The application of polyethylene glycol to radioimmunoassays used in haemostasis

THROMBOSIS RESEARCH 29; 333-341, 1983 0049-3848/83/030333-09$03.00/O Printed in the USA. Copyright (c) 1983 Pergamon Press Ltd. All rights reserved.

THE APPLICATION

OF POLYETHYLENE GLYCOL TO RADIOIMMUNOASSAYS USED IN HAEMOSTASIS

8.

J.

Haemostasis Royal

Free

Woodhams Unit,

and

F.8.P..

Department

Hospital

,

London

Kernoff, of

tiaematology, England

NW3 ZQG,

Accepted by Editor C.R.M. Prentice. (Received 25.8.1982. Received in final form by Executive Editorial Office 22.11.1982)

fiBSTRACT Use

of

polyethylene

antibody

glycol

6000

radioimmunoassays

in

for

the

secc.nd

stages

fibrinopeptide

A,

of

double

B-thromboglobulin

and platelet factor 4 facilitates the more rapid separation and allows precipitating antiserum free from bound antigen, At a final PEG 6000 concentration used in greater dilution.

of to be of

of free from bound antigen was complete within s %, separation 1 hour, and antisera couid be used in dilutions 3-g times greater PEG 6000 had a than those recommended by the manufacturers. negligible effect on the affinity of first stage antibodies for their respective antigens. Radioimunoassays using PEG 6000 were failure to adjust standards to sensitive to protein concentration, similar artefactcal

protein ly

concentrations low results.

as

those

of

test

samples

causing

I NTRODUCTI ON An essential from bound

step antigen.

precipitate

in

all radioimmunoassays Separation methods

antigen-antibody

absence of the antigen non-specific precipitation However , double antibody expensive step

of

and the

High molecular antigen-antibody from solution

Key

Words:

complexes

to

techniques - it

require

glycol, in,

have

is overnight

weight polyethylene interactions (1) (2).

Polyethylene B-thromboglobul

have

not

the

disadvantages for

(PEG) is precipitation

radicimmunoassay, platelet factor

333

advantages,

the

known

of second

free tc

of the sulphate.

being precipitation

to enhance both of macromolecules

fibrinopeptide 4.

of

including

charcoal, and avoidance such agents as ammonium

unusual incubation.

glycol and the

is the separation a second antiserum

several

stripping caused by associated with

time-consuming

assay

(RIAs) which use

A,

334

POLYETHYLENE GLYCOL IN R.I.A.

In this RlAs assay

study

used time

we

have

evaluated

in haemostasis, by increasing

the assay. which utilises

The

assays a first

the higher molecular (BTG and PF4), which

the

with the the speed studied stage

weight

use of PEG 6000 in three dcuble antibdy particular objective of shortening overall of precipitation in the second stage of

were an antiserum antigens

utilise

rabbit

haptanic raised

of

FPA:

Rabbit

in

RIA for rabbits;

fibrinopeptide and two

B-thromboglobulin and

goat

MATERIALS Radioimmunoassay

Vo1.29, No.3

and

antisera,

A(FPA), for

RlAs

platelet

factor

4

respectively.

AND METHODS

anti-human

FPA antiserum,

desaminotyrosyl

FPA

(DATFPA), and FPA standard were obtained from IMCO Limited, Stockholm,Sweden, and prepared for use as previously described (3). Anti-FPA antiserum was used at a dilution of 1 in 128o. Apart from differences in the method of separation of free from bound antigen, the assay was performed as previously described (3), first stage incubations normally being overnight at 4’C. Shorter incubations - e.g. 30 mins. at 37’C followed by 15 mins. at 4’C gave similar results. All dilutions for FPA assays were made in 0.05M tris buffer containing O.lM sodium chloride, @.l% sodium azide and 0.1% ovalbumin (TBS ovalbumin, pH 8.5). presented in Tables 2-7 Optimisation ‘Goat

of

anti-rabbit

antiserum. second

The

controls

separation

IgG

stage

according ation with

the

effects

incubation

of

assays were mean values. of

(Sigma)

to the standard al 1 components (tube

All being

was

free

as

out

bound

the

dilutions shown

system

in

duplicate,

FPA antigen

second

stage

concentrations

and

protocol, of the

frcm

used

different

times,

carried

of

results

using

PEG 6000

(Sigma),

of antiserum were studied Table 1, which compared

in present

(tube

PEG 6000:

precipitating

1)

with

a series

separof

2-5). TABLE

1

Tube 1

number

2

3

4

5

.1251-DATFPA

0.1

0.1

0.1

0.1

0.1

TBS ovalbumin

0.2

0.2

0.2

0.3

0.3

Ant i-FPA

0.1

0.1

0.1

-

-

Overnight Precipitating PEG

antiserum

6000

0.1 0.5

TBS Ovalbumin

Volumes of free

(ml) used in bound antigen

incubation -

0.5 c.1

experiments designed using PEG 6000

to

0.1 -

at 0.1

-

0.5

0.5

-

0.1

0.5

optimise

4’C

the

separation

The control tubes were included to check: possible precipitation of 125-lDATFPA-anti-FPA conjugate by PEG 6000 alone (tube 2); possible precipitation of 125-I-DATFPA-anti-FPA conjugate by the precipitating antiserum alone possible precipitation of 125-l DATFPA trapped or bound to the (tube 3);

second DATFPA uged

at

25OOg

before antigen was free

from

30

bound

mins. in

at

was

5% (w/v),

4OC,

counter

a Rackgamma In the final

antigen

precipitating

Separation precipitating

an

accomplished

incubation

antisertim

at

(LKB

version

of

using

period

of

Ltd.). the

were were

25 ml 20 ml

activated Sepharose was O.lM sodium bicarbonate groups were blocked by The Sepharose conjugate

also this

stored as l:g mixture

incubated centrifuged

natant was removed further two times. counted. Separation

of

the

phase

liquid

purposes. assay tune

at

free

0.1 and

temperature,

mixed with 1 ampoule 18 hours at 4’C. for mixing the was then and stored

Langmuir

Assay

of

FPA

from

gel for a further 2hours extensively washed with at 4OC as a 1:l slurry

was

in

using bentonite concentration

bound

FPA antigenusing

PEG 6000:

maximum in

binding

unactivated In the assay,

tube

This

precipitating

method

was

used

and

the

mix-

antiserum for

diluted 1 in 32 was added to each were incubated overnight at 4OC.

tubes

were

and

centrifuged

the

calculated

tubes

from

at

20C0

g for

in

comparative second stage After adding 30

mins.

at

4’C,

counted.

data

obta

i ned

from

standard

curves

(5).

clinical

prepared of

that l:Y

1:l slurry with TBS ovalbumin. was added to each second stage

ml antiserum the mixtures

pl’cts

and

a

of undiluted Any rema i n-

at 4’C. After adding 1 ml TBS ovalbumin, the g for 10 minutes at 4’C, 1 ml of the superand the washing cycle repeated a with a vacuum I ine, After removal of the final supernatant, the tubes k_,ere

without

affinity

using

collected

room

overnight at 200C

the 1 ml TBS ovalbumin, the supernatant aspirated, Antibody

of cf

100.

O.lM sodium bicarbonate and TBS ovalbumin, Preliminary experiments indicated with TBS ovalbumin. was obtained wher. the Sepharose conjugate was diluted

tures tubes

bound

concentration

1 hour 1 in

of

separation

free from bound FPA antigen using Sepharose-conjugated antiserum without PEG 6060: This method was used for comparative Sepharose 46 (Pharmacia Ltd.) was activated using cyanogen bromide

CNBr-activated lg L-glycine.

Sepharose, 0.2 ml of

Percentage

assay,

a final

of

a dilution

precipitation of 12.5-lthe tubes were centrifremoved with a vacuum

of

purposes. (CNBr) (4). antiserum in ing with

for

counting calculated.

PEG 6000 of and

and possible 4); After incubation, and the supernatant

phase antiserum by PEG (tube by PEG 6000 alone (tube 5).

1 ine

335

POLYETHYLENE GLYCOL T.NR.T.A.

vo1.29, No.3

blood for

samples

assay

as

using

described

PEC 6000:

Blood

by Woodhams

adscrption to remove cross-reacting first stage incubation tubes used

samples

6 Kernoff

fibrinogen. to prepare

were (3))

The protein standard curves

for assay of plasma samples using PEG 6000 was increased by the addition of 0.1 ml 7% ovalbumin or 0.1 ml FPA.-free horse serum, the latter being prepared by bentonite absorption of horse serum foIlo.+ed by extensive dialysis against 0.1 ml saline until no FPA was detectable in an assay using Sepharoseconjugated

antiserum.

Radioimnunoassays

of

BTC and

PF4:

Rabbi t

anti

-human

BTG cnt i serum

.jnd bTJ stan-

dard were a gift from Dr.Duncan Pepper. Reagents were prepared and the BTG assay performed as described by Bolton et al(6),apart from differences in the method of separation of free frcm hound antigen.Goat anti-human PF4antiserum was a gift from Dr.Karen Kaplan. PF4 standard was supplied by Dr.DuncanPepper. 10 ug PF4 was iodinated using 1mCi of carrier-free 125-l (Radiochemical Centre, Amersham, UK) by the method of Hunter & Greenwood (7). The label led PF4 has separated from unreacted 125-l by one passage only through a Sephadex

336

POLYETHYLEXE GLYCOL IN R.I.4.

(1.C x 15 cm). for BTG and PF4 containing C.15M

G25 coium dilutions (pH 7.5)

vo1.29, yo.3

No further purificaticn was performed. Al! assays were made in O.C5M scdium phosphate buffer sodium chloride, C.12 sodium azide, 10 iu/ml

(Weddell) and 2% horse serum, which was also used as porcine mucosai heparin eluart in the preparation of labellec ETG and PF4. First stage incubations in BTG and PF4 assays were overnight at LiGC. Optimal conditions fcr use c,f PEG 6000 in the second stages of both assays were established as described above for the FPA assay, and were similar tc these used in the FPA assay i.e. a f ina 1 PEG 6OOC concentration of 5% and an incubation period of I hour at rcom temperature. In tt-e final version of the BTG assay, the precipitating antiserum (goat anti-rabbit IgG, Sigma, titre 1 in 16) was used at a dilution of 1 in 100. In the PF4 assay, rabbit anti-goat IgG (Sigma, titre

1 in 16) was used at a diluticn of 1 in 150. Separation cf BTG using Sepharose-conjugated antiserum was carried out as for FPA, except that scdiusl phosphate buffer was substituted

free

frcm

described for TBS

bound above cvalbumin

RESULTS Effect of PEG 6000 concentration of seoaration of free frcm bound FFA antigen (Table 2): Within the final concentration range of 1.D - lO.O%, increasinc concentrations cf PEG 6000 caused enhancement of orecioitation Control Lubes of bound antigen (tube 1) with an optimal effect at 5%. showed negligible precipitation.

TABLE

2

-. Final of

Tube

concentration

PEG 6OCO

(w/v)

2

1

nurrbe r 3

4

5

1.0%

14.2

1.6

1.6

2.2

2.1

1.25%

3G.5

2.0

1.6

2.0

1.5

1.5%

39.0

1.5

1.6

2.2

1.5

2.5%

56.9

2.2

1.6

5.1

1.9

5.0/;

c3.e

2.0

1.6

2.c

2.0

10.0%

61.2

3.9

1.6

3.9

3.3

Percentage of bound FPA antigen precipitated using different Experimental design as shown in concentrations of PEG 6000. Table 1. Second stage incubation time 1 hr. at room temper1 : 100. ature. Precipitating antiserum dilution

Effect of precipitating antiserum dilution on separaticn of free from bound FPA antigen (Table 3): Inclusion cf PEG 6000 in the seccnd stage of the assay allaa use of the precipitating antiserum at a higher working dilution (1 in 1CO) than that recommended by the manufacturer for conventional methods cf separation (1 in 32).

Vo1.29,

%.3

337

POLYETHYLENE GLYCOL IN X.1.A.

3

TABLE _--

Tube Antiserum

dilution

1

2

3

number 4

5

1 in

32

62.6

1.6

1.6

1.6

1.6

1 in

SO

62.5

1.4

1.4

1.4

1.4

1 in

100

69.2

2.0

2.0

2.0

2.G

i

in

150

32.0

2.2

2.2

2.2

2.2

1 in

200

4.0

2.4

2.4

2.4

2.4

Percentage

of

antiserum Table Final

Effect antigen

in

bound

FPA antigen

different

dilutions.

1. Second concentration

stage of

precipitated

incubation PEG 6000

time 5%.

of second stage incubation time (Table 4): Using PEG 6000 the

pletion within separation of incubation.

using

Experimental 1 hr.

at

room

on separation >>a>=

the

of

as

shcwn

in

temperature.

free

react

1 hour (tube 1). Without PEG 600C (tube free from bound antigen could be detected

TABLE

precipitating

desiyn

ion

from

bound

reached

FPA

corn-

-

3), very little after 4 hours

4

Incubation (room

time

(hrs)

Tube 1

temperature)

2

3

number 4

5

0.5

57.6

2.0

2.0

2.0

1.6

1 .o

61.5

2.0

2.3

2.3

2.0

2.0

62.4

2.0

2.6

2.6

2.0

4.0

61.9

2.0

1.6

1.6

l.P!

Percentage second stage in Table 1. concentration

of

bound

FPA antigen

incubaticn Precipitating of

PEG 6000

precipitated

times. Exper imenta antiserum dilution

using

different

1 des i gn as shown 1 in 100. Final

5%.

Comparison of stanlord curves and affinity ccnstants obtained using PEG and non-PEG methods of separm%f free from bound FPF. antigen (Tables 5 d 6) : The PEG 6000 method of separation was compared tiith two other optimised methods. In the first, precipitating antibodies were conjugated to Sepharose 4e ; in the second, the antiserum was used in the liquid phase. Calibration curves obtained by all three methods were very similar (Table 5), as were

338

POLYETHYLENE GLYCOL IN R.I.A.

antibody cause first

affinity alteratior! stagz of

constants

(Table

of binding the assay.

6)

characteristics

TABLE

vo1.29, No.3

indicating that PEG 6000 did between FFF and anti-FPA

not in

the

5

--% BOUND

F”,I II PEG 6000

Sepharcse

63.2

59.0

54.7

0.52

56.7

56.2

45.0

1 .a4

49.2

53.8

36.4

2.05

35.7

38.8

33.5

3.13

29.8

31.6

21.4

4.17

27.8

25.3

20.3

6.25

20.8

17.4

16.8

8.34

15.5

15.8

11.4

2.3

5.0

1.2

pmol s/n1

0

BLANKS

Comparison methods of

of standard separation

curves of free

TABLE

Liquid

Phase

obtained using three different from bound FPA antigen.

6

PEG 6000

Sepharose

Liquid

phase

-

Ka L/M

x

10’

Comparison methods of

Assay

of

FPA

in

4.2

of affinity separation

clinical

plasma

4.5

3.7

constants (Ka) using different of free from bound FPA antigen.

samples

using

PEG 6000:

Initial

application

of the PEG 6000 method to clinical plasma samples produced significantly lower results at low levels (0.5 pmols/ml) than those obtained using Sepharose-conjugated antiserum. The possibility was studied that differences in protein concentrations between assay mixtures containing standards and those containing test samples might be a cause of these lower results. Addition of 0.1 m! of either ovalbumin (70 g/l) or FPA-depleted horse serum to standards used in the PEG 6000 assay caused a shift in the standard curve (Table 7), and produced results which did not significantly differ from those obtained using Sepharose-conjugated antiserum. (Hilcoxon signed rank test,

POLYETHYLENE GLYCOL ZN R.I.A.

vo1.29, No.3

68 plasma samples, FPA Within assay coefficient using

PEG 6000

coefficient

was

of

range of

13.4%

(mean

variation

was

standard

O-15 pmols/ml, variation for 1.04

8.8%

TABLE

normal

a single

pmols/ml, (mean

normal

n = 10).

3.18

339

pmols/ml,

deviate

sample Between n = 9).

1.26).

assayed assay

7

% Bound pmol /ml

FPA

No added

With

protein

added

protein

0

83.0

70.7

0.52

77.3

64.8

1.04

69.0

62.1

2.~8

56.0

51.3

3.13

46.4

44.4

4.17

40.4

39.7

6.25

31.2

30.8

8.34

28.4

27.1

Comparison of standard curves obtained with and without addition of ovalbumin. Similar results were obtained using FPA-depleted horse serum as a source of protein.

Application Optimal those of precipitation

of conditions the

the

PEG 6000 for

method

of

separation

BTG and PF4 assays PEG 6000 without

FPA assay. of 125-I-BTG, Precipitating

to

BTG and

PF4

assay:

similar using PEG 6000 were antisertim caused negligible

to

125-I-PF4, or first stage antigen-antibody antisera used without PEG 6000 were unable to complexes. In both cause antigen-antibody precipitation after a 1 hour incubation. antisera could be used 6-g times more dilute than suggested by the assays, As with the FPA assay, manufacturers for conventional methods of separation. the protein concentration of standards was increased by addition of 0.1 ml horse serum when assaying clinical samples. There were no significant differences between plasma BTG levels measured using PEG 6000 and those measured using Sepharose-conjugated antiserum (Wilcoxon signed rank test, 0.28). Affinity 64 samples, BTG range O-200 ng/ml, standard normal deviate constants (Ka) were similar (2.9 and 3.5 x lOlo L/M, respectively). Within assay coefficient was 10.3% (mean

of 76.5

variation ng/ml,

for a single n = 8). Between

sample assay

assayed coefficient

using of

PEG 6000 variation

was 7.2% (mean 57 ng/ml , n = 6). Mean recovery of BTG added to normal plasma in the range 60-2000 ng/ml was 97% (SO 21, n = 7) , with no trend to No comparison of different different recoveries at higher or lower levels. separation methods was made in the PF4 assay, but the range of results on normal plasmas (0.02 - 1.04 pmols/ml, mean 0.47, SD 0.27, n = 21) was similar

to

previously repeated values using other methods of separation assay coefficient of variation for a single sample assayed was 1.5% (mean 1.74 pmols/ml, n = 5). Eetween assay coeff i cwas 8.8% (mean 1.54 pmols/ml, n = 6). nean recovery of

Within (8, 9). using PEG 6000 ient of variation

340

POLYETHYLENE GLYCuL IN R.I.A.

PF4 added to n = 16) with

normal plasma in the no trend tc different

range O-15.5 recoveries

,b-01.29, No.3

pmols/ml at higher

was 112.9:: (SD or lower levels.

30.5,

DISCUSSION How PEG precipitates proteins is poorly understood. It has been suggested that it acts as an inert sponge which, by absorbing solvent, raises the effective concentrations and therefore activities of proteins in the surrounding mi I ieu (2). Larger molecular weight proteins are most sensitive to this effect and will precipitate at lower PEG concentrations. The activity of PEG increases than those used in PEG will antiserum

precipitate (10).

polymers

cause

of

excess

time

RlAs can antiserum.

By

suitably

for

precipitation

addition high

of

in

equally

of

second

applicable

of

disruption

in

to

of

stage

the concentration are expensive and

economy

molecules, and precipitating It had little effect on lack

the

the

RlAs

therefore,

use

based

of on

double

of has

haptens

antiserum.

and

larger

from different of antibodies

for

animal FPA or

equilibria

in

the

of the PEG method Failure to adjust test samples may

of

antibody

assay time - an samples - which

precipitating

antigen/antibody

stages of the assays. The only disadvantage be its sensitivity to protein concentration. to similar protein concentrations as those artefactual ly low results.

significant

of precipitating we have found use

overall clinical

antiserum affinity

the

of

a second

molecular weight in the presence

PEG concentrations,

effective means of shortening when results are needed from

advantage

indicating

without

PEG and other interactions

adjusting

be reduced by increasing However, these reagents

a more factor

PEG appeared

complexes

concentrations, of antigen-antibody

(1).

required also

additional

antigenic sources.

2). At concentrations higher agents such as ammonium sulphate,

of the large molecular weight antibody-antigen-antibody a double antibody RIA can be accelerated without of other components of the assay.

of

PEG to be important the

antigen-antibody

At lower enhancement

antibody

precipitation complexes precipitation The

with polymer size (1, this study, and as with

BTG,

first

appeared standards cause

to

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K.

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antibody

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4.

5.

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and

INGHAMS,

polyethylene

glycols.

WOODHAMS, 6.J. Fibrinopeptide

WILCHEK, affinity

PIWCKARD, conjugates. D.M. Weir

Equilibrium

Chapter

R.N. In: 17.

K.C. J.

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1974.

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radioimmunoassay

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M. and ANFINSEN, chromatography.

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Protides

nephelometry.

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and KERNOFF, P.B.A. A in human ptasma.

CUATRECASAS, P., purification by 636-643, 1968.

(Ed)

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Publications.

London,

edition. 1978.

POLYETHYLENE GLYCOL :N R.I.A.

vo1.29, No.3

6.

BOLTON, A.E., LClDLAM, C.A.; MOORE, S., Three approaches to the radioimmunoassay

Er. J. f?aematol., 33, 233-238, 7.

HUNTER,

\!.M. and

CREEF!WOOD,

human

growth hormone 495-496, 1962.

a.

5.

Preparation

cf

D.S.

and

CASH,

J.D.

B-thrombcglobulin.

1976.

F.C.

high

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specific

activity.

iodine-131

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iveture, 194,

The release, distribution DAWES, J., SMITH, R.C., and PEPPER, C.S. and clearance of human B-thrombcglobulin and piatelet factor 4. Thrcmb.

Rea.,

KAPLAN,

K.L.

Platelet 199-202, lc;.

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PEPPER, of

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COLLINS,

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CWEN, 4 as

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Flasma

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levels platelet

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D-thromboglobclin

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in vivo.

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W.P. @ARfJARD, G.J.R. and HENNAM, J.F. Factors affecting the choice of separation technique. In: Steroid ~mcunoassay. E.H.D. Cameron, S.G, Hillier, K. Griffiths (Eds.) Alpha Omega Publishing Ltd., Cardiff, Wales, 1975, pp 223-228.

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