A sephadex column radioimmunoassay of unconjugated estriol in pregnancy plasma

327

A SEPHADEX COLUMN RADIOIMMUNOASSAYOF UNCONJUGATED ESTRIOL IN PREGNANCY PLASMA

James E. Christner and Marion C. Fetter Ames Research Laboratory, Ames Company Division of Miles Laboratories, Inc. Elkhart, Indiana 46514 U .S . A. Received:

514174

ABSTRACT A rapid, precise, and accurate radioimmunoassay for unconjugated estriol in pregnancy plasma has been developed which makes use of the adsorptive properties of Sephadex. The estriol is extracted from plasma by adsorption onto a small column of Sephadex. After the proteins and other interfering materials are washed away, the estriol is equilibrated with a limiting amount of specific antibody. The Sephadex column serves as a means of separating the bound from the unbound estriol, the ratio of which is determined by adding to the system tracer amounts of tritium labeled hormone. The sensitivity is about 220 pg in the sample. The intra- and inter-assay coefficient of variation is S-6%. The method correlated well with one which involved purification of the estriol prior to quantitation by radioimmunoassay. INTRODUCTION The human fetus and placenta the biosynthesis

of numerous

amounts of estrogen during

together

steroids

and is responsible

The assay of urinary

used for many years for the assessment called “placental

of pregnancies

insufficiency.

complications

which fall into this category

estriol

in monitorlng

assays

Under normal conditions, three months of pregnancy.

Volwne 24, Number 3

A sudden

S

TDEOXDI

in the urine

estriol

has been

complicated

‘I The various

by

pregnancy

and the clinical value of urinary

fetal risk were reviewed the excretion

out

for the great

found in the maternal blood and excreted

the last half of pregnancy.

what is generally

form a unit which carries

by Bjoro (1).

rate rises steadily downturn

during

the last

can signal impending

September,

1974

328 fetal death.

Because of short-term

bility of excretion

rates, the determinations

samples to be meaningful complete 24-hour necessarily

fluctuations

(2) . The practical

urine specimen

on plasma,

must be done on serial 24-hour problems

are considerable,

delayed by the sample collection

the measurements

in urinary flow and varia-

the collection

of collecting

a

and the results are

period.

However,

inconsistencies

by doing

and delays

would be eliminated. Numerous reports have appeared immunoassays involve

for free plasma estriol

in the literature

(3-7) . All

describing

the reported

ratio-

assays

solvent extraction

of the sample and purification

of the extract by

either Celite or Sephadex

LH-20 column chromatography.

The values for

unconjugated consistently

plasma estriol in the last trimester of pregnancy

range from less than 2 ng per ml at the 20th week to greater

than 10 ng per ml at term and Tulchinsky 32nd week of normal pregnancy,

Recently,

numerous workers

derivatized

at carbon-6

albumin at that position

specific

radioimmunoassays

using an antibody through

carbon-6.

have reported the preparation conjugation

(8-11) . Antibodies

a high degree of specificity

prepared

and promised

which do not involve a rapid,

plasma estriol concentra-

(4) .

allowing

showed

We have developed

and Abraham found that after the

no unconjugated

tions below 4 ng per ml were found

estriol

(3-6) , fairly

accurate

to bovine serum against these antig@ns

the development purification

and precise

raised against estriol conjugated The estriol is first extracted

Of

Of

steps.

radioimmunoassay

to bovine serum albumin

from serum by adsorption

329

onto a small column of Sephadex.

The serum proteins

and some interfering

factors are then washed away, and the antibody is added and allowed to equilibrate attained,

with the hormone on the Sephadex. the antibody

effects separation

labeled

is washed from the column.

of antibody-bound

which is determined

After equilibration The Sephadex

from unbound estriol,

is column

the ratio of

by adding to the system tracer amounts of tritium-

hormone. MATERIALS

Unlabeled estriol, estradiol- 17p, estrone, and progesterone were obtained from Schwarz/Mann, Orangeburg, New York; 1,3,5 (10) -estratrien3,15a, 16a, 178-tetrol (estetrol) from Steraloids, Inc., Pawling, New York; estriol-16a-glucuronide from Ikapharm, Ramat-Gan, Israel; and estriol 3-sulfate from Sigma Chemical Co., St. Louis, Missouri. The purity of these estrogens was determined by thin-layer chromatography on silica gel G plates with at least three solvent systems; ethyl acetate, chloroformacetone (80/20, V/V) , and ethyl-acetate-hexane-ethanol-glacial acetic acid (72/13.5/4.5/1O,v/v). The spots were developed with iodine vapor. Less than 5% impurity could be detected by this method. All the above compounds except the estriol 16a-glucuronide gave only one spot. The estriol 16aglucuronide contained multiple impurities including an estimated 2% unconjugated estriol . The 6, 7-3H estriol was obtained from the New England Nuclear Corporation, Boston. It was greater than 94% pure as determined by TLC in the above three solvent systems. Other chemicals

used were reagent grade unless otherwise

indicated.

The anti-estriol antibody was obtained from Professor H. R. Lindner of the Weizmann Institute of Technology, Rehovot, Israel. The antibody was prepared by immunizing rabbits with estriol-6-carboxymethyloximebovine serum albumin conjugate and had a titer of 1: 5,000 in a radioimmunoassay using a dextran-charcoal separation technique (8) . It was stored at 4’ in undiluted form. METHODS Solutions The buffer used to dilute the antibody,

dilute plasma samples,

prepare standards, and run the assay consisted of 8.66 gm Na HP0 , 4.68 gm KH PO , 9.0 gm NaCl, 1.0 gm sodium aside, and l.Oam gelatin Knox, un f-3avo!ed) dissolved in charcoal-filtered, glass dietiUed water to give a total volume of 1 liter. The gelatin was first dissolved in a small amount of hot water before being added to the total mixture. The liquid scintillation counting solution consisted of 12 gm of 2,5diphenyloxazole (Packard Instruments, Inc.) , 0.3 gm of 1,4-bis-2- (4methyl-5-phenyloxozolyl) -benzene (Packard Instruments) , and 300 ml of Bio-Solve BBS-3 (a nonionic solubilizer obtained from Beckman Instruments) dissolved in 3 liters of scintillation grade toluene (Beckman Instruments). 6, 7-3H estriol was diluted in buffer to give about 13,000 cpm/ml . This solution was prepared fresh each day. Estriol stock standard was prepared in methanol at a concentration of 11 pg/ml. T&ris solution was stable for at least six months when stored in the dark at 4 . This solution was diluted in buffer to give a cos$centration of 22 ng/ml. The aqueous standard was stored in the dark at 4 and was used for one week. The more dilute standards were prepared daily by serial dilution in buffer. Preparation

of Columns

Small columns of Sephadex G-10 were prepared in 3 ml plastic syringe barrels (Pharmaseal Laboratories) fitted at the bottom with porous polyethylene discs (10.7 mm dia) cut from l/16 inch Vyon filter sheets (Atlas Minerals, Chemicals Division, Mertztown, Pa.) . The discs were stirred in 1%B rij-35 (Atlas Chemical Industries) for 60 minutes, blotted, and dried in a forced-air oven at 70’ for one hour before being used. Sephadex G-10 (Pharmacia Fine Chemicals) was allowed to swell in water overnight. After the fines were removed by repeated suspension in water, the supernatant water was removed as completely as possible, and the Sephadex suspended in 0.5 volume of the buffer twice and one volume once. The Sephadex was allowed to settle overnight in a graduated cylinder, the supernatant buffer was removed, and an equal volume of fresh buffer was added to give a 50%suspension. , While the suspension was being vigorously stirred, 2 ml of the slurry After the excess buffer had drained, was pipetted into each syringe barrel. the columns were ready for use. The columns were prepared each day. Procedure

for Radioimmunoassay

of Estriol

Estriol standard or plasma (0.2 ml) was added to 2.0 ml of the 6, 7-3H estriol solution, and 1 ml of this dilution was pipetted onto the Sephadex column. After the sample had flowed into the Sephadex, the

column was washed with 4 ml of distilled water. The syringe outlet was capped, 1 ml of buffer was added, and the Sephadex gel bed was thoroughly mixed with a Vortex mixer. The outlet was then uncapped, and the buffer allowed to flow out. The 6 ml of effluent collected to this point was discarded. The syringe was placed over a test tube, and 0.25 ml of appropriately diluted antiserum was added. After the antiserum had penetrated the gel bed, the column was allowed to stand for 1 hour at room temperature. The column was then washed with 1.75 ml of buffer. The effluent was mixed and 0.5 ml was pipetted into a scintillation vial along with 10 ml of scintillation fluid and 0.05 ml of 4% SnC12 in 0 .lNHCl . Standard curves were constructed relating the fraction bound (FB) to the cold estriol concentration where FB = Net cpm in sample Net cpm in absence of added estriol’ A dilution of antibody was used that would bind 40% of the tritiated estriol in the absence of added cold estriol . Because the gel bed is thoroughly mixed before addition of the antibody, about 8% of the added radioactivity was nonspedfically eluted along with the antibody-bound estriol. Routinely, the nonspecific counts were not subtracted in making the above calculations. RESULTS Extraction

Efficiency

In order to obtain the most reproducible extracted possible.

results,

from the sample onto the column as efficiently Of the added radioactivity,

of effluent with both the standards

the estriol must be and consistently

as

5.1% (f 0.3%) is lost in the first 6 ml

and the plasma samples.

No correction

was applied for this loss. Precision

and Sensitivity

of the Standard

The results from 22 standard curves pooled to give the results shown in Figure Figure deviation intra-assay

Curve run on separate days were 1 and Table 1.

1 shows the mean fraction bound and the intraassay

obtained from 22 standard curves standard deviation

run on separate days.

(Table 1) was calculated

standard The

from the differences

between the duplicate pg/ml

level,

determinations

the coefficient

The sensitivity

of variation

is about 5%.

which gives a response

of the fraction bound at zero concentration

to an estriol concentration lowest detectable

of about 20 pg.

concentration

would give correspondingly

TABLE I.

Mean F.B.

0

1.00 0.874 0.782 0.648 0.504 0.387 0.311

2.

3.

Two

mrrespond

Since a 1: 10 dilution is used,

the

plasma samples.

Less dilution

greater sensitivity.

Inter- and Intra-Assay

Standard Concentration (ng/ml)

(12).

in the sample would be 220 pg which is more

than adequate for last trimester pregnancy

1.

which is

different from that given by the zero concentration

standard deviations

0.063 0.126 0.252 0.505 1.01 2.02

At and above the 126

of the assay method is defined here as the detection

limit -- the smallest concentration significantly

at each level.

Precision

of Standard Curve

InterAssay S.D.’ (kF.B.)

IntraAssay S.D.2 (+F.B.)

Intra-Assay S.D. in ng/ml’ (? ng/ml)

0.018 0.023 0.018 0.013 0.011 0.012

0.024 0.013 0.006 0.010 0.009 0.005 0.006

0.007 0.006 0.013 0.021 0.030 0.115

Intra-Assay C.V.

11.1 4.8 5.2 4.2 3.0 5.8

Calculated from the means of the duplicate determinations done on 22 sets of standards. The pooled standard deviation from the mean of duplicate determinations done on each standard each day: 2 S.D. = + Jwhere d = difference between duplicates, and n = number of pairs (= 22) . The variation in ng/ml was read off the standard curve in Figure 1.

S

333

x'~EOLD-

0.9

0.8

0.7

0.4

0.3 t

1I

163

Figure

1.

0.126

1 I

t1

0.252 0.505 E3 Concentration (ng/ml)

I

1.01

I .-

2.02

Standard Curve. The points correspond to the means of 22 The bars correspond to the pooled standard standard curves. deviations from the mean of the duplicate determinations (see Table I) .

Inter-

and Intra-Assay

Precision

of Analyses

To determine the inter-assay performed

on each of five days.

with the intra-assay coefficients TABLE II.

Pregnancy Plasma #

192

223

Also, differences The results concentration

precision

of variation

precision,

of Plasma Samples five replicate assays were

The results are shown in Table II along for each day.

The inter- and intra-assay

were both in the 4-6% range,

Inter-assay Precision

of the Determination of Plasma Estriol

Date

X (ng/ml)

4/3/73 4/4/73 4/5/73 4/6/73 4/g/73

4.94 4.86 5.42 5.06 4.92

0.313 0.270 0.110 0.219 0.217

6.34 5.56 2.02 4.33 4.41

Inter-assay

5.04

0.225

4.45

4/3/73 4/4/73 4/5/73 4/6/73 4/g/73

11.1 11.0 10.7 10.5 9.96

0.475 1.74 0.449 0.507 0.709

4.29 15.8 4.20 4.84 7.12

Inter-assay

10.7

0.454

4.27

the intra-assay between

standard

the duplicate

are shown

in Table

deviation

results III.

S .D.

was calculated

obtained

The coefficient

range of O-31 ng/ml was 5-6%.

(ng/ml)

C.V.

(%l

from the

on 68 plasma samples. of variation

over

the

335 TABLE III.

Intra-Assay

Precision

of E3 Determinations

Done on Pregnancy Concentration Range

N

Mean (ng/mll

0.0 5 10 15

7 22 22 17

2.12 7.13 12.3 19.4

1.

-

4.9 9.9 14.9 31

See Table

Various

11.0

0.107 0.419 0.612 1.11

5.0 5.9 5.0 5.7

added to aliquots

and each level

IV, good

recovery

of a pool of

was assayed

in tripli-

and reproducibility

were

at each level.

TABLE N. E3 rig/ml Added

were

individuals,

As shown in Table

obtained

C.V. (%)

of Added Estriol

amounts of estriol

plasma from pregnant cate.

S.D.’ (+ ng/mll

I

- Recovery

Accuracy

Samples

Recoveries

of E3 Added

E3 ng/ml Recovered 13.0 11.9 12.4

to a Pregnancy

Plasma Pool

% Recovery

Average

118 108 113

113%

5.50

5.26 5.70 5.43

95.7 104 98.7

99.5%

2.75

2.62 2.62 2.84

95.3 95.3 103.

97.9%

1.38

1.30 1.36 1.58

94.3 98.3 114. Overall

102%

Average

=

103%

Aliquots

of the pregnancy

solution

mntaining

analyses

plasma pool

the indicated

were

amounts

were then done by the standard

estriol

concentration

Accuracy

The accuracy

samples

estriol

is purified

of the Sephadex

had been assayed by Celite

fled by FUA using 39 samples

With a Reference

of plasma samples

These

at different

“parallelism”

since the sample

This indicates

other

potentially

the test.

Such parallelism step was omitted. in the serum.

Method

from Dr. Dan Tulchinsky.

antiserum

(4) ,

2.

This is termed the standard

was obtained

that the various

components

steroids,

could not be obtained

curve.

over a 20-fold of serum,

do not interfere

This was found to be due to the presence

In the absence of serum proteins,

the estriol was spread

over a much larger

in distribution

resulted

in a difference

the antibody.

Therefore,

differences

of

the estriol bound while in their

band.

in the conditions in binding

in

when the gel-bed

to the column in a narrow band at the top of the column, presence,

on the

of the method to give the

is paralleling

cross-reacting

it is quanti-

The correlation

of the sample.

curve

before

is shown in Figure

is the ability

dilutions

including

proteins

The endogenous

obtained

As shown in Table V, good parallelism

mixing

procedure.

The

column FUA method was also tested

from Dr. Tulchinsky

test of accuracy

range.

estriol.

column chromatography

same results

dilution

of unlabeled

by a RIA method in which the extracted

a nonspecific

obtained

Another

1: 10 into 3H-E3

in the pool was 3.54 ng/ml.

- Comparison

by the analysis

diluted

This difference

of equilibration

were observed

among

with

s

337

YrmRoxDm

y = 1.02x,+1.23 r = 0.9144 Sy= f 2.06

4

8 E3 (ng/ml)

12 Tulchinsky

16

20

Comparison between results obtained by the Sephadex column RIA method and results obtained by Dr. Tulchinsky on 39 plasma samples from pregnant women.

solutions

containing

the gel bed,

various

concentrations

the equilibration

so that buffer standards

conditions

of serum proteins.

By mixing

for all samples become identical

could be used with plasma protein-containing

samples. TABLE V. Plasma No.

Parallelism

* Dilution’:

Between

Pregnancy

Plasma and Buffer

Standards

None

l/2

l/4

l/8

l/20

Mean

S.D.

C.V.

104 105

5.3 20.4

5.0 20.9

4.8 21.0

5.1 22.8

5.1 21.1

0.21 0.96

4.1% 4.5%

152

19.8

21.4

21.9

21.6

20.6 18.4 25.3

22.0

2.02

9.2%

1.

The extent to which the plasma was diluted with buffer diluted 1: 10 into the 3H-E3 solution.

before

being

Specificity The cross-reaction known to be present determined. the estriol account

with certain

in pregnancy

for the observed

contained

in Table VI. unconjugated

quantities

Gross-Reaction

estriol

Estriol Estradiol- 178 Estrone Estetrol Estriol-3-sulfate Estriol- 16a-glucuronide Progesterone Reaction

of Various

which

Steroids

% Cross

Steroid

=

was

As stated previously,

cross-reactivity.

TABLE VI.

% Cross

and neutral steroids

plasma in appreciable

The resuIts are shown 16a-glucuronide

phenolic

Reaction

100 0.63 < 0.3 6.6 1.6 1.7 C 0.06

ng of estriol giving 0.5 FB ng of test steroid giving 0.5 FB

could

It had been noted during results

were

obtained

goes

with pregnancy

cross-reactivity, Therefore,

up 3-fold.

material being

removed

known that charged strength

solutions

reactivity

plasma samples

if the 4-ml water

the extent of reaction

it is likely

while

at least part of the interfering

are removed

the uncharged

16a-glucuronide

from Sephadex

species

It is

is due to the contamination

by low ionic

remain adsorbed

cross-reactivity

of the water wash which

of

with the antibody

by the water wash is estriol-3-sulfate.

molecules

The extent of estriol the omission

of the test that high

If the water wash is omitted in the determination

wash was omitted. estriol-3-sulfate

the development

confirms

(13).

is unaffected

by

the idea that its cross-

by estriol.

DISCUSSION Through

the use of the adsorptive

linked

Sephadex

estriol

has been simplified

reproducibly

G-10,

the radioimmunoassay

extracted

use solvent

extraction

but often introduces for extraction

considerably.

which

Also,

By washing

from the column.

compounds

are ionizable

derivatives

is used to separate

siderably

the Sephadex

simpler

eliminating

Presumably,

of estriol.

radioimmunoassay

to run than conventional

methods,

the need to

to correct cross-reacting

these interfering

Finally, estriol

and

cumbersome,

it is not necessary

the antibody-bound column

cross-

is efficiently

the column with water,

are removed

Although

The estriol

not only is manipulatively

interferences.

losses.

of the tightly

of plasma unconjugated

from the plasma thereby

derivatives

column

properties

the Sephadex

from the unbound. method is conthe precision

is

equal to or better

than that reported

extract

from the plasma and dextran-coated

the estriol

separate bility

the antibody-bound

of the present

extraction, entirely there and,

test would

purification,

within one container

since

identical

the standards

conditions,

However,

1: 1 (V/V) .

women

unconjugated

Also,

by organic

losses

solvents,

are necessary.

procedure

extracted

is adequate of pregnancy.

from plasma that

the sensitivity

which would

the

could be ex-

allow the use of the test to

in plasma from normal non-pregnant

(7). The accuracy

estriol

was determined

added to pooled

recovery

was obtained

physiological

column before

of values.

at concentrations

involved

purification

Excellent

The slight

positive

the recovery

from pregnant covering

the Sephadex

it was quantified

antibody.

by determining

plasma obtained

Also,

values.

a method which

specific

Therefore,

estriol

required.

the last trimester

is efficiently

since

are run under essentially

for extraction

in plasma during

by at least five-fold,

measure

being

of the test with the present

estriol

to

steps are done

introduced

and plasma samples

we found that estriol

has been diluted

with

interferences

to

The reproduci-

to be improved

with no transfers

no corrections

The sensitivity for unconjugated

be expected

use solvents charcoal

from the unbound.

and antibody-equilibration

are no unpredictable

tended

tracer

for methods which

the expected

Good range of

column method was compared of the estriol

by radioimmunoassay

correlation

women.

of

was obtained

bias may indicate

on a Celite

using

a non-

over

a broad

the presence

range

of un-

S known

estrogens

cross-reacting

water-wash

remaining

the greatest

tested for cross-reactivity, The estriol: estetrol

extent.

mester of pregnancy

is about 8 (6) .

concentration

be increased

should

sulfate is present that of estriol

step.

would

The contribution will

Although fete-placental

estriol

estriol (2))

function

unconjugated

within two hours.

Therefore,

is observed,

should

is tested.

been used to assess many potential

rapidly

Also,

be quickly

downturn

determination

uncon-

(4) and any reflected

in 0.1 ml of pregnancy

if a slight

coula

column method

column radioimmunoassay

estriol

a second

estriol

to the estriol

of sample collection. very

2.4 times

sample purification

until a pure standard

from the blood

is able to measure

day.

a separate

has traditionally

The Sephadex

estriol-3-

estrogens

of the Sephadex

the assay in plasma offers

in fete-placental

concentration.

concentration

results

such as ease and rapidity is cleared

estriol

Since

the apparent These

to

the last tri-

the apparent

of estriol-16-a-glucuronide

urinary function

estriol

deterioration

l-6%,

by about 4%.

remain a question

reacted

plasma at a concentration

to the method incorporating

concentration

jugated

higher

ratio throughout

Therefore,

reacts

be increased

for the somewhat

advantages

after the

estetrol

by no more than 1%.

in term pregnancy

(14) and it cross

concentration

as compared

on the column

step,

Of the estrogens

account

341

!rB~OXDrn

in the

method plasma

in plasma estriol can be run the same

~.4RrtorxMII

342

Although

the data presented

estriol,

the procedure

of other

compounds

investigating

these

described such

here

relates

to the assay

may be generally

as steroids

of free plasma

applicable

and hormones.

to the assay

We are currently

possibilities. ACKNOWLEDGEMENTS

The authors

are grateful

for Women for his kind values Lindner

for unconjugated for his generous

assistance estriol.

to Dr.

D. Tulchinsky

in supplying We would

gift of anti-estriol

plasma

of the Boston samples

also like to thank

Hospital

and reference Professor

antiserum.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

13. 14.

Bjoro, K., ACTA ENDOCR., Suppl. 161, 5 (1972) . Klopper, A., AMER. J, OBSTET. GYXC, 107, 807 (1970) . Lipsett, M. B., Loriaux, D. L . , Ruder, H. J., Knab, D. Rxnd J . CLIN. ENDOCR . 35, 887 (1972) . Tulchinsky, D. and Abraham, G. E., J. CLIN. ENDOCR. 33, 775 (1971) . Wu, C. H. and Lundy, L. E., STEROIDS Is, 91 (1971). E., J. STEROID Giebenhain, M . E . , Tagatz, G. E. and Gurpide, BIOCHEM. 3, 707 (1972) . Powell, J. E. and Stevens, V. C., CLIN. CHEM. l9_, 210 (1973) . A. and Zeitlin, A., Lindner, H. R., Perel, E., Friedlander, STEROIDS 19, 357 (1972) . S. J. and Wotiz, H. H., STEROIDS 21, 259 Walker, C. S., Clark, (1973) . Kuss, E . and Goebel, R. , STEROIDS 19, 509 (1972) . D. C. and Preedy, J. R. K., STEROIDS 21, Wright, K., Collins, 755 (1973) * Feldman, J . and Rodbard, D., PRINCIPLES OF COMPETITIVE PROTEIN-BINDING, ASSAYS, Editors, W. D. Odell and W. H. Daughaday, Lippincott Co., Philadelphia, 1971, p . 158. Janson, J. C., J, CHROMATOGR. 28, 12 (1967) . Smith, 0. W. and Hagerman, D. D., J. CLIN. ENDOCR. 25, 732 (1965) .