0022-1554/79/2708-1216$02.00/O

Jouas*i

THE

Copyright

OF

HISTOCHEMISTRY

The The

AND

Histochenucal

© 1979 by The

of Antibody

Gel Electrophoresis-Derived Application

Department

ofSurgery,

J.H.,

Section

G.H.T.);

By combining the sites niques;

N. C. PEDERSEN School

Oncology,

ofMedicine,

virus

School

components

of FeLV

Key

J. HIGGINS,

are

components

words: Feline concanavalin

are

H.

assay

antigens;

leukemia

twice

induced

are

(GEDELISA)

are

into

nique

may

experimental

infection

or

an in

m.w. was

offeline

Purification FL74

cells

purified

(16).

leukemia

Tissue

on a Ficoll-gradient

1.096

to

1.105

transcriptase

high

particles. Polyacrylamide

gel

SDS

according

was

tions.

performed

Five

percent

protein

Electron

(FeLV): was

(15). A single

contained

activity.

fluid

NaHCO3, proteins the

stacking

to and

showed PAGE

L#{228}mmli (8) 13%

Ficoll

resolution

only in the

under

virus

gels

25 cm

virus of condi-

long

NaCl,

and

unbound

fractions Mo.)

specificity

ofa

were

added

and

stored

given

serum,

100 ,il of coating

tubes NaCl

for

bound and

2 hr

at

of 1:25

EDTA, 20).

detect

50

with

0.05%

proteins

Tween

37#{176}Cwith to

mM

1:100

antibody

specifically

100 l

bound

pH

of cat

or goat

dilution 7.4,

was IgG,

washed

Subsequently,

in serum

Tris-HC1,

Unbound

were

20.

buffer

0.1%

washed 100

BSA, out

gd of

as

1:500

Pa.)

Inc.,

A binding FeLV for IV)

per

Oberlin,

assay:

components, 1 hr

at 37#{176}C with

100 gil of serum

zur

Con

tubes

Ohio).

To detect were 5 g dilution

coated of Con buffer.

Con

A binding

as described A (Sigma, After

sites above

St. washing

Louis, out

A, 100 gd of serum dilution buffer containing 5 g of horseradish peroxidase (Sigma, Type VI) were added to each tube and incubated at 37#{176}C for 60 miii. If present, the multivalent Con A did bind horseradish peroxidase which itself is a glycoprotein. By subsequent incubation with substrate solution as described above,

immunosorbent

Nationalfonds

three

Louis,

for

of the times for wide well strip was

anti-goat

Laboratories,

Grade

the Schweizerischer Forschung.

or

Instrument

incubated

I Supported by a grant from F#{246}rderung der Wissenschafthchen

To

Concanavalin

(5).

enzyme-linked

St.

antibody

The M

Tween

Cochranville,

were

electrophoresis-derived

0.05%

anti-cat

Mo.

Gel

above

soaked

Gel

Lancer,

with

dilutions

oratories,

and

carbohydrates

at

above.

for

thick

tubes. of 0.15

2 mM

NaN3,

on separated

mm

was

fractions.

establish

incubated

sera

used in a Hoefer slab gel apparatus (Hoofer Scientific Instruments, San Francisco, Calif.). The gels were stained with Coomassie brilliant blue for proteins and by the PAS method 1.5

described

and for calculation

gel

were

electropho-

IgG peroxidase conjugate (Cappel Labwere added to the tubes for 30 rain at 37#{176}C.After a final washing, bound peroxidase was visualized by incubating the tubes with 150 il offreshly prepared 50 mM citric acid, pH 4, containing 2 mM H202, and 0.2 mM 2.2’-azino-di(3-ethylbenzthiazoline sulfonic acid), ABTS; (Boehringer Mannheim, Indianapolis, Ind.) as described by Saunders and Bartlett (14). After 8 to 10 mm the enzyme reaction was stopped by addition of 150 l of 0.1 M HF adjusted to pH 3 with NaOH. The reaction product was measured at 414 nra in a Gilford Stasar II spectrophotometer (Gilford

was

presence

reducing

were

described

density of and reverse

intact

5-mm

incubated

of the 2 ml

tubes

0.02%

entire

width

Proteins

buffer (2) (0.1 M pH 9.6, 0.02% NaN3) for 36 hr at 4#{176}C. The separated FeLV diffused from the gel slices during this period and bound to

anti-FeLV M

as

were loaded

in

out excess SDS. The 80-mm each of4 mm in width. Each

20 strips

To

stained

reaction

(12 x 55 mm,

use.

FeLV

of 8 mm

standards.

were

the

into

were

with

(0.15

m.w.

to wash

into tubes

surface

the

wells

immunologic

horizontally

fractions

and

tech-

ofpurifled

Additional wells

the

vertically

cut

gel

was grown

and

peak with concentration

electrophoresis:

with

A binding

hundredg

well.

in order

at -30#{176}Cuntil

artificially

FeLV

collected

microscopy

95616 (N.C.P.)

feline

immunoenzyme

electrophoresis,

in H2O

to polystyrene

METHODS virus

culture

Concanavalin

additional

with

cut

then

wide

the

After

10 mm

diluted

in

95616

gel electrophoresis for different

30 g of FeLV

comparison

by the gel electrophoresis derived ELISA Concanavalin A (Con A) binding components detected using free horseradish peroxidase. As this techbe of interest for application in immunocytochemistry (9), procedures are given in some detail. AND

California

California

Three

with

resed

how

MATERIALS

Davis,

Davis,

specific

principle,

80-mm

loaded

characterized and

easily

by

of California,

gel electrophoresis;

polyacrylamide

assay(GEDELISA):

acquired

Its

79-135)

ofCalifornia,

same

and

detected.

leukemia virus A receptors

naturally

(BR

antibodies

Knowledge of the specificity of an antibody is an important prereqfor immunocytochemical studies. Widely used techniques for characterization of antibody specificity in vitro are the Ouchterlonydouble-diffusion test (12) and immunoelectrophoresis (4). Together with the more recently described immunoreplicate technique introduced by Showe, et al. (17), these procedures have the disadvantage that only precipitating antibodies can be detected. Procedures which directly detect antibody-antigen complexes in gels after sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the antigens (11) can circumvent this problem. However, they are time consuming and use considerable amounts of specific antisera. Antibody specificity can also be characterized by combining SDS-PAGE with the high sensitivity of an enzyme linked immunosorbent assay (ELISA) as could be demonstrated in an anti-myosin system (10). In a modified technique we describe here how antibodies against feline either

THEILEN

polyacrylaniide

on the

Based

(GEDELISA) Virus

University

(ELISA)

Technique

Assay

Leukemia

University

dodecylsulfate

uisite

virus

by a New

Medicine,

Medicine,

characterized.

also

G.

D

of Veterinary

of Veterinary

the high resolution of sodium of enzyme-linked immunosorbent

sensitivity

leukemia

Specificity

Enzyme-Linked Immunosorbent to Antibodies Specific for Feline

of Clinical

Department

1979 U.S.A.

Printedin

Inc.

Demonstration

H. LUTZ’,

(H.L.,

Vol. 27, No. 8, pp. 1216-1218,

CYTOCHEMISTRY

Society,

1216

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OF

CHARACTERIZATION

ANTIBODIES

BY

1217

GEDELISA

to the previously described but poorly defined protein in the murine system (6). It seems likely that the p17 represents the p15(E) present in murine leukemia viruses (6). A gp35 has been defined in the avian system (1) and the nature of the other proteins awaits further study. Using GEDELISA with sera from different cats with natural FeLV infection, all of these proteins were antigenic. Goat-anti-FeLV serum OS 264 (obtained from the National Cancer Institute, Bethesda, Md.) reacted with all of the above proteins (Fig. la). Cat serum 7099 (Fig. ib) was collected from a cat which was naturally infected with FeLV and which was persistently viremic for 10 months (13). This serum recognized the gp73, p47 and to some extent plS and p12. Serum 7636 (Fig. ic) was collected from a cat relate

a

a 451

b

Cat Serum 07519

which

was

able

a no

exposed

FeLV

to an

as judged

viremia (13).

FeLV

infected

serum produced cells reflecting

This

tion

cat.

by the indirect

It never

showed

detect-

assay

immunofluorescence

a strong

immunofluorescence

for reac-

with FL74 an active immune response (3). The reaction of this serum was directed against p15 and/or p12 as well as against antigens in the region of p15(E), p47 and p60. The profile of the Con A binding assay (Fig. id) demonstrates that Con A was bound by the gp73 and by components with an apparent m.w. of

1.511

main

a as Cat Serum

6011

07636 C

34,000,

and

24,000

results

indicate

daltons.

DISCUSSION Our detected

1.600

FeLV

minor

-

BUsing

Assay

d

a as 24

L

.

. -

liii

PSLEOJLAft 91T GEDELISA

which

24,000

fore,

II

I

(

profiles

activity

was

localized

A binding

purified

major

bands

10,000, gp73,

with

in

most p27,

values detected. protein described (FOCMA)

likely pl5,

of 58,000, The in the feline coded

of

apparent

m.w.

representing

pl2,

and

plO.

47,000, origin range

of

those

fractions

gradient

of the

purified

of 73,000, the

17,000,

these

different daltons

oncornavirus-associated by the feline sarcoma

27,000,

known

In addition,

36,000, of 60,000

in

region

might

cat

serum

staining

7636 only

(Fig.

the

for

from

the

Figure

45,000

and

immunity. (Fig.

be

also

60,000,

in the m.w.

regions

explain

of

role in FeLV

antigens

these

blue

types

lc)

In the antibodies

regions

of 34,000

few

proteins

could

the

presence

of glyco-

le),

corresponding

be

immunologic

techniques

of binding

antibodies

and

GEDELISA

might

of antisera

We thank

used

the and

re-

GEDELISA

complement-fixing

mea-

restricted

is not

to the

antibodies.

be of some value in immunohistochemistry.

gel

1. Bolognesi

I. Isolation

There-

in determining

the

major

14,000

with

15,700,

12,000

proteins relate cell virus

revealed

FeLV

proteins

may

FeLV 15,000,

components apparent and

is to

5

and m.w.

8,500

were

unknown. the

membrane (7), while the

The

previously antigen

p47 may

for

helpful

discussions.

DP,

Bauer

and

CITED

H: Polypeptides

physical

and

ofavian

chemical

RNA

analysis.

tumor

Virology

viruses 42:1097,

1970 2.

Engvall (ELISA), istry

which

Dr. F.A. Troy

LITERATURE

3.

components.

separation

with

can

antibodies

ACKNOWLEDGMENTS gradient

RESULTS Electrophoretic

id)

m.w.

in the

of precipitating

the

8:871,

E, Perlmann quantitative

P: Enzyme-linked assay ofimmunoglobulin

immunosorbent

assay

G. Immunochem-

1971

Essex M, Jakowski RM, Hardy A: Feline oncornavirus-associated Antibody

Con

and As

the

an important

reacted

10)

with

and Coomassie Blue staining for protein. The molecular weights were calculated on the basis of log m.w. vs. relative mobility of standard marker proteins. The 14,000 and 17,000 dalton proteins probably correspond to p12 and p15(E), respectively.

contained

GEDELISA,

to immunohistochemical

different

specificity

FeLV using 3 different sera: a) goat-anti-FeLV OS 264 (obtained from the National Cancer Institute); b) cat serum 7099 collected from a cat persistently infected with FeLV; c) cat serum 7636 collected from a cat which had recovered from FeLV infection; d: Profile of the Con A binding assay performed as described in the text; e: Polypeptide composition of FeLV after SDS-polyacrylamide gel electrophoresis

enzymatic

la)

by Coomassie

(Fig.

in

play

daltons.

detection

‘‘IL

I

.

la-c.

(Fig.

found

Similar

* Fia.

serum

sures

‘I

..

I

may

were and

the

action.

kJL II

proteins

goat

detected proteins

a as

I

FeLV

daltons

17,000

Con A

by

are specific not only for the major but associated components. As can be concluded

which

minor ic,

that

titers

in cats from 54:637, 1975

WD,

Jr,

cell leukemia

Cotter

SM,

membrane cluster

Hess

P. Sliski

antigen.

households.

III. J Natl

Cancer Inst 4. Grabar P, Williams CA: M#{233}thode immuno#{233}lectrophoretique d’analyse de m#{233}langes de substances antigenique. Biochem Biophys Acts 10:193, 1953 5. McGuckin WF, McKenzie BF: An improved periodic acid Fuchsin sulfite staining method for evaluation ofglycoproteins. Cbs Chem 4:476, 1958 6. Ihle JN, Hanna MG, Jr, Schafer W, Hunsmann G, Bolognesi DP, H#{252}perG: Polypeptides of mammalian oncornaviruses. ization of p15 and reactivity with natural antibody. 60, 1975 7. Kahn AS, Deobagkar DN, Stephenson JR: Isolation terization of feline sarcoma virus-coded precursor

Blot Chem 253:8894, 1978 8. L#{228}mmli UK: Cleavage of structural

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proteins

during

III. LocalVirology and characpolyprotein.

the assembly

63:

J

1218 of the 9.

ET

LUTZ head

Limacher

noreactive

of bacteriophage W,

Manser

T4.

E, Lutz

Nature

H, Wild

227:680,

1970

P: Identification

of immu-

14.

10.

to antibodies raised against the NH1-terminal parathyroid hormone. J Histochem Cytochem 27.1)000, 1979 Lutz H, Ermini M, Jenny E, Bruggman 5, Joris F, Weber E: The

15.

11.

size of the by indirect chemistry Olden K,

16.

12.

dodecylsulfate-polyacrylamide Ouchterlony 0: Antigen-antibody

13.

sites

fibre populations in rabbit skeletal muscles immunofluorescence with anti-myosin 57:223, 1978 Yamada KM: Direct detection of antigens

Microbiol

Scand

Pedersen Orser B,

NC,

Theilen

32:231,

Chen

CH,

gels.

Anal reactions

Biochem in gels.

78:483, 1977 Acts Pathol

1953 G,

Allison

17.

Keane

MA,

C: Studies

Fairbanks of naturally

L, Mason transmitted

T,

feline leukemia Saunders GC, immunoassay

AppI

as revealed sera. Histo-

in sodium

AL.

Environ

virus Bartlett

infection. Am J Vet ML: Double-antibody

for the detection Microbiol 34:518,

Has 38:1523,

of staphylococcal

solid-phase

1977 enzyme

enterotoxin

A.

1977 Tamura TA, Takano T: A new, rapid procedure for the concentration of C-type viruses from large quantities of culture media: ultrafiltration by Diaflo membrane and purification by Ficoll gradient centrifugation. J Gen Virol 41:135, 1978 Theilen GH, Kawakami TG, Rush JD, Munn RJ: Replication of cat leukemia virus in cell suspension cultures. Nature 222:589, 1969 Showe MK, J.sobe E, Onorato L: Bacteriophage T4 prehead proteinase. II. Its cleavage from the product of gene 21 and regulation in phage-infected cells. J Mol Biol 107:55, 1976

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