Journal of Histochemistry & Cytochemistry

Journal of Histochemistry & Cytochemistry http://jhc.sagepub.com/

The use of avidin-biotin interaction in immunoenzymatic techniques. J L Guesdon, T Ternynck and S Avrameas J Histochem Cytochem 1979 27: 1131 DOI: 10.1177/27.8.90074 The online version of this article can be found at: http://jhc.sagepub.com/content/27/8/1131

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131$02.0O/O

0022-1554/79/2708-1 JOURNAL

THE

Copyright

OF HISTOCHEMISTRY

© 1979 by The

The

AND

Histochemical

Use

Vol. 27, No. 8, pp. 1131-1139,

CYTOCHEMISTRY

Society,

Inc.

of Avidin-Biotin JEAN-LUC Unite

1979 in U.S.A.

Printed

Interaction

GUESDON,

THERESE

d’Immu,wcytochimie,

in Immunoenzymatic

TERNYNCK

STRATIS

AND

D#{233}partement de BiOIOgie

Techniques

AVRAMEAS

Mol#{233}culaire,

Institut

(MS

Pasteur,

79-164)

Paris,

France

was covalently attached to antibodies, antigens and enzymes, and the effects of this labeling antigen and antibody binding capacity and on enzymatic activity were tested. Based on avidinbiotin interaction, the labeled proteins were used in quantitative enzyme-immunoaseay and enzymeimmunohistochemical staining procedures. Two procedures were developed. In the first procedure, named the Bridged Avidin-Biotin (BRAB) technique four steps were used sequentially in order to quantify or detect an immobilized antigen: 1) incubation with biotin-labeled antibody; 2) incubation with avidin; 3) incubation with biotin-labeled enzyme; 4) measurement or histochemical staining of the enzyme. The technique is based on the observation that avidin possesses four active sites. In the second procedure, named the Labeled Avidin-Biotin (LAB) technique, biotin-labeled antibody and enzyme-labeled avidin are used sequentially. Enzyme-associated antigen is then quantified or revealed immunohistochemically. The optimal conditions for enzyme-immunoassay and enzyme-immunohistochemical staining using BRAB and LAB procedures were established. Biotin

on the

Detection, antibodies

localization by

and

quantitation

immunoenzymatic

of

techniques

antigens

and

the

necessitate

the

the

action been

of used

tion

(7),

avidin-biotin in various gene

but

cellular

constant:1015 as bacteriophage

mapping

(13-15,

(16) and immobilization work, based on the

tion, two procedures for scribed. These procedures

linking were

procedure, enzyme,

enzymes applied

inter-

antibodies

an antigen, with the

eliminate incubation

antibody, avidin the biotin-labeled

excess labeled and washing,

substitution

antigen-binding

ing de-

activity

the biotin-labeled antigen. After washing

tein, of

the

secondarily procedure

antibody

(2,

19,

with

biotin

The remaining for another

residues

added to the system is similar to that

(Fig. 1A). described

21)

Concanavalin

and

tetravalent

profoundly

on

with

the

affected

antigen

that

was

antigen, even

in antibody

was

not with

capacity was enzymes

peroxidase, decreased

antianti-

and, after washing, further incubation

biotin, noted. remained

Aspergillus (Escherichia (E.

coli

and with

molecules

modified.

such

Dependloss

The

of up to

catalytic either un-

niger glucose coli $-galactoalkaline

phospha-

observations, the optimal conditions and in enzyme-immunohistochemistry LAB procedures were established.

MATERIALS

for

was of

sites pro-

The principle for bivalent A (3).

In

AND

METhODS

grade I (RZ = 3) and grade II (RZ = 0.6) and A. niger glucose oxidase grade I were obtained from Boehringer (France). E. coli alkaline phosphatase (BAPSF) was purchased from Worthington Biochemical Corp. (Freehold, N.J.). Purified E. coli f-D-galactosidase was obtained by courtesy of Dr. Agnes (Institut Pasteur, Paris). Rabbit y-globulin fraction II (Ig) was purchased from Miles Laboratories (Kankakee, Ill.) and bovine serum albumin (BSA) from Provite (Amsterdam, The Netherlands). AVidin, chromatographically prepared from egg white, was a product of Sigma Chemical Co. (St. Louis, Mo.). Antisera, antibodies and their Fab fragments: Rabbit and sheep antisera against the various proteins used in the present work Proteins:

associated

free active biotin-labeled

or was Based

to as

to

was added. After enzyme was then

will

react

be referred

(Fig. 1B). In the followto as the Labeled Avidin-

groups

capacity

of antigen-labeling

enzyme-immunoassay using BRAB and

anti-

sites,

antibody. as acceptors

degree

(horseradish was slightly

sidase), tase).

antigen After

associated

of amino

of antibody binding of biotin-substituted

changed oxidase)

and for of intra-

will

(LAB) technique. effects of introducing biotin into antibody, molecules were studied. It was found

on the

40%

procedure

(BRAB) technique. biotin-labeled antibody (or avidin were used. The labeled

or measured be referred

will

by biotin,

with the antigen (Fig. 1A). The proceprinciple that avidin possesses 4 active

all of which

enzyme

extensive

the enzyme associated dure is based on the not

the

selective

This step, after a further incubation and washing, by the histochemical staining or the measurement

with immobilized can then operate

washing,

stained histochemically ing, this procedure

M) has inactiva-

to proteins are to the quantitation,

this

to react with the avidin was added.

Biotin The enzyme

biotin-labeled antibody (or antigen), and native unlabeled avidin were used.

To quantify or to detect body was allowed to react

added. followed

17),

(2,

of macromolecules biotin-avidin interac-

by enzyme-immunoassay, of antigens and the localization by enzyme-immunohistochemistry cellular immunoglobulins. In the first biotin-labeled

strong

body was allowed enzyme-labeled and

immunological binding

exceptionally

(dissociation systems such

and

absorption of cells (18). In the present

the noncovalent for antibody-enzyme

description,

Bridged Avidin-Biotin In the second procedure, gen) and enzyme-labeled

use of enzyme-protein conjugates. For the preparation of such conjugates, a number ofprocedures using various cross-linking agents have been described. In general, these conjugates were prepared by covalently coupling the enzyme marker to the antibody (1, 5, 20, 22), but reaction has also been used 19, 21). Recently, the noncovalent

following

Horseradish

1131

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peroxidase

GUESDON,

1132

TERNYNCK

AND

AVRAMEAS

Biotin-labeling of proteins: Biotinyl-N-hydroxysuccinimide (BNHS) was used for introducing biotin moieties into proteins. It was prepared according to the procedure described, by Jasiewicz et al. (16) by using N-N-carbonyl diimidazole (Aldrich Chemical Co., Milwaukee, Wisc.) to form an intermediate biotin derivative which was subsequently 2

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