L-01.

-hTO?IBOSIS RESEARCH PrinTed rn Great

MEASUREMENT

OF

A COMPARISON TION

PLASMIN-ANTIPLASMIN

OF

INHIBITION

LATEX AND

COMPLEX

AGGLUTINATION,

LATEX

HEMAGGLUTINATION

Dept.MedicaI - Onderwijs Gasthuisberg University

Press,

IN

197s

Ltd.

PLASMA.

AGGLUTINATESTS.

Research

Research en Navorsing

of Leuven,

(Received 28.10.1977; Accepted by

3%39-39 j,

INHIBITION

and D .Callen A .Bini* for Thrombosis and Vascular

Center

12, pp. ??rgamon

Britain

Belgium.

in revised form Editor P.J. Gaffney)

15.11.1977)

ABSTRACT Plasmin-antiplasmin quantitction

of

complex

was

measured

complex-associated

in human

neoontigens.

plasma

Simple

by

immuno-

chemical techniques based on latex agglutination, latex agglutination inhibition and hemagglutination inhibition, were used. In the latex agglutination test polystyrene particles coated with Normol plasmas had ticomplex-specific antibodies were used. ters of less than 2 to 4, which, after activation with urokinase, rose to 256 to 1024. Since a titer of more than 8 may be considered as abnormal, the test could thus reveal the generation in plasma of 2 to 4 percent of the amount of plasmin-antiplasmin present in maximally activated plasma. This sensitivity cient for routine screening of fibrinolytic activation in False

positive

rheumatoid lystyrene

results factors.

particles

are

however

In the were

latex

coated

obtained

with

agglutination with

purified

sero

complex is suffivivo.

containing

inhibition

test

po-

plasmin-antiplasmin

complex and samples to be tested were preincubated with complexspecific antibodies. This test has a similar sensitivity to the direct agglutination test and is not sensitive to rheumatoid factors. However, its performance is more time consuming and purified antigen is required for coating the polystyrene particles. The hemagglutination inhibition test is comparable to the latex agglutination inhibition test but is less sensitive because for unexplained reasons higher titers are found in normal plasma.

*o

n I eave Italy.

of absence

from

Mario

Negri

389

Institute,

Milano,

390

PLASWIN-ANTIPL.ASXIX

COXPLEX

Vol.l2,?io.3

INTRODUCTION Previous the

work

fibrinolytic

plasmin

from

system

complex

molecules

(1).

activation

of

By the

Since

using

system

ga;sti

specificity

hemagglutination in

allows

agglutination,

and

The

with

plasma

latex Plasma 1000

and

was obtained kallikrein

Germany)

(Abbott

bating

far

and antiplasmin removal both

The plasma for

30

units per

at room

less

(1000

CTA

units

as described

of

of the

we have

fi-

investi-

immunochemical inhibition

and

of the plasmin-antiplasmin

but

this

plasminogen

titrated

blood,

(Trasylol,

(4) as described

did

Ill.)

of plasminogen previously.

CTA

The

instances

the

results.

units

ml plasma

depleted

coated

Leverkussen,

not alter

per

IgG

and antiplasmin. in most

1000

and

patients

with

Bayer,

by adding

plasma

different

in bath

Plasma

by removal

three

(determined

Chicago,

temperature.

than

from

precaution

prepared

rn normal

factors

of aprotinin

North

was prepared

was

of

in plasma

METHODS

depleted

was

Laboratories,

plasmin-antiplasmin

min)

ml,

plasma

of antiplasmin

proteins

in vivo

formation

agglutination

in serum

plasma,

in plasma

added

30 min

that

activation

was determined

by centrifugation

Urokinase-activated kinase

AND

of rheumatoid

inhibitor

were

precursor

directly

rapid

quontitation

complex

concentration

particles),

the

its

fractions.

in urokinase-activated a high

and

of

blood.

plasmin-antiplasmin

serum,

with

importance

latex

the

from

was shown

of in vivo

of simple

for

it

activation

the plasmin-anti-

be quantitated

be of clinical

MATERIALS Plasma

test

detection

and sensitivity

inhibition,

human

could

in

distinct

was associated

which

during

emerge

agglutination

could

as latex

structures

that

immunochemically

system

in blood

such

complex

a latex

which

has shown

new it

complex,

a method

techniques,

render

fibrinolytic

brinolytic the

laboratory

in blood,

which

plasmin-antiplasmin (2).

this

of uro-

and

incu-

in both

plasminogen

(3) and

subsequent

residual

content

of

5 percent. complex

of urokinase elsewhere

was purified per

(4).

ml plasma We

used

from and

urokinase-activated incubation

urokinase

rather

at 37°C than

strepto-

kinase

for

activation

because

kinase-activated

plasma

a cti va tar

.

activity

purified

were

preparations

found

to

contain

obtained small

from

amounts

strepto-

of

piasminogen

Antisera Rabbits using

were

immunized

a conventional

antiplasmin the ml

fresh

for

2 h at

fuga

by

plasma

was

ml

to

addition

serum, and

of

it

0.1

specific

addition

immune

complex,

raised

against for

mg purified

each

the

serum

render

After

temperature

plasmin-antiplasmin

rabbit

the

the

plasmin-

neoantigens

in

plasminogen

and

mixture

stirred

precipitate

was

removed

by

0.15

centri-

tion.

Latex

agglutination

test

Polystyrene

particles

tories,

Detroit,

rabbit

antiserum

rabbit

immunoglobuiins

fate

at

40%

tine

-

Mich.)

of

according

M

bovine

serum

albumin

ZO,IJI

of

were

mixed

and

OS the

cuba tion

Latex

the

as

M

1 .5

times

added

in

was was

Amsterdam,

with

The

diluted at

room

clearly

Difco

in

at

0.1%

until

20,ul

per

M

gly-

10 ml

The titer

agglutination

use.

and

buffer. The

sul-

0.1

NaN3

same

The

ommonium

Netherlands) the

from

(5).

4°C

with

temperature.

visible

Plotz

with kept

Labora-

fraction

and

mixed

containing

serially 5 min

Singer

precipitation

suspension

9.0,

from

immunoglobulin of

by

0.81

was

ofter

mg (control) drops expres-

5 min

in-

,

Polystyrene

0.03

(Poviet,

Latex

the

suspension pH

samples

dilution

agglutination

complex

with

procedure

latex

latex

for

(Bacto

prepared

buffer,

rotated

highest

the

The

NaCl

,um

coated

to

(w/v)

or

with

0.81

were

6.5%

0.15

of were

satumtion.

20,ul

sed

absorbed

subsequent per

room

purified The

procedure.

complex

complex

with

inhibition particles

follows.

NaCl the

buffer original

concentrations

The pH

test were

particles 9.0

coated were

containing

volume. between

with

washed 0.02%

Purified 0.1

purified

plasmin-antiplasmin

twice NaN3

in

0.02

and

plasmin-antiplasmin and

1

mg

per

ml,

M

glycine

resuspended complex

ond

the

mixture

in was

392

vol.l’,so.

PLASFlIX-AXTIPLAASlYIB COXPLEX

stirred

gently

with

the

buffer

0.1

9.0

kept

hexanoic

the

20,ul

buffer

units

showed

Human

necessary

30

min

at

inhibition

blood

and

plasmin

group

for

were

complex

latex

0

Rh

in

for

Rheumatoid

factors

Rheumatoid

complex

after

using

1 month.

coating

5 min

per

mode

in

dilutions

4 fold

higher

of the

than

latex.

mixtures

were

the agglutination

as the

was

degmdation

were

were

highest

was

dilution

incubation.

tanned

The

(6).

between

quantitated

The

(6).

with

a latex

cells

with by

were

stored

of plasmin-anti-

0.03

with

products

coated

described

concentration

was

determined

and

the procedure products

solution

complex

fibrinogen

cells

degmdation

within

the

factors

Marburg/Lahn,

and

this

0.12

mg per

reagent

reagent

exactly

(Behringwerke,

Germany). RESULTS

AND

DISCUSSION

agglutination The

from

less

256-l

024

than

was expressed

NaCl

M 6-amino-

of these

of these

and

M

were

agglutination 20~1

washed

albumin

0.05

50~1

suspension

negative

fibrinogen

used

as described

the

samples

at a concentration

of agglutination

Plasmin-antiplasmin

Latex

ml.

temperature

titer

serum

containing

per

then

inhibition

et al.

at 4°C

of the

9.0

was - 0.15

10 mg bovine

to obtain

room

The

plosmin-antiplosmin

Merskey

pH

suspension M glycine

dilutions

of antiserum

above.

Hemagglutination

and

aprotinin

antigen-coated

as described

purified

NaCl

concentration

with

which

Serial

Kl

in 0.1

NaN3

use.

50~1

for

The

resuspended

M

1000

with

incubation

read

until

and

temperature.

0.1%

- 0.15

acid

minimal

mixed

buffer,

at 4°C

incubated

After

room

containing

M glycine

were

2 hr at

incubation

pH

ml and

for

3

plasmin-antiplasmin

titer

of the

than

2 up to 4,

(Table

previously same.

the antisem.

This

I). found

The

titer

whereas

improved Serum

whereas

mtio

had the

complex

urokinase-activated

of normal the

in normal

titer

plasma

plasma

plasma

was two

had a titer

to four

of urokinase-activated

is probably

explained

same plasmin-antiplasmin

times plasma

by better titer

varied

lower was

absorption

as plasma.

of

of

ml.

PLASMIX-ASTIPLXSYI?;

TABLE

393

I

PLASMIN-ANTIPLASMIN

COMPLEX

TITERS Hemagglutination

Latex agglutination

Latex agglutino tian

Sample

COXPLES

inhibition Normal

I serum

Norma

Urokinase-activated plasma Serum

with

toid

(2-4.

2-8

i2-8

2-8

plasm0

rheuma-

factors

Plasminogen antiplasmin

256

- 1024

128

256

- 512

(Z-4

and depleted

inhibition 4 - 32

- 512

64 - 256

4

2-4

(2-Z

2-4

plasma

As previously

observed,

o false

positive

activity

could

complex

by passing

IgG.

Thus

applied

tion

rent Latex

with

5 mM

the

neoantigenic

it appears

that

the

of potients latex

column

The

agglutination

test which

rheumatoid

of insolubilized

dithioerythritol

factor

human

destroyed

in

rheumatoid

caused

of plasmin-antiplosmin

both

the

the plasmin-antiplasmin

agglutination

a feature

factors,

titer

expression

latex with

the

(2).

arthritis

test factors

should

cannot in

their

the

be

blood.

be checked

complicates

easily

for

the

routine

pre-

applica-

test.

latex

reagent

could

be kept

for

several

months

at 4°C

without

appo-

loss of reactivity. agglutination Polystyrene

were

changing

of the plasma and

rheumatoid

reagent

a small

of rheumatoid

The

latex

over

a positive

of the

without

with

the plasma

to the plasma

Conversely,

of patients of the

be removed

factors

complex.

sence

agglutination

Treatment

rheumatoid

plasma

particles

agglutinated

of antigen 4000). between

by the

in the

The

inhibition

coating

agglutination

2 and

8 and

that

coated

with

purified

complex-specific solution inhibition

was 0.1 titer

plasmin-antiplasmin

antiserum

when

the

mg per

ml or more

of normal

plasma

or urokinase-activated

plasma

complex concentration

(titer

2000

or serum

between

128

to

varied and

512

(Table

I).

Serum

glutination. plasma

of patients

Furthermore

and

rheumatoid

rheumatoid

it appeared

normal

plasma

factor

reacted

plasmin-antiplasmin

with

that

factor

mixtures

or urokinase-activated very

complex

similarly,

was

not

did

inhibit

ag-

of urokinase-activated plasma

indicating hampered

not

and

that

serum

the

containing

quantitation

by the presence

of

of rheumatoid

factor. Tanned

red

cell

Optimal

agglutination

antiplasmin

complex,

concentration was

The

the

tion

inhibition

activating

for

of normal The

room

solution titer

not

titer

in the

of the at room

the

red cells

Plasma normal

with

plasmin-

plasma

might

in

that

did

not

in plasmino-

the

hemagglutina-

a plasminogen

be responsible

0.01

a

was 4 - 32 and

factors

plasma

when

ml or more

depleted

suggesting

cells

temperature

mg per

Rheumatoid

tests

red

purified

was obtained

of normal

than

other

from

with

of 0.06

of agglutination.

released

2 hours

coated

64 - 256.

plasma

Treatment

phosphate titers

but

activity

discrepancy.

coating

had a lower

test

cells

antiserum,

inhibition

inhibition

gen and antiplasmin

at

in the

agglutination

with

red

by complex-specific

of urokinase-activated

interfere

inhibition

of tanned

of antigen

used.

that

hemagglutination

for

this

M diisopropylfluoro-

however

did

not

result

in

lower

plasma.

tanned

red

temperature

cells

could

without

be kept

apparent

for

up to 1 month

loss of

at 4’C

and

1 day

reactivity.

Conclusions The

present

methodological

workable

technique

complex

in human

including

latex

hemagglutination

test

is the

simplest

for

positive

results

latex

its

routine

plasma.

agglutination

undertaken

determination

of the

and simple

latex

inhibition to perform

preparation. are

was

Rapid

agglutination,

cell

antigen

for

study

obtained inhibition

(5 min The

therefore

time)

d isa d vantage

with and

sera

inhibition used.

incubation

of this

containing

tanned

red

cell

to provide

a

plasmin-antiplasmin

immunochemical

agglutination

were

in order

and

The and

latex

tanned

is that factors.

hemagglutination

red

agglutination

requires

method

rheumatoid

methods,

no purified false The inhibition

tests

are

not

procedures

sensitive which

purified

take

antigen

The

ratio

activated plasma

of

for

between

16

plasma.

can

The

moderately

titers 64

normal

128

considered

of

the

factor In

but

addition

are

two

they

grade

plasma

latex

tests.

A

methods

of

is

This

complex

the

regional

test

and

the

of

these

or

plasma

abnormal.

activation

low

in

amount of

inhibition

plosmo

rheumatoid

to perform.

of to

sensitivity

to detect

I

be

severe

hemagglutination

of

1 hour

is

2 to 4 percent

sufficient

norma

the

8 or

va ted

presence

step

require

coating.

urokinase

of

the

about

with

sence

detect

to

in

fibrinolytic

due

to

the

in to

high

system The

normal the

acti-

enough

to

may

be in-

but

sensitivity

higher

pre-

maximally

therefore

activation.

lower

titer

corresponds

present is

maximally

titers

of

found

the

in

1

REFERENCES

1.

COLLEN,

2

III

and

7,

235,

1 COLLEN,

D.,

latex

888, 6.

J.M.

to

for

and

serologic

red

cell

quantitative

-antipIasmin

hemagglutination estimation

in-

of

complexes

CAMBIASO, rapid

human MERTZ,

C .L.

quantitative

plasma.

in

E.T.

and

human PLOTZ,

of

Science

thrombin-

human

plasma

MASSON,

properties

The

Intex

rheumatoid

P.

of

the

7,

21,

purification 170,

69, fixation

arthritis.

plasmin1977. from

1095,

of a new

Eur.J.Biochem.

C.M.

diagnosis

:

Plasminogen

some

plasma.

and

estimation

Eur.J.Clin.lnvest.

chromatography.

Identification in

tanned

the

1970.

fast-reacting 209,

test-i. Am.

1976. Appiica-

J.Med.

21,

19.56,

MERSKEY, sensitive

the

F.,

affinity

inhibitor

SINGER,

in and

by

0.

plasmin

COCK, test

D.G.

COLLEN,

tion

DE

A f or

1975.

complex

plasma

F.

plasmin-alpha!

agglutination

DEUTSCH, human

5.

COCK, (TRCHII)

Thromb,Res.

antiplasmin

4.

DE

immunoassay

antithrombin

A

3,

and

D.

hibition

C., method

Proc.Soc.Exp.BioI.Med.

LALEZARI, for

P.

measuring 131,

and

JOHNSON,

fibrinolytic 871,

1969.

split

A.J. products

A

rapid,

in

human

simple, serum.