Comparative characterization of monoclonal antibodies to carbonic anhydrase

Vol. 125, No. 2, 1984

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

December 14, 1984

Pages

690-697

COMPARATIVE CHARACTERIZATION OF MONOCLONAL ANTIBODIESTO CARBONICANRYDRASE P. J. Linser a,b , M. S. Perkinsa, F. W. Fitch'

and A. A. Mosconab

aC. V. Whitney Laboratory of the University of Florida, St. Augustine, FL 32086

Route 1, Box 121,

bLaboratory for Developmental Biology, University of Chicago, Chicago, IL 60637 University

'Department of Pathology, of Chicago, Chicago, IL

60637

Received October 12, 1984 Monoclonal antibodies (Mabs) were generated to avian carbonic anhydrase-C and characterized; their reactivity with human, murine, bovine, chicken and fish erythrocyte carbonic anhydrase-C, and with humancarbonic anhydrase-B was investigated by ELISA and electroblot techniques. Reactivity of the Mabs with native and SDS-denatured carbonic anhydrase was compared. Mabs that recognize antigenic determinants shared by all the carbonic anhydrases examined were identified. The results demonstrate the potential usefulness of these particular probes for investigating various aspects of function, evolution, development and regulation of this important, but not well understood group of enzymes. 01984 Academic

Press,

Inc.

Carbonic anhydrase (CA) is an enzyme with a wide phyletie (7, 8, 13,14).

Recently,

there has been increasing

developmental biology and functions 10).

In higher vertebrates

of their

there exist

expression (13,

several genetically

14).

detailed

Investigation

distinct

physiological

had lead to CA diversity

(14).

might also provide clues concerning regulation different

cells,

Abbreviations:

and identify

of the various

forms of CA in

for molecular-genetic

SDS-PAGE,sodium dodecyl sulfate

0006-291X/84 $1.50 1984

stage at which

Such comparative studies

model systems suitable

gel electrophoresis.

0

roles

CA, Carbonic anhydrase; CA-C, CA-B, Carbonic anhydrase-C, B;

Mabs, monoclonal antibodies;

Copyright All rights

isozymic

of this problem could

be advanced by comparative studies to define the evolutionary gene duplications

in the

of this enzyme in neural tissues (6,

forms of CA, but not much is known about their and control

interest

distribution

by Academic Press, in any form

of reproduction

Inc. reserved.

690

polyacrylmide

Vol. 125, No. 2, 1984 analysis.

BIOCHEMICAL

However,

immunological

such

probes

capable

We have generated their

ability

man.

Our results

retained

across

this

relationships

be useful

MATERIALS

AND METHODS

and for

to CA and describe

vertebrates

of CA within for

from

here fish

to

of CA are

some of the Mabs can a single

investigating

future

ranging

characteristics

Furthermore,

forms

of specific CA forms.

(Mabs)

immunological

spectrum.

of CA enzymes mechanisms.

various

certain

isozymic

regulatory

different

antibodies

CA from

species

Mabs should

on the availability

of identifying

show that

different these

depend

monoclonal

to recognize

distinguish Hence,

studies

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

species

(man).

structure/function

exploration

of developmental-

Carbonic Anhydrases: Carbonic anhydrase-C (CA-C) was purified from chicken, mouse, and flounder erythrocytes by affinitychromatography (12). Purified human CA-C and CA-B were generously provided by Dr. Thomas Maren, University of Florida. Monoclonal Antibodies (Mabs) to CA-C were produced against chicken CA-C by the method of Kohler and Millstein (4) with some modifications (9). C57B1/6J mice were injected i.p. with 50 ug of CA-C emulsified in Freund's complete adjuvant and boosted after two weeks. Six weeks later they received 25 ug CA-C i.v. After 3.5 days splenocytes were fused with hybridoma SP2/0 (9). Fusion, selection in HAT medium, and cloning procedures were as Positive hybridoma cultures were detected by ELISA assay described (9,ll). (1) and cloned by limiting dilution (11). From these initial clonings, the hybridoma cell lines described here were selected and recloned. Mabs were produced in quantity by the ascites fluid method. Mab isotypes were determined using a screening kit from Zymed Laboratories Inc. Quantitative ELISA assays were performed using horseradish peroxidase-conjugated goatantimouse Ig (Boehringer Mannheim Inc. (I)]. Electrophoresis and Electroblotting: SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed in 10% gels (5). Purified CAs or soluble proteins from a 100 kxG supernatant of chicken retina lysate were run in duplicate gels: one gel was fixed and stained with Coomassie blue following electrophoresis; the other was electroblotted onto nitrocellulose (2). The blot was immunostained and CA was located by the indirect immunoperoxidase method (15).

RESULTS From 97 hybridoma antibodies From these,

to CA-C,

cultures 10 were

the 6 Mab-secreting

that

subjected

were

found

to at least

hybridoma 691

lines

by ELISA

assay

two rounds described

here

to produce

of recloning. were

selected

Vol. 125, No. 2, 1984

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1

Monoclonal

antibodies

1sotype

to CA-C

2H10-6 2HlO-9 2A2-1

IgG2b IgG2b IgGl IgGl I&l I&l

7C6-1

7F4-1 7F4-2

and expanded. I.

These

are

IgGl.

The designations

6 hybridomas

Specificity with

of the Mabs was examined

retina

(2,15). rather

subjected

The retina

contains

erythrocytes

associated

with

the

Electroblots

were

conjugated

second

product

mixture

were

than

comparison

with

Furthermore, chicken

antibody with

CA-C showed

reaction

was different

reaction

with

Species

for

roughly

equalize

from the

titers,

mouse,

evaluated

by ELISA

assay.

the CAs.

fluid

chicken

chicken,

all

this

for

as CA-C by

Mabs;

(Fig. only

Mabs.

However,

CA-C.

the

Mab 2HlO-6

(Fig.

At higher

erythrocytes.

Then,

dilutions,

distinct 692

purified Thus,

these

intensity

Mabs

of the

gave the weakest

The relative

by ELISA after

all

assay

initial

against

dilution

erythrocyte

as human erythrocyte

low dilutions,

1).

1).

of the Mabs with

and man as well

peroxidasereaction

identified

all

was used

the problems

with

contained

determined

At very

only

(hemoglobin).

run in parallel

pattern

analysis

1)

that

tissue

the peroxidase

were

reaction

reacted

to eliminate

of each of the Mabs was examined.

flounder,

with

(Fig.

gels

the different

CA-C by this

and four

from chicken

protein

6 cases

band

SDS-denatured

Specificity

antigen

In all

a single

of each Mab in ascites

original

of a single

the same staining

chicken

of CA-C (6,lO);

of SDS-PAGE gels

detect

they

proteins

of the enzyme)

blue-stained

electroblots

if

each of the 6 Mabs and then

(15).

Coomassie

can specifically

titers

with

in Table

by SDS-PAGE and electroblotting

concentration

reacted

was associated

Soluble

level

source

listed

two are IgG2b

by determining

to analysis a high

Mabs are

type;

of proteins.

(the

high

of these

of the IgG secreting

are

CA-C in a complex

neural

and isotypes

to

CA-C from CA-B was

6 Nabs showed

patterns

the

of specificity

reactivity

Vol. 125, No. 2, 1984

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A

67 k

w

30k

--0

-

20k

Extract of chicken Fig. 1. Binding of anti-CA-C Mabs to electro-blots. retina was electrophoresed concurrently on two SDS-polyacrylamide gels. Gel A received 100 ug of retina extract in lane 2 and molecular weight markers in lane 1, and was stained with Coomassie blue. Gel B received 100 ug/lane retina extract. Gel B was electroblotted, the nitro-cellulose paper was cut into strips corresponding to the lanes, and these were immunostained with labs, followed by reaction with HP-GAM IgG. Mabs were derived from ascites fluids and diluted 1:500. The control strip (lane 7) was not reacted with Mab, but was otherwise treated identically to the rest. Lanes in Gel B: l6- Mab lab 7C6-1; 2- Mab 2a2-1; 3- Mab 7F4-2; 4- Mab 7F4-1; 5- Mab 2HlO-9; 2HlO-6; 7- control. All the Mabs bind selectively to CA-C (30K molecular marker, bovine CA-C).

emerged w

reactivity

in Fig. low

and each antibody

2.

with Chicken

reactivity

CA-C.

is

Titration

shown).

with

High

and electroblotting.

for

flounder

reactivity

for

as having

II). reactivity

with

was seen with with

all

and 7C6-1

CA-C were

was evident

low,

intermediate

Representative

by Mabs 7F4-2

reactivity

all

results the Mabs.

or

are

shown

Typical

assayed

against

mouse

of the low

variety

(not

Mabs 2A2-1 Mab 2A2-1

and 7C6-1 for

against

mouse CA-C,

and

human CA-C.

we tested

CA-C (Pharmacia),

CA (Table

exemplified curves

Mab 7F4-2 Next,

a given

be described

CA-C showed high

Intermediate

human CA-B.

could

the Mabs against

following Intensity

enzyme

the CAs listed denaturation

of immunoblot 693

with staining

above, SDS, gel

as well

as bovine

electrophoresis

was compared

to the

Vol. 125, No. 2, 1984

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE II Carbonic

Mab

F Low Low Low Low Low Low

2HlO-6

2HlO-9 2A2-1 7C6-1

7F4-1

714-2

anhydrase M

C High High High High High High

Low Low High Low Low Low

HC

Hb

Low Low High Low Low High

Low Low Low Inter. Inter. Inter.

Comparison by ELISA assay of lab reaction with non-denatured CA-C from flounder (f), chicken (c), mouse (M), human (Hc), and human CA-B (Hb). Designation of w, intermediate or J~J reactivity was based on titration curves (see Results and Fig. 2).

reactivities

of the Mabs with

immunoblot

analysis

and showed

lowest

reactivities

the native

using

Mab 7C6-1.

reaction

intensity

in immunoblot

tests

enzyme in ELISA.

This with

of the

with

the flounder denatured

I

2

3 4

5

6

7 6

9

IO

I

2

3 4

5

6

7

9

IO

6

Mab reacted

Fig.

I

-

I

Fig. all

enzyme.

CAs with

3 4

5 6

7

6

9

IO

2

3

5

7

6

9

IO

6

the CAs tested The relative

different

2

4

3 shows an

Nabs

2. Titration by ELISA assay of Mabs 2A2-1 (m), 7F4-2 (O---O); and (a---d) against CA-C of chicken (C), mouse (M), and human (Hc), and human CA-B (Hb). The initial dilution of each Mab was 2A2-1, 1:300; 7F4-2, 1:900; 7C6-1, 1:6OO. The scale on the abscissa represents serial 1:3 dilutions following the initial dilution. 7C6-1

694

Vol. 125, No. 2, 1984

C

F

a

BIOCHEMICAL

M

Hc

AND BIOPHYSKAL

Hb

RESEARCH COMMUNICATIONS

b

94k 67 k

43 k 30k

20 k

Fig. 3. Electroblotting analysis of reaction of different CAs with Mab 7C61. Two ug per lane of CA-C from flounder (F), chicken (C), mouse (M) and human (Hc) and human CA-B (Hb) was electrophoresed in parallel in 2 SDS-gels; molecular weight markers (A, left lane) included bovine CA-C (30K). Following electrophoresis, gel A was stained with Coomassie blue, Gel B was electroblotted (6 v/cm overnight). The electroblot was immunostained with Mab 7C6-1 (diluted 1:500). Note that the CA from different sources, including bovine CA-C, reacted with this antibody, though with different intensities.

could

not

always

enzymes.

be predicted

For example,

Mab 7C6-1

human CA-C and CA-B,

while

than

2).

with

CA-C (Fig.

reactivity highest

with titer

From these the antigenic

results

reacted

in ELISA

of ELISA assays strongly

assay

On immunoblots

chicken of all

from

CA-C (Fig.

it

reacted

while

non-denatured

on immunoblots

with

more strongly

Mab 2HlO-6

l),

with

showed

in ELISA

both

with

a relatively

assay

this

CA-B low

Mab had the

the Mabs tested.

results

it

is

determinants

clear

that

recognized

the CAs tested

by the panel

differ

with

respect

of Mabs described

to

here.

DISCUSSION We describe anhydrase-C mammalian

(CA-C). CA (3),

immunologic

This but

relatedness

man, and for

comparing

and SDS-denatured isozymes

6 Mabs generated

CA.

of human origin,

characteristics.

it

work

against extends

represents between

the first

indicate

to possess that 695

study

such diverse

CAs examined

found

erythrocyte

carbonic

on Mabs against

use of such Mabs for

of immunologic

The various

Our results

a previous

CAs from

retention

were

chicken

the

vertebrates

reactivities here, shared

including

probing as fish

between

native

two different

immunological

CA antigenic

determinants

and

Vol. 125, No. 2, 1984 recognized enzyme, other

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

by several since

hand,

tertiary

they

are

recognized

the reactivity

structure

of other

of the antigen.

involving

study. with

chicken

between

to the

of fish

mammalian

and saurian

library

could

not well

help

understood

apparently

influenced

increased

or decreased

of those as probes

the evolution that

On the by the following

Mabs to react

in studies

translation,

of CAs were

several

CA-B and avian

divergence

CA.

of the

with

on CA gene and in

studies.

finding

CA-B isozyme

sequence

or denatured

and --in vitro

CA-C and human CA-B than

relatedness led

the

it

useful

mRNA isolation

concerning

However,

Mabs is

The ability

immunocytochemical-developmental Conclusions

by the primary

native

in that

CA makes them especially

expression

defined

in both

of the enzyme

SDS denaturation denatured

of our Mabs are

with

in mammals (13,

classes.

family

fish

CA-C suggests

Thus, 14)

the gene

apparently

vertebrates

but

The Mabs described

in clarifying

the aim of this

of our Mabs reacted

CA-C.

and terrestrial

not

the evolution

before

of this

an evolutionary

duplication

occurred

here

more strongly

which

after

the

the separation and others

important

work

and as yet

of isozymes.

Foundation Science

was supported

No-l-733 Foundation

Development

Award

by Grants

(A. A. Moscona), grant RDA-1-15

from March No.5-425

No. PCM8209360 from

of Dimes-Birth

(P. J.

Linser);

(A. A. Moscona);

the University

of Florida

Defects National

and by Research to P. J. Linser.

REFERENCES 1.

2. 3. 4.

5. 6. 7.

Engvall, E. and Ruoslahti, E. (1979) In R. M. Nakamura, W. R. Dito, and E. S. Tucker III (eds.). Immunoassays in the Clinical Laboratory, Alan R. Liss, Inc., New York, pp. 89-97. Erickson, P. F., Minier, R. S. and Lasher, R. S. (1980) J. Immunol. Methods 51: 241-49. EricksonFR. P., Kay, G., Hewett-Emmett, D., Tashian, R. E. and Claflin, J. L. (1982) Biochemical Genetics 20: 809-19. Kohler, G. and Milstein, C. (1975) Nature 256: 495. Laemmli, U. K. (1970) Nature 227: 680-685.Linser, P. and Moscona, A. A. (1981) P.N.A.S. 78: (11) 7190-94. Maren, T. (1967) Physiol. Rev. 47: 595-781. 696

of

in our

ACKNOWLEDGMENTS This

had

Vol. 125, No. 2, 1984

8.

9. 10. 11.

12.

13. 14.

15.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Maren, T. H. and Sanyal, G. (1983) Toxicol. 3, in Ann. Rev. Pharmacol. press. McKearn, T. J. (1980) In R. H. Kennett, T. J. McKearn and K. B. Mechtol (eds.). Monoclonal Antibodies, Plenum Press, New York, pp. 36869. Moscona, A. A. and Linser, P. (1983) Curr. Top. Dev. Biol. 18: 155-188. Oi, V. T. and Herzenberg, A. (1980) In B. B. Mishell and S.M. Shiigi (eds.). Selected Methods in Cellular Immunology, Freeman and Co., pp. 351-372. Osborne, W. R. A. and Tashian, R. E. (1975) Analytical Biochem. 64: 297-30. Tashian, R. E. (1977) In Rattazzi, M. C., Scandalios, J. and Whitt, G. S. (eds.). Isozymes: Current Topics in Biological and Medical Research, Vol. 2. Alan R. Liss, New York, pp. 21-62. Tashian, R. E., Hewett-Emmett, D., and Goodman, M. (1980) In Peeters, H. (ed), Protides of the Biological Fluids, Vol. 28, Pergamon Press, Oxford, pp. 153-156. Towbin, H., Staehelin, T. and Gordon, J. (1979) P.N.A.S. 76 (9): 435054.

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