Selection of high-affinity and high-specificity monoclonal antibodies for 1α,25-dihydroxyvitamin D

SELECTION OF HIGH-AFFINITY AND HIGH-SPECIFICITY MONOCLONALANTIBODIES FOR la,25-DIHYDROXYVITAMIN D E. Barbara Mawer, Jacqueline 1. Berry, Juana Bessone, Shraga Shany, h University of Manchester Medical School, Department of Medicine, Manchester Ml3 9PT, U.K. and *Department of Chemical Pathology, Hope Hospital, Salford M6 8HD, U.K. Received (Revised

7-31-85 10-21-85)

ABSTRACT

The preparation of high-affinity and high -specificity monoclonal antibodies to Ia,2&dihydroxyvitamin D is described. Monoclonal antibodies were derived from Balb-c mice imnunised with either la-hydroxy-25,26,27acid or trinor-24-cholecalcioic with la-hydroxy-26,27-dinor-cholemouse calciferol-25-oxime, and spleen cells were hybridised with myeloma cells. From six fusions nine monoclonal antibodies (MAb's) were selected from 676 antibody-secreting hybrids. Antibodies varied widely in their ability to bind la,25-dihydroxyvitamin D3 (50% displacement of radioligand ranged from 25 - 900 pg); two had particularly useful characteristics for la,25#Qdroxyvitamin D assay. MAb 5F2 has high affinity (Ka = 1.39 x IO M I and does not discriminate between la,25-dihydroxyvitamin D and D , thus enabling the two forms to be measured together. &b 163 is highly specific, having no crossreactivity with 25-hydroxy-, 24,25-dihydroxyor 25,26-dihydroxyvitamin D at concentrations found in normal human serum; this MAb has the potential to eliminate lengthy extraction procedures the involved in currently available assays for la,25-dihydroxyvitamin D. INTRODUCTION Assays currently vitamin

D,

by the

cross-reacting

food

need

to

of vitamin

August,

present

September

1985

metabolite

(1,25(OH)2D)(1). preparation at

between

in

serum

necessary

higher

to

concentrations, D

An

being

being

1,25(OH)2D3 Steroids

the

from

problem

therapy form.

do not

Volume

remove

derivat-

endogenous

assays

are

(24,25(OHj2D)

additional

derived

of

namely

the la,25-dihydroxylated

former

and the latter in use for

hormonal

(25.26(OHj2D).

D2 and D3, the

antisera

active

24,25-dihydroxyvitamin

D

distinguish

supplementation

polyclonal

sample

D (25(OH)D),

25,26-dihydroxyvitamin

ives

the D

extensive

metabolites

25-hydroxyvitamin

is the

for

la,25-dihydroxyvitamin

complicated

and

available

or Most

cross-react

46, Numbers

2,3

with

binding

assays

varying

equally

other

ing highly

specific

and provides

these methods

for

the

clones

antibodies

to problems

supply

to produce

sensitivity

would

in

cross-react

have the sensitivity

to

or no cross-reactivity

D2 or D3.

The elegant

technique

production

of monoclonal

antibodies

in culture

for

paper we describe

the required

1,25(OH)2D2

1,25(OH)2D

and show little

assay development

In this

MAb's with

(4)

already

in protein-

and presents

and 1.25(OH)2D3,

of vitamin

a constant

improving

(5,6).

the assay of

metabolites

by hybrid

with

is tedious

concentrations,

secreted

(t&b’s)

employed

(2,3)

for

and Milstein

receptors

cross-react

preparation

1,25(OH)2D2

picomolar

of Kohler

thus

and

antibody

with

detect

1,25(OH)2D3

reproducibility

An ideal

with

for

extents,

between-batch

1,25

some of the tissue

1,25(OH)2D2;

the

offers

radioimnunoassay

of antibody and precision. MAb's to

of

of defined

1,25(OH)2D

characteristics,

One of us has exploited

progesterone

the selection and

the means of select-

and testosterone

and characterisation

specificity

for

the

of

assay of

(OH)2D. EXPERIMENTAL

Reaaents Unlabelled 25(OH)D3 was obtained from Roussel, Paris, 25(OH)D2 from Upjohn, Kalamazoo, 24,25(0H) D3 from Roche Products Ltd., 25,26(0H) D from Dr. J. Redel, Par ? s, and 25,26(OH)2D2 from Dr. 0. Willia&s~ Cambridge England. 1,25(OH) D and 1';~;;;;2D3N J ant were kindly suppliid by Hoffman La R&Be Inc., la-hydroxycholecalciferol from Leo Laboratories, Princes Risbo;ough,'U.K. All vitamin D sterols were stored under nitrogen at -20" and concentrations for standard solutions were determined by U.V. spectrophotometry, using the apprgpriate extinction coefficients (X max 26! nm). 1,25(OH) [26,27-methylHID (180 Ci/nanol) was purchased from Amersham Inte 6 nationa1,U.K. la-iydroxy-25,26,26-trinor-24-cholecalcioic

S

TPIIOXDD

743

acid, kindly provided by Dr. Milan Uskokovic and la-hydroxy-26,27-dinorcholecalciferol-25-oxime, given by Dr. L. Fraher, were used to prepare imnunogens. Bovine serum albumin fraction V (BSA) was purchased from Sigma,U.K. The buffer used in the antibody evaluation procedure was 0.07 M sodium phosphate, pH 6.0,and contained 0.1% gelatin, unless specified to the contrary. Dulbecco's phosphate-buffered saline (without calcium was purchased from Flow Laboratories, Rickmansworth, and magnesium) U.K. Normal mouse serum used in the second-antibody assay method was obtained from non-immune Balb-c mice. Sheep-antimouse I gG- antiserum was produced by imnunising a Kerry-Gritstone cross-bred ewe with 500 clg mouse IgG (Sigma, U.K.) at monthly intervals and collecting serum lo-14 days after the last injection. Synthesis

of Imnunogens

(Fig.

1)

-la-Hydroxy-25,26,27-trinor-24-cholecalcioic acid (la(OH)D3-acid) and la-hydroxy-26,27-dinor-cholecalciferol-25-oxime (la(OH)D -oxime) were each conjugated to BSA using the mixed anhydride reastion (7). Ultraviolet spectroscopic analysis indicated that the complexes contained 36 and 30 steroid residues per mol BSA, respectively.

lwhydroxy-25,26,27-trinor2kcholecalcioic

Fig.

1.

Structures

la-hydroxy-26,27-dinorcholecalciferol-25-oxime

acid

of the immunogens.

Immunisation Balb-c mice received S.C. injections of 100 pg of the BSA conjugate of either la(OH)D -acid or la(OH)D -oxime emulsified in Freund's complete adjuvant fallowed by multiple3i.p. injections of 10 ug conjugate Four days prior in Freund's incomplete adjuvant at monthly intervals. to the fusion, 10 ug conjugate in phosphate-buffered saline was given i.v. to the mouse selected on the basis of high antibody titre or of high specificity for 1,25(OH)2D3. Hybridization

and Antibody

The method for preparation in detail elsewhere (4,5).

Production of monoclonal antibodies has Briefly, spleen lymphocytes

been dgscribed (2 x 10 cells

S

744

?P-EOXDI

were fused with 1 x lo7 P3-NSI/l-Ag4 mouse myeloma cells (donated by Dr. C. Milstein) using polyethylene glycol 4000 (Merck, Darmstadt) and fused cells were distributed into 6 x 96 well plates (Costar, layers' of non-immunised mouse Northumbria Biologicals5Ltd.1 on 'feeder After 6-10 days, hybrid colonies were spleen cells (1 x 10 /well). tested for antibody secretion and positive cultures were cloned at least twice by limiting dilutiqn (8). Hybrids chosen for further study were injected i.p. (1 x 10 cells) into pristane-primed Balb-c mice and antibody in ascitic fluid was isolated lo-14 days later; the antibody was stored at -20'. Antibody

Detection

Assay

A preprecipitated double-antibody assay was performed as previously described for progesterone (4) to detect antibody in culture medium from hybrid cells. Briefly, 100 ul culture medium, mouse antiserum, or ascitic fluid was incubated with 50 ul sheep-antimouse IgG-antiserum (1:20) and 50 ul normal mouse serum (1:400) for 18 h at 6". The antibody complex was precipitated by centrifugation at 1700g for 20 min and resuspended with 2503u1 sodium phosphate buffer plus 10 ul ethanol containing 13 fmol [ H]- 1,25(OH) D . After 2h at 6' the samples were diluted with 500 ul ice-cold phosshate buffer (pH 6.0, 0.07 M) and bound fractions were separated and counted for radioactivity. Assessment

of Antibody-secreting

Hybrids

Hybrids were classified as antibody-secretors when binding of C3H]-1 25(OH) D was greater than 10% above that in lymphocytes culturid as c&&ols. Where there were large numbers of antibodysecreting hybrids, a preliminary assessment of sensitivity and specificity was carried out to select the better antibody-secreting hybrids for large scale production in vivo. Dilutions of antibody were chosen to give 25% binding of C3RR(OH) D and were incubated with sheep-antimouse IgG-antiserum as des#bed above. Displacement of radioligand by li25(OH)2Da was compared with displacement by at least 4 concentrations of e ch potentially interfering metabolite in the appropriate range 6-100,OOOpg per tube. Incubation and separation were performed as described above. Detailed

Assessment

of Monoclonal

Antibodies

in Ascitic

Fluid

Titres were established by diluting ascitic fluid with phosphate buffer containing no gelatin. Sensitivity and specificity were determined from full displacement curves derived from all available vitamin D derivatives (see Reagents) using the preprecipitated doubleantibody method described above. 8 at a given titre was defined as 4e mass of 1,25(OH) D in pg/tube r8[ uired to displace 50% of bound [ HI-1,25(OH) D . 'S$ecificity was expressed in terms of crossreactivity, c Elsulated according to Abraham (9) from the mass of each vitamin D sterol required to displace 50% of bound C3H]-I 25(DH) D Affinity constants were calculated by the method of S&chard'(%)) as modified by Chamness and McGuire (11).

S

745

TlilEOXDI

RESULTS Six fusions

were

acid

3

and

results

are

fusion

varied

itmnunogen,

performed,

derived

was the

produced

titre

hybrids

from produced

selection

each group of

produced

after

of

large

antibodies

the

using to

the

the

same

number of

The most striking

total,

hybrids

compared

of

with

antibody-secreting

6 necessitated

based on

the

antibody-secreting

number

1, 2 and

la(OH)D3-

at the time of

fusions

the fusion.

8-90% of

fusions

titre

relationship

proportion

The

with

la(OH)D3-oxime;

Mouse antibody

a direct

oxirne.

of the better

with

within

higher

in

inmtunized

1.

la(OH)D3-acid, the

mice

ionization

bore

much

with

Table

hybrids

with

0.3-1.0s

in

widely,but

antibody-secreting result

from

shown

the

3

their

a

B50

preliminary

and on

their

specificity. TABLE 1 DETAILS OF FUSIONS AND HYBRIDS PRODUCED IMMUNOGEN

la-hydroxy-26,27-dinorcholecalciferol-25-oxime

la-hydroxy-25,26,27trinor-24-cholecalcioic acid

FUSION

1

Ab TITRE AT FUSIONa NO. HYBRIDS PRODUCED (MAXIMUM 576 PER FUSION) NO. Ab-SECRETINGb HYBRIDS NO. STABLE MONOCLONAL ANTIBODIES CHARACTERISED

6

2

1:450

1:3000

3

1:15000

4

1:6000

5

1:2000

1:2500

533

399

576

478

573

572

42

104

520

5

3

2

9

2

1

0

1

2

a.

Titre (dilution of Ab binding 50% [3H]-l,25(OH)2D3) antiserum from mouse at the time of fusion.

b.

Binding

>lO% above lymphocytes

cultured

as controls.

of polyclonal

746

?E-EOIDI -

=

Fig,

2

secreted

by hybrid

having

no

preliminary

2F5 from fusion

cross-reactivity

25,26(OH12D3 The

the

shows

even with

antibody

the extent

with

characterisation 6.

with

of 22 and 1.3X,

antibody

was highly

specific

24,25(OHj2D3

25(OH)D3,

concentrations

cross-reacted

The antibody

of an

and

of 94, 23 and 29 ng,respectively. and lu-(OHID

1,25(OHj2D2

respectively.

The B5D for

to

[3H]l,25(Otl)2D3

was 85 pg.

lOOByBO 80-

60-

20-

0 10° Fig.2.

10'

Displacement antibody.

From

curves

the preliminary Tables

the imnunogen

laIOH)D3acid had little

able cross-reaction

to vitamin

selection,

characterisation,

B50, but it

I lo3

lo2

2 and 3. (Table

other

D metabolites

nine MAb's From MAb's 21, 3Ell

cross-reactivity with

d lo'p#tube

vitamin

with

10'

using

hybrid

were chosen for produced

from fusion 1,25(OH)2D2

D metabolites;

in

2F5

further

response

to

1 had the lowest and

consider-

208 from fusion

2

747

TABLE 2 CHARACTERISATION OF MONOCLONALANTIBODIES DERIVED FROM IMMIJNISATION WITH la-HYDROXY-25,26,27-TRINOR-24-CHOLECALCIOIC ACID 1

1

1

2

2

6

3Ell

lH6

4Fl

3F6

2D8

167

1:25000

1:2500

1:5500

117500

FUSION MONOCLONALANTIBODY TITRE" DISPLACEMENT(B50)

b

40

270

700

900

1: 45000 1: 20000 60

80

SPECIFICITYC 1,25(OW2D3

100

1,2510H12D2

13

25(OH)D3

100

100

100

100

100

82

51

45

58

6

9

20

12

15

0

24,25(OH)2D3

11

21

63

75

27

0

25,26(OH)2D3

25

68

73

la(OH )D3

0.6

a.

The dilution

of antibody

b.

The concentration radioligand.

of 1,25(OH)2D3

C.

Cross-reactivity,

% compared with

0.7

needed to bind

4

0.6

0 0.5

2

50% C3H] 1,25(OH)2D3.

in pg required l,25(OHj2D3.

to displace

50% of

h h also

possessed

other

the

metabolites

with

of

characteristic

vitamin

antibody

of no cross-reactivity

03, a behaviour

which

2F5 from

fusion

from 2F5; however,this

cell

did not survive).

had also

been detected

in the polyclonal

used for

the fusion

or

hybrid

useful

24,25(OHj2D3.

(Table

3)

fusions

which

gave

MAb

3A8 (fusion

MAb's with

high-affinity

antibody

interesting

because of its

For comparison,data and

for

from

mice

lower

the

similar it

polyclonal

a calf

thymus

antibody

than either

greatly

superior

the polyclonal

has

those

for (12)

1,25(OH12D2

antibody

with

being

the

particularly

1,25(0H)2D2. a

sheep

l

polyclonal

currently

use for

in

31 has a similar

B50

cross-reaction

interfering

or the receptor.

25(OH)D3

5,

MAb167 [Table 2) has a higher

cross-reactivity

mouse

from

from fusion

MAb 5F2 (Table

and

the

la(OH)D3-oxfme

with

with

Table 3

receptor

of with

to

B50 's resulted

shown in

to the thymus receptor.

has

itmnunized

5F2 (Ka = 1.39 x 1O1' M-l)

are

produced

These characteristics

reactivity

high cross-reactivity

observed

(no MAb was

41, hhwas similar

1,25(DHj2D assay in our laboratory. to

was also

serum antibodies

had shown no cross-

Fusions

1 and 2.

antibody

line

6 (Fig.21

with

B50 but

metabolites

S

749

?faIrorr,m

TABLE CHARACTERISATION

OF MONOCLONAL ANTIBODIES DERIVEDFROM IWJNISATIONWITH la-HYDROXYCHOLECALCIFEROL-26.27-DINOR-25-OXIF1E ~._ _ _______ . -.-._- .._ - __. _- - _

FUSION

4

MONOCLONAL

3

ANTIBODY

5

3A8

TITRE'

1:2000

DISP~ACEMENT~B50)b

5

5F2

Calf e Thymus Receptor

Sheep d polyclonal

6012

1:100000 1:60000

1:200000

1:6

100

25

43

23

1,25KW2D3

100

100

100

100

1,25WH)2D2

29

95

22

2

119

25(OH1D3

15

8

13

30

0.2

24,25(OHj2D3

43

11

27

21

0.05

43

16

24

3

2

9

SPECIFICITYC

1

a (OHlO

a. The

dilution

of

b. The concentration of radioligand.

of

c.Cross-reactivity,

d. Raised conjugate, (kindly e.Prepared

antibody

needed 1,25(OH)2D3

X compared

with

0.05

4

0.6

to

bind in

100

50%

pg required

C3H] to

0.1

1.25(OH)2D3 displace

50%

1,25(OH12D3.

in

sheep to la,25-dihydroxycholecalciferol-25-hemisuccinate-~SA Currently in use in our laboratory to assay donated by Dr. J. O'Riordan). in

our

laboratory

1.25(OHj2D2simultaneously.

and

used

to

assay

1,25(OH)2D,

1,25(01i)2D3

J

and

Mb 5F2 was selected developlnent curves

for

of an assay for

in the

further

investigation

1,2510Hj203

The curves

useful

range for

assay of

ligand

at 25 pg/tube.

a view to the

and 1,25(OHj202.

range 1.25 - 160 pg/tube

are shown in Fig.3.

with

for

these

are virtually

1,25(OH12Dwith

Standard

two vitamin

superimposable

D sterols and cover

50% displacement

of

bound

80-

80.

40.

201,2502 1,25D,

OI

l-25

Fig.3.

2.5

5

10

Standard curves t0 1,25(Oh)2D2 at a dilution of 1:lOOOOO.

20

40

and

80

a

180pg/tube

1,25(OH)2D3using MAb5F2

751

DISCUSSION

The measurement binding

present

extraction

to prepare

for

that

difficult

monoclonal

to

antibodies

nine

stable

standardize.

a series

monoclonal

lengthy

assay.

with

There

is

I,26(OH)2D2

and

proteins

are tedious

In this

paper we

have shown

to overcome

these

of 676 antibody-secreting

antibodies

related

receptor

have the potential

and have produced

to the

equally

measurement;

receptor-

necessitating

prior

cross-reacts

or

of structuraTly

concentrations,

techniques

use in routine

and

by radioinrnunoassay

the presence

in much higher

antiserum

1,25(OH)2D3

ulties

by

and HPLC separation

no available

which

1,25(OH12D

assay is complicated

metabolites

that

of

diffic-

hybrids

to 1,25(OH12D3

have

from

been char-

acterised. Two different

insnunogens were compared

but the characteristics to

the imnunogen

produced

from

reactivity

with

specificity

of the resultant

used. the

same

30

immunised

cross-reactivity illustrates antibodies the

and

showed

major

in finite

produced

on

the

advantage

quantity a large

of

the

specificity

167.

polyclonal

being

in a rare

animal

D

antiserum

Antibodies

of very

metabolites. technique

animals produced. can

The

one mouse from more

monoclonal

number of

were cross-

characteristic

antibody

scale.

not be related

to 0% with

this

vitamin

in a large

a suitable

4Fl

only

desirable

of HAb's

la(OH)D3-acid,

of the

however,

interfering

can be raised of

using

to that

could

differing

from 20% with

the fusion;

with a

chances

produced

of widely

of MAb 167 was similar

the preparation

antibodies

inznunogen;

25(OH)D3 varied

of the mouse used for than

Antibodies

in

thus

This in

that

increasing

Such an antibody

then be

produced

low

from

imnortalised imnunisation

S

752

h(OH)Dj-oxime

~4th similar 5F2

also

to the majority

was

produced

T331OXDI

varied

in

of those

which

resulting

differed

cross-reactivity

same

imnunogen

differed

widely

while

showing

broadly

similar

is

with

difficult,

relationship

also

specificities,

therefore,

between

to

the ilrrnunogen

were

but MAb the desired

derived

from

the

from

fusion

1,

had displacement

that

been

confer

predicted

specificity

Ia(

which

reactivity

on

antibodies

substituted

feature

that

values

conclusions

antibodies

the sterol

MAb.

the

However,

substituents,

metabolites

cross-reacted

with

the

it

might

of the sterol synthetic

and

would

derivative little

crbss-

conversely,

strongly

except

region

was an

sidechain

the Both

chain,and

showed very

imnunogen,

on

produced.

side

A ring

from either

interaction

firm

the unrestricted

has no sidechain

with

suggesting

the

draw

and the

have

side-

with

167,

important

of most of the antibodies. The

most

striking

of antibody-secreting hybrids

fusions

that

has yet

which

the

total

resulted

10 from oxime fusions;

number

of

from la(OH)D3-

no explanation

for

this

been found.

a rapid

early

from the vitamin

produced

screening

second-antibody

might

Although

666 secretors

number of hybridomas

preprecipitated interference

only

in the innnunogens was in the number

produced.

in each case,

but

The large

difference

hybrids

was similar

difference

media

in having

in B50; e.g.,those

to B5A through

meant

3A8,

from lu(OH)D3-acid,

fundamentally

inmaunogens were linked

acid

like

from 40 to 900 pg of 1,25(OH)2D3.

It

chain

some,

1.25(OH)2D2. Antibodies

high

varying

specificity;

method

method D-binding

have masked specific

was

in

some of

had

antibody

fusions

to be developed.

used to

globulin

the

present binding.

eliminate in

serum

A any and

Preliminary

S testing ing

for

affinity

hybrids

TEEOXDR

and specificfty cloned

were

and

ensured

that

grown in vivo

only

for

the most

promis-

large-scale'

antibody

production. The

purpose

with

suitable

Until

now

of the present

characteristics only

one

1,25(OHJ2D3; this reported with

further,

other

We have displacement

described

is

in having

enormous

advantages

receptor

needs a source

age,

receptor.

(12).

assay for

1,25(OH)2D

of bound

method used for MAb 167,

although

of 1,25(OH)2D3

the

polyclonal

for

1,25(OH12D2

titre

calf

MAb 5F2 radioligand

the characterisation

over

higher a useful

to been

strongly

differing

in

valuable

antisera

higher

the

B50 than range,

described

than in

low

because

dilution,

of a routine

charcoal

separ-

the second-antibody the

MAb 5F2, still and,

The

of a partic-

on the development

rather

than

supply.

from animals

a

far

MAb possesses

and unrestricted

and involving

so

1,25(OHj2D3

and

a B50 slightly

glands

Work is now in progress

with

MAb's

has to be made at extremely

utilising

not

two show particularly

having

of high

of fresh

and free

has

Mb's

cross-reacted

Compared to the receptor,

and the preparation

1:6 - 1:8

ation

affinity

in terms

raise

that

nine

which

all

displacement,

thymus

described

to

1,25(DH12D.

assay. to

equal

the calf

account

characterising of

superior

for

(13).

1,25(OH12D

and has the requisite

ular

in

a range of Mb's

assays

described

preliminary

specificity, for

been

the antibodies

succeeded

characteristics

was to produce

improved

has

D metabolites

and

MAb 5F2

in a

and

vitamin

for

attempt

was

study

of

present permits its

zero

study. assay cross-

S

754

reaction an can

with

assay be

in

simplified

other which

v itamin the or

TmEOfDI

D

metabolites.

h

may

perm

it

the

fication

development

of

procedures

omitted. ACKNOWLEDGMENTS

This study was supported by awards to the Departments of Chemical Pathology and Medicine from the North Western Regional Health Authority (Research Infrastructure) and by a Project Grant awarded to EBM by the Medical Research Council. Dr. S. Shany was a Bruno Mendel Fellow of the Royal Society. We are grateful to Professor J.G. Ratcliffe and Dr. D-C. Anderson for their helpful suggestions and to Dr.G.N. Smith for chemical advice and assistance. Excellent technical assistance was provided by Mrs. S.M. Hall and Mr. P. Cundall. NOTES

1.

2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

AND

REFERENCES

Systemic and trivial names of vitamin D secosterols abbreviations used in this paper are as follows: (5Z,7E)-(3S)-9,10-seco-5,7,10(19),22-ergostatetraen-3-o1;

and their vitamin D v?;amin

la(OH )D3-acid; la-hydroxy-26,27-dinorcholecalciferol-25-oxime, la(OH )D3-oxime. Jongen, M.J.M., Van Ginkel, F.C., van der Vijgh, W.J.F., Kuiper, S., Netelenbos, J.C. and Lips, P., CLIN. CHEM. 30, 399 (1984). Fraher, L.J., Adami, S., Clemens, T.L., Jones, G. and O'Riordan, J.L.H., CLIN. ENDOCRINDL. 19, 151 (1983). Kohler, G. and Milstein, C.,mATURE 256, 495 (1975). White, A., Anderson, D.C. and Daly, Jr, J.CLIN.ENDOCRINOL. METAB. 54, 205 (1982). WhXe, A., Gray, C. and Corrie, J.E.T., J. STEROID BIOCHEM.(1985). Erlanger, B.F., Borek, F., Beiser, S.M. and Lieberman, S., J. BIOL. CHEM. 228, 713 (1957). Goding, m., J. IMMUNOL. METHODS39, 285 (1980). Abraham, G.E., J. CLIN. ENDOCRImL. METAB. 29, 866 (19691. Scatchard, G., ANN.N.Y. ACAD. SCI. 51, 660 (1949. Chamness, G.C. and McGuire, W.L., SmOIDS 26, 538 (1975). Reinhardt, T.A., Horst, R.L., Orf, J.W. and Ellis, B.W., J. CLIN. ENDDCRINOL. METAB. 58, 91 (1984). Perry, H.M., Chappel, J.Cy Clevinger, B-L., Haddad, J.G. and Teitelbaum, S.L., BIOCHEM. BIOPHYS. RES. COMM. -’112 431 (1983).