Radioimmunoassay of thromboxane B2 in plasma: Methodological modifications

THROMBOSIS RESEARCH 51; 533-541, 1988 0049-3848/88 $3.00 t .OO Printed in the USA. Copyright

(c) 1988 Pergamon

Press plc.

Al1 rights reserved.

RADIOIMMUNOASSAY OF THROMBOXANE B2 IN PLASMA: METHODOLOGICAL MODIFICATIONS

P.G. Rogasi, R. Paniccia, M. Coppo, D. Prisco, M. Boddi, J. Chent G.F. Gensini, R. Abbate Clinica Medica

1, University

of Florence,

Italy

in original form 12.5.1988 by Editor P.M. Mannucci) (Received by Executive Editorial Office 28.6.1988)

(Received 2.5.1988; Accepted

ABSTRACT Thromboxane commercial

82

(TxB2) determination

3H-RIA

kits.

in plasma are not detectable this the

study is the

is usually performed

However, the without

evaluation

by

low amounts of TxB2

previous extraction.

The aim of

of 1) plasma protein interferences

on

binding and separation

charcoal

steps of bound from free analyte and 2) Our results in different experimental conditions.

efficacy

indicate that plasma proteins do not influence the antibody the efficacy of precipitation but significantly reduce dextran-charcoal,

so that the

protein amount increase

buffer. Our findings plasma

TxB2

supernate radioactivity

(r= 0.99 pcO.001).

in the samples counts concentration in plasma of

using present

suggest

without

that, in

extraction,

rises with the

Such greater

determines a lower when the calibration

binding, of kit

number

of

estimation of TxB2 curve is set up in

order to measure

low amounts

it is useful: 1) to refer to a

Chinese *from the Cardiovascular Institute and Fu Wai Hospita1 Academy of Medical Sciences. Peking - China; recipient of a Research Award from Italian Government. ____________________~~_~_~__~~~~~~_~~~____~_~____~~~~~__~~~~~~__~~~~~~~~~~~~~ Key words: Radioimmunoassay,

Thromboxane

82.

533

Vol. 51, No. 5

RADIOIMMUNOASSAY OF PLASMA TxB2

534

calibration

2) to use curve set up in buffer-diluted plasma, charcoal concentration allowing the lowest stripping

uncoated 3) to perform al1 steps at 4OC.

the and

INTRODUCTION is the most widely employed method for the Radioimmunoassay (RIA) quantitative determination of thromboxane 82 (TxB2) levels in biological mainly in plasma or serum and in urine (1,2). The large amounts of fluids, TxBP present in serum are easily measurable after adequate dilutions, whereas the

lower

amounts in urine

need extraction

and

concentration.

As

plasma

RIA raises some difficulties because of the low TxBE samples are concerned, - near to the detection limit of the commonly used RIA - present in levels plasma (3). In order to concentrate

TxB2 and to remove al1 plasma

interfere

with

the assay,

in many laboratories

performed.

However, these procedures

introduce

substances which could

extraction

some drawbacks

procedures

are

(4): 1) recove-

ry, which has to be estimated

on each plasma sample, may be low and variable;

2) impurities

solvents and

deriving from

gases

column leakages and so forth, may contaminate tions

and chromatographies

remove plasma

used for evaporation,

from

the sample, even though extrac-

materials

possibly interfering

in

the assay; 3) capacity, rapidity and handiness of the method are diminished by pretreatment of samples. These concerns could be avoided by performing the

assay

on

plasma

chromatographic widely

used

samples,

purification.

RIA kits (NEN,

present in unextracted

previous some

solvent

extraction

commercially

or

available

ABT) fail to detect the low amounts of TxB2 (5). This failure could be due to interferences

plasma

exerted by plasma components

without

Unfortunately,

in some step of the assay.

The aim of our study was to set up a suitable and reliable procedure for

the

assay

the

of

variables procedures)

TxB2 in unextracted (incubation

plasma.

For this purpose we have studied

temperature

and bound from free analyte separation on the various phases of the TxB2 RIA in the (6-11).

possibly interfering

presence of plasma proteins

MATERIALS

AND METHODS

Materials:

American Biomedical Technologies (ABT) 3H-RIA kit for TxB2 was purchased from AFB Clinical Pharmacology Inc (Berlin). Liquid scintillation fluid: Aquassure, New England Nuclear (NEN, Boston, MA, USA). Charcoal: activated powder "Norit A", Serva (Feinbiochemica, Heidelberg, West Germany). PGD2, PGE2, bicycle-PGE2, PGF2 alpha, 6-keto-PGFl alpha, (UpJohn, Kalamazoo, Mi, USA). Blood sampling and preparation _ were drawn,

after a three-day

prostaglandin-free

oral acetyl-salicylic

plasma.

Nine mL of blood

acid treatment

(lg/die),

Vol. 51, No. 5

RADIOIMMUNOASSAY OF PLASMA TxBP

from 15 healthy volunteers, of

10 ug/mL phenoprophen

platelet-poor 4oc,

were

plasma,

into cold polypropylene

535

syringes containing

in 0.037 mmol/L EDTA solution.

obtained

by centrifuging

1 mL

Fourteen volumes of

at 3200 x g for 30 minutes at - 10% suspension

treated with one volume of the proper charcoal

(w/v) in phosphate buffer (20 mmol/L, pH 7.4, diluted 1:12 with twicedistilled water) vortexed and centrifuged at 4OC for 10 minutes at 3200 x g. Lastly it was two-fold Experimental 1)

leached and stored at -2OOC.

procedure two influence ---of plasma on the standard curve, 10, 25, 50, 100, 250, 500 and 1000

In order to evaluate the

series

of

standard

TxB2

pg/mL were prepared. in TxB2

and in buffer,

plasma: 100 vl_ of each standard dilution, both in were transferred into the assay tubes; 100 uL of 3H-

and 100 uL of anti-TxB2

taining 100

The first series was set up in assay buffer, the second

prostaglandin-free

plasma

dilutions:

antibody were then added.O-standard

100 uL of buffer or prostaglandin-free

uL of antibody)

Al1 the tubes were thoroughly (25OC)

100

and "NSB" tubes (containing either

100 uL of plasma + 100 uL of buffer and temperature

plasma,

tubes (con-

uL of tracer and

200 uL of buffer

or

100 I.ILof tracer 1 were also prepared.

mixed and allowed to incubate overnight

together with total count tubes (containing

at room

700

uL

of

buffer or 100 FL of plasma t 600 uL of buffer and 100 uL of tracer). The

day after 500 UL of dextran-coated

bath under continous

charcoal

tube. After a contact time of 10 minutes 1000 x g for 10 were

minutes

transferred

scintillation triplicate.

into

cocktail After

beta counter minutes/vial.

suspension

(placed in an ice

stirring at least 1 hr before) were added to each in a liquid were

(+ 5') the

refrigerated

centrifuge vials scintillation

added.

Al1 the

a 3-hour dark adaptation

(Beckman Instruments

Inc.,

assay

tubes were centrifuged (4OC). and 5

procedure

was

performed

al1 the vials were counted

Fullerton,

The binding capacity of the antibody was calculated

at

The supernates ml of liquid

Calif.,

USA)

in

for

in a 10

according to the formula:

0 standard - NSB x100 Total counts 2)

For assessing the various charcoals -in the separation phase we compared supplied by ABT kit with albumin-coated charcoalcharcoal pH 7.3) uncoated charcoal 0.5% (w/v) in phosphate buffer (10 mmol/L,

dextran-coated containing different

bovine albumin concentrations

(0.4% w/v) - and uncoated charcoal in phosphate

prepared at four

buffer (described above):

1, 5, 10 and

50 g/L. Each charcoal preparation, stored at 4OC for 24 hrs and placed in an ice bath under continuous stirring at least 1 hr before, was added to six NSB tubes (containing 100 uL of prostaglandin-free plasma, 100 JJL of buffer and 100 uL of 3H-TxB2) and to six 0-standard tubes (containing 100 ~.JL of prostaglandin-free plasma, 100 uL of 3H-TxB2 and 100 flL of antibody). After a 10 - minute

contact, centrifugation

according to the procedure described

and counting above.

procedures

were

performed

536

Vol. 51, No. 5

RADIOIMMUNOASSAY OF PLASMA TxB2

3) The uncoated

influence

efficiency

was

by on the separation charcoal the Üñcoated this by ABT. To supplied

of plasma protein concentration

charcoal

(5

g/L)

was investigated

and

compared with dextran-charcoal containing 100 ul of up a series of NSB samples plasma/buffer at increasing ratios (O%, 30%, 50%, 70%,

set purpose we prostaglandin-free

lOO%), 100 uL of buffer and fixed amounts (100 uL) of labeled analyte and a of 0-standard samples containing the same increasing concentrations series 100 uL of tracer and 100 ML of antibody, in of prostaglandin-free plasma, The two sample series were incubated overnight at 25OC. The day sextuple. after the samples (triplicate NSB and 0-standard) were treated either with uncoated

charcoal

or

with dextran-coated

charcoal,

following

the

usual

For calculating the binding a group of total count tubes procedure. containing the same plasma protein final concentrations was set up, incubated and counted. Al1 the procedure was performed in sextuple. 4)

Finally

we

manufacturers

investigated of

the -effect of temperature on the assay. the ABT assay kit allow the antigen-antibody reaction

the addition of cold charcoal

in both the phases of the

assay, we

prepared four groups of samples: A, B, C and Cl . Each group consisted of NSB tubes (50 VL of prostaglandin-free tracer) and six 0-standard of

buffer,

separation uncoated of

100 of

tubes (50 uL of

uL of tracer, bound

charcoal

prostaglandin-free

100 uL of antibody).

from free analyte was obtained

(5 g/L).

prostaglandin-free

plasma + 150 VL of buffer,

Moreover we set

100 uL

six of

plasma t 50 PL

In al1 the groups by

and

In order

keeping the tubes at room temperature.

to evaluate the influence of temperature

The

addition

of

the cold

up six total count tubes (50 uL

plasma t 650 uL of buffer and 100 GIL of tracer), whose

average radioactivity was used for calculating the antibody capacity. Group A tubes were allowed to incubate at 4OC and the separation phase (addition of uncoated charcoal and centrifugation) was performed keeping the tubes at 4OC. In group B both incubation and separation, were performed keeping the tubes at 25OC. Group C tubes were incubated at 25OC, whereas separation step was carried out at 4OC. separation at 25OC. In

addition,

temperature overnight

In group D incubation was performed at 4OC

in order to furtherly confirm the influence of the

and

incubation

on NSB, we set up four series of six NSB tubes and incubated them

respectively

at 4OC,

treated with uncoated charcoal only variable. Statistical

analysis.

statistical

analysis

15OC,

25OC and 37OC.

Al1 these samples were

at 4OC, so that incubation temperature was the

Results are given as mean t standard deviation. The was performed by following tests: Wilcoxon rank- sum test for unpaired data and linear regression analysis (12-13).

RESULTS AND DISCUSSION 1)

Influence

of plasma on the standard curve. A standard curve

set

up

in

Vol. 51, No. 5

RADIOIMMUNOASSAY

prostaglandin-free

plasma and the

suggested

by the manufacturers

Standard

curve

in

plasma

OF PLASMA TxB2

537

usual standard curve set up in buffer

as

are shown in Figure 1.

does not superimpose

the

curve

in

buffer

but

parallels

it, i.e. for each dilution of TxB2 standard higher radioactivity is observed in plasma than in buffer. This can account for the underestimation, or undetectability, of TxB2 in unextracted plasma samples when the standard reference curve is set up in buffer. The binding capacity of the antibody in the

two

values

conditions are

The differente that

a

by

an

different.

3-fold higher

in radioactivity

variation

explained

is not significantly

approximately

(~0.001)

On

the

contrary

in plasma than in

levels of the two reference curves,

in the binding of the

antibody

is ruled

impaired activity of the dextran-coated

out

can be in the

,

charcoal

presence of plasma proteins. In fact the very high levels of that the separation of bound from free TxB2 is not complete.

NSB

NSB

buffer. provided

indicate

2000

1500

P) 3 .z 1000 ài P Y c 0” 0

500 o plasma 0 buffer

+/

g 10

1

25

I

50

I

100

Thromboxane

Comparison

FIG.1 plasma;

(0)

of two standard curves:

I

I

250

500

10’00 (rm/mU

62

(0)

set

up in prostag 1andin-free

set up in buffer.

2) Assessment --of various charcoal preparations -in the separation . phase. The binding capacity of the antibody and efficiency of the separation of bound from free analyte in undiluted plasma performed with dextran-coated charcoal, charcoal-albumin in

Figure 2.

and uncoated charcoal Uncoated charcoal

at

various concentrations

at 5 g/L is more efficient

are

than dextran

shown and

Vol. 51, No. 5

RADIOIMMUNOASSAY OF PLASMA TxB2

538

albumin-coated

in precipitating

charcoal

free

TxB2 as demonstrated

by

the

lower NSB values. The efficiency is more evident the more concentrated is the charcoal concentration dependent, On the other hand a stepwise, (Figure 2). decrease in the binding capacity of the antibody is observed charcoal.

cpm

(x1001

%

10.

L

9.

I

30.

8.

x

x

7. 6.

e 20. ._ u E ._ m

m 5. v) 24. 3.

10.

2. 1.

1

aten

unco char

COi

1 WL 1 510 uncoated charcoa I

charcoal

charcoal Fig. 2 Use of various -Coating: dextran (Ij, albumin (IJ).

separation

higher

antibody.

The decrease of binding capacity is probably due to the disruption

of antigen-antibody

complexes

experimental

uncoated

charcoal,

step.

the

separation of free TxB2,

of

the

improving

these

(10-50 g/L)

in

Actually,

In

concentrations

preparations

impair the binding capacity of

by charcoal at high concentrations

conditions

uncoated charcoal at 5

g/L

even the

(7,8). concentration

seems to be the most adequate separation procedure, because it implies a minima1 stripping of bound analyte. However, NSB values, even lower, are stil1 exceeding

10% of total counts.

3) --Effect of plasma concentration ----in the assay system on binding capacity and separation efficacy. When samples are treated with uncoated charcoal the NSB values slightly increased (from 210 cpm at 390 cpm) in relation to the increase in plasma/buffer ratio (from 0% to 100%) (r=0.99, pdO.001). Differently when samples are treated with dextran-coated charcoal NSB values markedly increase (from 360 cpm at 921 cpm) with the increase in plasma/buffer ratio (from 0% to 100%) in the assay system (r=0.99 pcO.001). In comparison with the samples treated with uncoated charcoal the radioactivity for each plasma concentration is higher and the slope of the line is steeper. The divergente of the two lines indicates that the better efficiency of uncoated charcoal

Vol. 51, No. 5

RADIOIMMUNOASSAY OF PLASMA TxB2

becomes more evident The

binding

increasing

the concentrations

of plasma (p
capacity of the antibody does not show

using either charcoal the contrary,

preparation

539

significant

at any plasma concentration

when uncoated charcoal

buffer, the binding is significantly

variations

(about 33%). On

is added into the tubes containing

only

lower (p-=O.OOS).

4)

Effect of incubation and separation temperature. Results are shown in -Table 1. The procedure enKely performed at 4OC gives lower NSB and higher

binding

values

than those obtained

manufacturer's

instructions

with

and

group

separation

D

performing

the assay

(group A VS group B).

of group B with group C gives

temperature.

according

to

the

The comparison

of group A

clue

effect

on

the

of

The addition of cold charcoal to the tubes kept in an Table 1

Effect of incubation

and separation

temperature

on NSB, binding and

NSB/TOTAL COUNT ratio Group Incubation temperature

(OC)

A

-B

-C

-D

4

25

25

4

Separation temperature (OC) 4 25 4 25 ____-__-________-__-~~-~~-~~-~~--~--~~-~~-~~-~~~~~~~~~~~~~~~~~~~~ NSB

(cpm)

X 250 316 336 245 SD 10.04 ll.67 19.38 9.31 _________________________-_____-__-__-___________________________

% Binding

x

34.45

31.56

34.48

30.78

SD 1.96 1.18 1.08 1.30 __-__-________-_____-~~-~~-~~-~~-~~-~~-~~-~~-~~~~~~~~~~~~~~~~~~~~ NSB/TOTAL COUNTS % 5.9 7.4 7.9 ________________c___~_~~_~____c_____~~_~_____________________ NSB A VS B

p

eo.001

A VS C

p

CO.001

A VS D

5.8

BINDING p

CO.001

p

-=O.OOl

ns

ns

B VS D

p

-=O.OOl

B VS C

p

CO.05

ns p


p CO.001 C VS D p
bath

and the following

centrifugation

at 4OC give higher binding values

without affecting NSB values. On the contrary the comparisons of group A with group C and of group B with group D give information on the effects of incubation temperature. The in the absente of any effect on incubation at 4OC gives lower NSB values, the binding capacity of the antibody. a positive linear correlation Moreover,

was found between NSB

values

and

FWIOIMMUNOASSAY OF PLASMA TxBP

540

Vol. 51, No. 5

p-=O.OOl).At incubation temperature of 4X, incubation temperature (r= 0.88, 15oc, 25OC and 37OC NSB values were about 210, 390, 410 and 860 cpm respectively. These data could appear quite surprising as one would expect an influence of the incubation temperature on the formation of antigen-antibody and of the separation temperature on the i.e. on the binding, precipitation of free TxB2, indicated by changes in NSB values. The effect of

complexes

the lowering of separation accounted complexes and some with

temperature

for by a decrease by the charcoal.

on the binding of the antibody can

in the disruptive

capacity exerted on the

be

ag-ab

As the relationship between incubation temperature it is conceivable that at a higher temperature

NSB values is concerned,

form of aspecific binding between TxB2 and plasma proteins does occur, This hypothesis less precipitable by charcoal.

formation of complexes

could account for higher values of NSB when separation

is performed

at 25OC.

CONCLUSIONS The

results of our experiments

indicated

by the manufacturers

suggest some modifications

to

the

procedure

of the ABT assay kit so allowing direct assay

of TxB2 in unextracted plasma samples. These modifications are: appropriate dilution of plasma with assay buffer, equal in unknown

1) the samples

and in the

(5 g/L)

standard reference curve; 2) the use of uncoated charcoal

for separation method;

3) the 4OC

By using these modifications and

accuracy

temperature

of the experimental

(Table 2) remain satisfactory,

RIA in unextracted

for incubation procedure,

and separation. the

specificity

whereas the sensitivity

of

the

plasma samples improves.

Table 2 -Characteristics of the modified TxB2 RIA _______-_____-_______^__________________~~_~~~~~~~~~~~~__~_~~____~~~_~______ SENSITIVITY Detection limit: 10

PRECISION pg/mL

CV -intra-assay:

6.60%

-inter-assay:

10.10%

SPECIFICITY: Cross-reactivities

PGDP

1.70%

PGE2, PGF2 alpha

0.07%

6-keto-PGFl

alpha

0.05%

Bicyclo-PGE2

0.05%

ACCURACY

pg added: 10 25 50 100 250 500 1000 (ti= 7) pg assayed: 16 22 39 85 227 427 1071 _______---_____-__________________________~~~~~__~~~~~~_~~______~__~_________

ACKNOWLEDGMENTS This

work was supported

in part by grants from the Consiglio

Ricerche ( N. 87.00382.56 1, Project Atherosclerosis, Ministero Pubblica Istruzione ( 12.01.5625 1

Nazionale delle

Rome, Italy and from

Vol. 51, No. 5

RADIOIMMUNOASSAY OF PLASMA TxB2

541

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