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Laccase as a New EnzymaticLabel for Enzyme ImmunoassayOlga V. Skorobogat'ko a , Andrey L. Gindilis a , ElenaN. Troitskaya a , Alexander M. Shuster a & AlexanderI. Yaropolov aa A. N. Bach Institute of Biochemistry, RussianAcademy of Sciences , 117071, Moscow, RussiaPublished online: 22 Aug 2006.

To cite this article: Olga V. Skorobogat'ko , Andrey L. Gindilis , Elena N. Troitskaya ,Alexander M. Shuster & Alexander I. Yaropolov (1994) Laccase as a New EnzymaticLabel for Enzyme Immunoassay, Analytical Letters, 27:15, 2997-3012, DOI:10.1080/00032719408000307

To link to this article: http://dx.doi.org/10.1080/00032719408000307

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ANALYTICAL LETTERS, 27(15), 2997-3012 (1994)

LACCASE AS A NEW ENZYMATIC LABEL FOR ENZYME

IMMUNOASSAY.

KEY WORDS Laccase; Immuno-laccase Conjugates; Enzyme

Immunoassay .

Olga V.Skorobogat'ko, Andrey L. Gindilis, Elena N.Troitskaya,

Alexander M.Shuster, Alexander 1.Yaropolov.

ANBach Institute of Biochemistry, Russian Academy of Sciences, 117071 Moscow, Russia.

ABSTRACT

A new immunochemical reagent is suggested containing as an

enzyme marker laccase obtained from the cultural liquid of basidial

fungi Coriolus hirsutus. The feasibility of immuno-laccase conjugates in

different versions of immunoanalysis (sandwich, competitive and indirect

enzyme immunoassay) was demonstrated. The assay based on antibodv-

laccase conjugates is simpler than that employing antibody-peroxidase

2997

Copyright 0 1994 by Marcel Dekker, Inc.

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2998 SKOROBOGAT’KO ET AL.

conjugates, since in the former case air oxygen is used as the second

substrate of the enzymatic reaction.

INTRODUCTION

Enzyme immunoassay is widely used in biological sciences,

medicine, food industry, etc.l92. One of the most important advantages

of enzyme immunoassay is its high sensitivity which is achieved as a

rule, due to high catalytic activity of enzyme markers conjugated with

different ligands (antigens, antibodies, cofactors, biotin, protein A, etc.).

This means that selection of the marker enzyme and method of

conjugate synthesis is of prime importance for successful realization of

enzyme immunoassay. The choice of enzymes that can be employed as

markers is rather wide 2; however the most often used enzyme is

horseradish peroxidase 3. Despite of a number of this enzyme undoubtful

advantages resulting from catalytic and physicochemical properties,

peroxidase is also characterized by certain short comings such as strong

background coloration of the reaction mixture caused by the presence of

hydrogen peroxide and formation of inactive intermediate compound of

the enzyme in the course of catalysis, as well as some others

comprehensively described in the literature 1945.

We suggest to use as a marker enzyme laccase obtained from the

cultural liquid of basidial fungi Coriolus hirsutus. Laccase catalyzes the

oxidation of polyphenols, polyamines and some inorganic ions in the

presence of oxygen 5 9 6 9 7 . The enzyme represents a glycoprotein with

molecular mass of 55,000 consisting of a single polypeptide chain and

containing 17% of carbohydrate moieties per protein molecule. Previous

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LACCASE 2999

investigations 8 showed that laccase possesses considerable pH and

thermostability. The values of Km determined for laccase and peroxidase

for a common series of chromogenic substrates are close to each other

and equal to OJ8-OJ9 mM and 0.2 mM respectively 4~7,899. The value of

Km for oxygen determined for laccase from different fungi is about 10-5

M, so that the enzyme is saturated with oxygen in the air atmosphere.

Laccase has following advantages as a marker enzyme compared

to peroxidase:

a) it accepts oxygen as the second substrate and hence is not inactivated

during the reaction;

b) it is less sensitive to the presence of metal ions varying oxidation

state in the reaction medium;

c) laccase based immunoassay can be carried out with standard reagent

and equipment used in peroxidase based analysis.

The aim of the present work was to develop laccase conjugates

suitable for immunoanalysis and to demonstrate their feasibility in

different versions of enzyme immunoassay in comparison with

immunoperoxidase conjugates of similar composition.

MATERIALS AND METHODS

Buffers

The most frequently used ,uffers were: 0.0 M potassium

phosphate buffer (PBS) pH 7,4, (buffer "A'); 0.01 M PBS, pH 7,4,

containing 415 M sodium chloride (buffer "B); 0,Ol M PBS, pH 7,4,

containing 0,15 M sodium chloride and 0,05% Tween-20 (buffer "C");

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3000 SKOROBOGAT'KO ET AL.

0.01 M PBS, pH 7.4, containing 0,15 M sodium chloride, 0,05% Tween-

20, 05% ovalbumin and OJ% cow fetal serum (buffer "D).

Enzvmes

Laccase was separated from the cultural liquid of basidial fungi

Coriolus hirsutus by precipitation with ammonium sulfate. Homogeneous

enzyme preparation was obtained by using a two-step purification

procedure 8 involving ion exchange chromatography on

Di+ethylaminoethylcellulose (Whatman Chemical Separation, Ltd, Kent,

UK) and gel-filtration on Toyopearl HW-55 (Toyo Soda MFG, Co., Ltd,

Japan).

AntiFens and Antibody

Homogeneous pig insulin was a generous gift from

Dr.IA.Donetsky.

Goat antimouse immunoglobulins and their conjugates with

peroxidase were obtained from Sigma Chemical Company, USA.

Mouse and rabbit class immunoglobulins, rabbit antimouse and

rabbit antihuman immunoglobulins were obtained as described 10.

Monoclonal antibodies E10A2 against insulin were kindly supplied

by Dr.T.V.Cherednikova and purified by affinity chromatography on

insulin-Sepharose 4B column.

Calculation of the Amount of IgG

Immunoglobulin concentrations were determined from adsorption

at 280 nm assuming extinction coefficient ts cm2 fmg and molecular

mass 150,000 11.

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L ACC AS E 3001

Preparation of insulin-SeDharose 4B

The affine sorbent insulin-Sepharose 4B was prepared according

to a standard procedure recommended by manufacturer (Pharmacia Fine

Chemicals AB, Uppsala, Sweden), which includes covalent attachment of

insulin (10 mg) to CN-Br-activated Sepharose.

Affinity-Purification of Monoclonal Antibodies against Insulin

Ascitic fluid (02-LO ml) containing 5-10 mg of monoclonal

antibodies was applied to a chromatography column (5 x 10 mm) packed

with insulin-Sepharose. Unadsorbed proteins were eluted with buffer "B.

Monoclonal antibodies were then eluted with 0.l M glycine-HC1 buffer,

pH 3,O. The eluate was immediately neutralized by addition of 02 M

TRIS-HCL buffer, pH 8.0. The fractions, containing monoclonal

antibodies were dialyzed against 0,02 M TRIS-HCL buffer, pH 7.4 and

stored at 40C in the presence of 0,l M NaN3.

Assav of Laccase

Catalytic activity of laccase was measured spectrophotometrically

using pyrocatechol 8 (Reachim, Russia) at 410 nm or 2,2'-azino-di(3-

eth~~benzthiazoline-6-sulphonic acid 374 (ABTS) (Sigma Chemical

Company, USA) at 405 nm as substrates. Before use pyrocatechol was

purified by sublimation in vacuum.

Svnthesis of conjugates bv periodate method

To a solution of 6 mg laccase in 1 ml of distilled water solution

of sodium periodate was added to a final concentration 0,12 M. The

mixture was incubated for 20 min in dark at room temperature and

diaked against 0.01 M potassium acetate buffer, pH 45, at

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3002 SKOROBOGAT'KO ET AL.

4OC. The enzyme solution obtained in this way was gradually

supplemented with antibodies until enzyme/antibody molar ratio reached

the value of 2.5 and incubated at room temperature for 2 h, the pH

value of the mixture being kept at 68-9.0. After that sodium borohydride

was added to the solution in the amount of 1 mg per mg of laccase and

the mixture was incubated for 2 h at 4OC. The obtained conjugate was

dialyzed against 5 mM potassium phosphate buffer pH 65 at 4 C for 16-

18 h. The conjugate was stored in 50% aqueous glycerol at -18OC 12.

Preparation of solid-phase sorbent with antigens

Polystyrene plates (Dynatech) were incubated with various

antigens such as pig insulin, rabbit antimouse immunoglobulins or rabbit

antihuman immunoglobulins. Antigen concentration was 10 mg/l in 0,05

M carbonate buffer, pH 9.0.

Sandwich Enzyme Immunoassay Procedure for Determination of Mouse

Polystyrene plates with adsorbed rabbit antimouse

immunoglobulins were incubated with known concentration of mouse

immunoglobulins at 37OC in buffer "D. After 2 h the mixture was

removed and plates were rinsed with buffer "C". Solution of laccase

conjugate with goat antibodies against mouse immunoglobulins (2

pg/ml) was placed into pits and after 1 h incubation at 37% plates

were rinsed with buffer "C" as described above. Catalytic activity of

immunosorbed laccase was measured spectrophotometrically by means of

Dinatech MP 600 immunoassay reader (USA) using ABTS as a

substrate.

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LACCASE 3003

Enzyme Immunoassav Procedure for Determination of Insulin by

Competitive Method

Plates with adsorbed insulin were incubated with solution

containing known concentrations of freely dissolved insulin and laccase

conjugate with monoclonal antibodies against insulin (2 pglml) for 1 h

at 37OC. After that the solution was removed and plates were rinsed

with buffer "C" as described above. Catalytic activity of bound enzyme

was measured using ABTS as a substrate.

Indirect Enzyme Immunoassav for Determination of Monoclonal

Antibodies against Insulin

Plates with adsorbed insulin were treated with monoclonal antibodies

against insulin followed by the twofold titration of the latter in buffer

'ID. After 2 h incubation at 37OC the plates were rinsed with buffer "C"

and incubated with laccase conjugate with goat antimouse

immunoglobulins in buffer " A for 1 h at 37OC. The catalytic activity of

immunosorbed enzyme was measured as described above.

Comparison of Absolute Sensitivities of Immunoperoxidase and

Immunolaccase Coniueates

Plates with adsorbed human immunoglobulins were treated with

different concentrations of immunoperoxidase and immunolaccase

conjugates (goat antihuman antibodies conjugated with marker enzyme)

in buffer " A and incubated for 1 h at 37OC following by rinsing of the

plates with buffer "C" and determination of enzymatic activity as

described above.

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3004 SKOROBOGAT'KO ET AL.

The plates with adsorbed rabbit antibodies against human

immunoglobulins were treated with various known concentrations of

human immunoglobulins in buffer 'ID, incubated for 1 h at 37OC rinsed

with buffer "C" and incubated with conjugates of laccase and peroxidase

with antihuman antibodies for 1 h at 37% Catalytic activity of

immunosorbed enzyme was measured as described above.

Definition of the Sensitivity of Dose Response Curves

The sensitivity of dose response curves for enzyme immunoassay

technique was taken as the minimal amount of investigated substances

which gave peroxidase or laccase activities bound significantly above that

non-specifically bound in the absence of investigated substances

(background).

RESULTS AND DISCUSSION

Homogeneous laccase preparation obtained from cultural liquid of

basidial fungi Coriolus hirsutus contained 17% of carbohydrate moieties

per protein molecule as determined by phenol-sulfur method. Thus the

polypeptide/carbohydrate ratic. in laccase (17% for 55,000 total

molecular mass) and peroxidase (11% for 40,000) are practically the

same. For this reason the synthesis of laccase-antibody conjugates was

performed using the non-modified periodate method.

Synthesized conjugates of laccase with different kinds of

antibodies exhibited high enzymatic activity, viz. 75-80% of initial activity

of laccase taken for synthesis. Clearly the oxidation of carbohydrate

moieties of laccase does not lead to significant changes in active site of

the enzyme which could cause a decrease in catalytic activity. Hence the

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LACCASE 3005

periodate method can be used to synthesize immunolaccase conjugates

with high retention of catalytic activity. Laccase conjugates prepared in

this way retain their immunological and enzymatic activity for at least 1

year when stored in 50% aqueous glycerol at -18OC.

Several authors 13914 showed that immunoperoxidase conjugates

synthesized using periodate method are quite heterogeneous in terms of

molecular mass which is explained probably by the high reactivity of

aldehyde groups formed during the reaction of the enzyme with m-

periodate. However for practical purposes fractionation of heterogeneous

conjugate preparations is not necessary 1 so immunolaccase conjugates

used in our experiments were not subjected to any separation procedures.

As a rule the choice of the type of enzyme immunoassay to be

used is dictated by aims of the investigation. For example the indirect

enzyme immunoassay is usually employed for quantitative determination

of specific antibodies against pathogens in serum l2. Sanctsich enzyme

immunoassay is mostly often used for antigens with several epitops 12 and so on. In this connection we investigated the feasibility of

immunolaccase conjugates in different types of enzyme immunoassay. In

the case of the sandwich enzyme immunoassay the mouse

immunoglobulins were used as antigens whereas rabbit antimouse

antibodies and laccase-bound goat antimouse antibodies were employed

as first and second antibodies respectively. Experimental protocol is

described in details in Materials and Methods. Results are presented in

Fig.1. The data show that this type of immunoassay provides reliable

determination of antigen concentrations as low as 2,5*10-= M which

demonstrates high sensitivity of the immunolaccase conjugate taken at

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3006

2.0 D

405

I .o

0.0

SKOROBOGAT’KO ET AL.

-12.0 -1 1 -10 -9 -8

Ig DgGl

FIG. 1. Scheme of the sandwich enzyme immunoassay employed for determination of mouse immunoglobulms concentration using laccase conjugates with goat antimouse antibodies. Conjugate concentration - 2 pg/ml, substrate - ABTS, incubation time - 30 min at room temperature.

concentration of 2 yg protein/ml. According to previous reports l5,

similar concentrations of conjugates of alkaline phosphatase and

peroxidase are used in immunoassays based on these marker enzymes.

Owing to recent progress in hybridome technology the least of

widely used techniques has being supplemented with the indirect enzyme

immunoassay which is successfully used for the detection and

quantitative determination of antibodies against bacterial and viral

antigens 12.

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LACCASE 3007

0.75

405

0.60

0.45

0.30

0.15

0.00 I I I I I I

-9.5 -9.0 -8.5 -8.0 -7.5 -7.0

lg [mon ABI

FIG. 2. Calibration curve for determination of monoclonal antibodies against insulin concentration by mean of the indirect enzyme immunoassay. Conjugate concentration - 2 pg/ml, substrate - ABTS, incubation time - 30 min at room temperature.

In the present work this method was used for quantitative

determination of monoclonal antibodies against insulin (see Materials

and Methods for experimental details). The sensitivity of the analysis

was 1 ng of antibodies per sample at the concentration of the detecting

conjugate 2 p g protein/ml (Fig.2). Thus, the suggested reagent can be

successfully used for the hybridome screening as well as for

determination of different antigens such as, Plasmodia, Trypanosoma,

Vibrio cholerae, ect., whose sensitivity of the assay was expressed in

nanomoles of the substance 16.

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3008

1.5

D 405

1

0.5

0.0

SKOROBOGAT’KO ET AL.

-9.5 -8.5 -7.5 -8.5

FIG. 3. Calibration curve for determination of insulin concentration by the competitive enzyme immunoassay. Conjugate concentration - 2 pg/ml, substrate - ABTS, incubation time - 30 min at room temperature.

One of the most sensitive versions of enzyme immunoassay is the

competitive method allowing determination of picogramm amounts of

analytes. However, the specificity of this method is markedly dependent

on the specificity of antibodies used 15. We showed that the competitive

method is applicable for quantitative determination of insulin using the

laccase conjugate with monoclonal antibodies E10A2 (Fig.3). The data

shows that the method provides reliable determination of insulin

concentration as low as 1 ng/ml. Evidently, this is due to the high

specificity of monoclonal antibodies E10A2 which are characterized by

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LACCASE 3009

405 1'5 1 D

1.0 -

0.5 -

0.0 / I I I I 1

0 50 100 150 200 250

t, min

FIG.4. Time dependence of the accumulation of the chromogenic product during incubation with immunolaccase (1) and immunoperoxidase (2) conjugates. Conjugate concentration - 2 pg/ml, substrate - ABTS, investigations provided at room temperature.

the value of Kd=5*10-10 M 17, It should be mentioned that the

competitive enzyme immunoassay of insulin using monoclonal antibodies

E10A2 and peroxidase as a marker enzyme allows one to determine 3

ng/ml of insulin at immunoperoxidase conjugate concentration 5 pg

protein/ml 17.

A comparative investigation of immunoperoxidase and

immunolaccase conjugates showed (Fig.4) that in the case of laccase the

formation of chromogen during ABTS oxidation is much higher than in

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3010 SKOROBOGAT'KO ET AL.

D405

0.5

0.0 I -10.5 -9.0 -7.5

lg [IgGl

FIGS. Determination of the human immunoglobulins concentration by sandwich enzyme using conjugates of antihuman IgG withimg::rJ) and antihuman IgG- peroxidase (2). Substrate - ABTS, incubation time - 30 min at room temperature.

the case of peroxidase (i.e., after 120 min incubation the absorbance of

the reaction mixture with laccase was three times higher than that with

peroxidase).

In contrast to peroxidase which forms inactive intermediate

compound in the course of the reaction, laccase is not inhibited by

reaction products 478. Comparison of absolute sensitivities of conjugates

also confirms this conclusion (Fig.5). The data shows that sensitivity

limits for immunolaccase and immunoperoxidase conjugates differ by the

factor of three and are equal to 7.7*10-11 M and 23*10-10 M

respectively.

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LACCASE 301 1

It should be noted that the background oxidation of the

chromogenic substrate is much less pronounced in the presence of

oxygen as compared to hydrogen peroxide. In blank experiments

preincubation of the solid phase with buffer followed by the treatment

with irnmunolaccase reagent lead to an average change in absorbance at

405 nm of 0,l units/h. The data obtained can be explained by the

presence of hydrogen peroxide in the chromogenic mixture used in the

case of peroxidase-catalyzed reaction.

In conclusion laccase from Coriolus hirsutus can be successfully

used as a marker enzyme for enzyme immunoassay. We have confirmed

experimentally advantages of laccase as compared to peroxidase both in

absolute sensitivity of the immunoassay and catalytic properties

demanded from the marker enzyme.

1.

2.

3.

4.

5. 6. 7. 8.

9. 10.

11. 12.

13.

REFERENCES T.T. Ngo and H.M. Lenhoff, Eds., in "Enzyme-mediated immunoassay", Mir, Moscow, 1988, 444. BJ. Gould and V. Marks in Monosotopic immunoassay, Acad.Press, N-Y, 1988, 3. E. Ishikava, S. Hashida, T. Kohno, K. Tanaka in Monosotopic immunoassay, Acad.Press, N-Y, 1988, 27. B. Porstman, T. Porstman in Monosotopic immunoassay, Acad.Press, N-Y, 1988, 57. J.H. Bovaird, T.T. Ngo, Clinchem., 2, 2423, (1982). A.M. Mauer, E. Harel, Phytochem., a, 193, (1979). A.M.Mauer, Phytochem., 26, 11, (1978). A.L. Ghindilis, E.O. Zhazhina, YuA. Baranov, A.I. Yaropolov, VS. Gavrilova, Biokhimiya, 53, 735, (1988). C. Blake and B.Y. Jould, Bich.Bioph.Res.Commun, m, 533, (1984). BAL. Hurn and S.M. Chantler, Methods in Enzymology, 70, 104, (1980). Y.L. Palmer and A. Nisonoff, Biochem.2, 863, (1964). PX. Nakane and A. Kawaio, J.Histochem.Cytochern., 22, 1084, (1974). S. Avrames, T. Ternynck and J.L. Guesdon, ScandJ.Immunol., 8, 7, (1978).

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14. A. Yu. Karulin, B.B. Dzantiev, A.V. Zherdev, A.M. Egorov,

15. A. Jeanson, J.-M. Cloes, Bouchet M. and T. Reirtier,

16. 17. T.V. Cherednikova, M.Yu. Rubtsova, M.V. Demcheva, E.M.

Biotekhnologiya, I, 324, (1989).

J.Immunol.Methods, 111, 261, (1988). A. Voller and D. de Savigny, J.Immunol.Methods, &, 1, (1981).

Gavrilova, A.M. Egorov, Biotekhnologiya, 5, 612, (1989).

Received June 15, 1994 Accepted July 31 , 1994

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