiments, the total concentration (c,v) of native colipase is equal to 5 10--7 M and the total concentration (IgGT) of monoclonal antibody is equal to !.25 10 -7 M. The value of the dissociation constant (k,) is about 10-9 M; therefore it can be assumed that two molecules of colipase are bound to each molecule of mor~oclonal antibody.

lmmunoaf-finity chromatography Purified antibody m A b 72. ! 1 (30 mg) produced at tke laboratory was coupled to BrCN-activated sepharose 4B (Pharmacia Fine Chemicals, Uppsala, Sweden) according to the manufacturer's instructions. The immunoaffinity column (i.6 x 8 cm) was prepared by suspending the immobilized antibody in 0. ! M sodium bicarbonate. The column was loaded with extracts of pancreatic tissue or pancreatic juice containing an average ameunt o f c e ! i p a ~ co..'respop.ding to !5 r ~ n 20 ~ units or about one milligram of cofactor. Adsorption of colipase to immetfilized antibody was achieved by recycling the eluate through the column with a pump working at a constant flow rate of 20 ml per hour. After i 5 h~ adsorption of procolipase on imm,._:,~adsorben: was almost complete (95-100%). The column was rinsed by passage of 60 ml of ~odiu.rn bicarbonate containing 1 m M benzamidine, and finally with 20 ml of sodium bicarbonate without benzamidine. Procolipase adsorbed to immobilized antibtxly was eluted with 0.1 M glycineHC! buffer pH 2.8 or 3.4. The pH of the eluted fractions containing colipase was immediately brought to 8.4 with a ! M Tris buffer. After each chromatography cycle, the immunoadsorbent column was washed with 20 ml of 0.1 M acetate buffer pH 4 containing 0.5 M NaCI and further equilibrated with 0.1 M sodium bicarbonate. All experiments were caJried out at 4°C. Samples submitted to immunoaftinity chromatography were prepared as follows: NaCI was added to sotuuons of crudc extracts of porcine and equine pancreas to bring the ionic strength to the same value as sodium bicarbonate 0.1 M and passed through a 0.22 lain Millipore p.ltcr. The column was loaded with an aliquot fraction (10 ml) of this solution containing about one milligram of colipase.

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C Dezan et al

Lyophilized human or porcine pancreatic juice was dissolved in distilled water such that the ionic strength of the solution was equal to that of sodium bicarbonate 0.1 M and the solution was added with benzamidine (final concentration of 1 raM). Chromat%m'aphy was performed on an aliquot fraction (25 ml) containing about i mg of colipase.

lipases are fully inhibited by m A b 72.11. Experimental results indicate that pure porcine and equine cofactors are similarly sensitive to immunoinactivation by m A b 72.11 and that, in both cases, the value of the microscopic dissociation constant of the antigen-antibody complex, ka, determined as described in Materials and methods, is about 10 -9 M (fig 1, curve c). This value is in accordance with that previously determined for porcine procolipase by radioimmunoassay (1.5 10 -9 M). Analy~,is by gel electrophoresis in SDS of the procolipase-antibody complex revealed that the integrity of the cofactor was not affected after 1 h of incubation (not shown), m A b 72.11 has no effect on the trypsin-activated forms of porcine or equine cofactors (fig 1, ,.;.otted line). Parallel immuno-inactivation studies were carded out with partially purified preparations of porcine and equine colipases obtained from homogenized pancreatic tissue. Results shown in figure 2 indicate that colipase activity, in these preparations, is highly sensitive to inhibition by m A b 72.11. The value of the dissociation constant of the inactive antigen-antibody complex estimated from the immuno-inactivation curve is around 10-9 M, a value similar to that found with pure porcine and equine procolipases. It then appears that colipase present in crude preparations of porcine and equine pancreas is in the form of procolipase and that interaction with m A b 72.11 is not significantly affected by the presence of lipase and other proteins.

ELISA titrations Assays were carried out on native or denatured antigen (procolipase or trypsin-activated colipase) as described previously [321 with Nunc maxisorb microplates (Nunc, Roskilde, Denmark).

Rcsults lmmuno-inactivation studies

Pure porcine or equine pancreatic procolipase was mixed with increasing amounts of m A b 72.11, in the absence of triacylglycerol emulsion, and residual cofactor activity was measured. Immuno-inactivation curves are shown in figure 1. As observed from figure 1, the activity of porcine and equine proco-

•~ 50 8

_.100 ~ ~

v

0

,

!

,

2

3

i

4 5 10-7 [MAb 72.11 1 mol~/liter

~ 50 °--

Fig 1. Effect of increasing concentrations of antiporcine procolipase monoclonal antibody 72.11 on the activity of pure porcine (0) and equine ([2) procolipases. Procolipase (2.5 ptg) was preincubated in 500 lal of 0.1 M sodium bicarbonate and 1 mM benzamidine containing 0.5% bovine serum albumin with increasing amounts of mAb 72.11 ranging from 5 to 40 l.tg for 1 h at 37°C and residual colipase activity of an aliquot fraction of 200 l.tl was measured in the triolein-deoxycholate lipolysis system containing colipase-free pancreatic lipase (40 units) under standard conditions. Curves a, b, c, d are theoretical inactivation curves calculated as described in Materials and methods, assuming microscopic dissociation constant of the inactive antigen-antibody complex, kd of 10-7, 10-8, 10- 9 and 10- R° M, respectively. The dotted line represents the experunental curve obtained with pure porcine (©) and equine (ll) trypsin-activated colipase.

e~

0

i

1

|

i

2

3

t

l

4 5 10-7 IMAb 72.11 ] mole/liter

Fig 2. Effect of increasing concentrations of mAb 72.11 on colipase activity of crude extracts of porcine (0) and equine (E3) pancreatic tissue. Experiments carded out as in figure 1, with aliquots of extracts containing 2.5 lxg of colipase. Theoretical inactivation curves (full linc) were calculated assuming a value of kd, the microscopic dissociation constant of the antigen-antibody complex, of 10-9 M.

Cross-reactivity of antiprocolipa.~e monoclonal antibody Inhibition of colipase activity by m A b 72.11 was also studied using various samples of porcine and human pancreatic juice. Experiments carried out with three samples of porcine pancreatic juice indicate that in all cases, about 9 5 - 1 0 0 % of the activity was inhibited after incubation with antibody (fig 3, curve a). In contrast, large differences are found in experiments carded out with different samples of human pancreatic juice. Immuno-inactivation experiments indicate that m A b 72.11 reacts with the human cofactor (procolipase form). However, in some samples of human pancreatic juice, colipase is partially in the activated form and, therefore, is not sensitive to immuno-inactivation by m A b 72.11 (fig 3, curves b and c). To investigate the interactior, of denatured procolipase with m A b 72.11, the inactive antigen-antibody complex formed between pure porcine procolipase and m A b 72.11 was mixed with increasing amounts of antigen inactivated by heating at 90°C or treated by DTT and iodoacetate (RCM-colipase). Procolipase displaced from the complex was determined by measuring colipase activity (fig 4). Experimental results shown in figure 4 indicate that porcine procolipase is displaced from the complex by either one of the preparations of denatured antigen. The microscopic dissociation constant of the complex formed between denatured porcine procolipase and the monoclonal antibody is slightly higher than that of the 100

1421

100

/ (a)

/,:'/ (b)

//

(c)

50 -7

-6

Log (total concentration

-5 of inactivated coEpsse)

Fig 4. Displacement of native porcine prccolipase from antigen-antibody complex formed with mAb 72. i l by antigen inactivated either by heating (©) or by reduction wi',b DTT (0). Native procolipase (5 10--7 M) was mixed wi~ mAb 72.11 (1.25 10--7 M) to obtain 50% inhibition of colipase activity. Inactivated porcine procolipase was then added to the mixture and activity of an aliquot fraction of 200 lal was measured. The increase in colipase activity corresponds to active procolipase (free native procolipase) displaced from the complex. Curves a, b, c (fuii lines) represent theoretical displacement curves calculated, assuming that the microscopic dissociation constant of the complex formed between inactivated porcine procolipase and antibody is 10-1°, 10-9 and 10-8 M respectively and that of the complex formed with native antigen is 10-9 M. The curve (dotted line) that fits experimental points corresponds to a value of the microscopic dissociation constant of denatured procolipase of 3.5 10-9 M.

"'"~'~~. .~. .r~

~ 50

°-,

\ '"..............~k "i ' ................• ............................................................................................. • N~.,

(b)

(a) i

i

i

~

1

2

3

.4, [ M A b 72.11

i

5 10 -7

l

mole/liter

Fig 3. Effect of increasing concentrations of mAb 72.11 on colipase activity of porcine (O) and human (4 and A) pancreatic juice. Experiments caiiied out as in figure 1 on aliquots of pancreatic juice containing 2.5 lag of colipase. Values of curve (a) represent average values found in three independent experiments that differed by less than 5%. Curves (b) and (c) were obtained with two different samples of human pancreatic juice.

complex formed with native antigen (fig 4). Denatured co lip ase ~. ., t.l y. p. ~ l l :l -.a.t.. ,.t l v a:t.¢.u. . . .~ ~_.--.~ loilill I. .., .O.U ,a l U .,,, llvt displace the procolipase-mAb complex. Displacement experiments were also performed under the same conditions to investigate the reactivity of m A b 72.11 to the porcine activation pentapeptide. Porcine procolipase was mixed with m A b 72.11 as described above (see Materials and methods) and the pentapeptide was added at concentrations ranging from 5 10 -7 to 10 -4 M. No significant displacement of procolipase from the complex was observed indicating that affinity of m A b 72.11 for the activation pentapeptide is, by no means, lower by at least three orders of magnitude than that of the antibody for the native antigen protein (109 M -t).

Immunoaflinity chromatography Advantage was taken of the affinity of m A b 72.11 for the precursor forms of porcine, equine and human procolipase to isolate cofactors by immunoaffinity chromatography. The column prepared with m A b

-4

i 4"~'~,._

C Dezan et al

72. ! ! coupled to sepharose was loaded with a crude preparation of porcine colipase containing about 16 000 units. The non-adsorbed fraction contained 1500 units representing activated colipase. Proteins adsorbed on the column were eluted by passage of 20 ml of 0. ! M glycine-HCl buffer, pH 2.8 and about 12 000 cofactor units were recovered in eluate. Analysis of this fraction by gel electrophoresis revealed the presence of one single protein component with mobility corresponding to porcine procolipase. Further characterization of this fraction was made by end-group analysis (valine as amino terminal residue) by the dansylation method [33] and by immunoinactivation with mAb 72.11. Very similar results were obtained with a crude sample prepared from horse pancreatic tissue. The immunoadsorbent column was alse used to isolate porcine and human colipases from pancreatic juice. A sample of porcine juice containing 20 000 colipase units, 95% of which was in the procolipase form as indicated by immuno-inactivation with mAb 72.11, was placed on the column and adsorbed colipase was eluted with glycine buffer as in previous experiments performed with crude extracts from pancreas. In the case of human pancreatic juice, about 85% of the 18 000 cofactor units placed on the column were adsorbed. The fraction eluted with glycine buffer contained 15 000 units, but, only 20% of the activity was inhibited by mAb 72.11 indicating that human colipase was in the activated form in eluate. In a second experiment, elution of human colipase was carded out with the glycine buffer at pH 3.4. Under these conditions, the same amount of co!ipa~e wag r~envered .from the column but more than 90% of the cofactor was sensitive to immunoinactivation by mAb 72.11 and, therefore, was in the precursor form. Homogeneity of human colipase isolated from pancreatic juice by immunoaffinity chromatography was controlled by reverse phase high performance liquid chromatography (fig 5) and polyacrylamide gel electrophoresis. The precursor nature of this fraction was ascertained by end-group analysis, by the dansylation method, which indicated the presence of a single residue of alanine. ELISA titrations

The immunoreactivity of pure equine and human procolipases with mAb 72.11, in ELISA, was compared to that of the porcine cofactor. Assays carded out by coating the plates with amounts of colipase ranging from 10 to 30 ng per well showed that procolipases from the three species reacted similarly to antiporcine procolipase monoclonal antibody. Reactivity of denatured procolipase to mAb 72.11 was also studied under the same conditions. Results indicated that the reactivity of porcine, equine ~nd human

0.02

,Ja 0.01