Antiurinary follicle-stimulating hormone serum in radioimmunoassay

Antiurinary follicle-stimulating hormone serum in radioimmunoassay

Antiurinary follicle-stimulating hormone serum in radioimmunoassay SADAYUKI KAZETO, MYROSLAW Buflalo, M. New Antiurinary laboratory compared co...

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Antiurinary

follicle-stimulating

hormone serum in

radioimmunoassay SADAYUKI

KAZETO,

MYROSLAW Buflalo,

M. New

Antiurinary laboratory compared correlation

M.D.* HRESHCHYSHYN,

M.D.

York

follicle-stimulating hormone (FSH) serum which was produced in our was employed in radioimmunoassay for urinary FSH, and the results were with those obtained with the use of antipituitary FSH serum. The was good.

URINARY FOLLICLE-STIMULATING hormone (FSH) has been measured by means of radioimmunoassay with the use of antiserum to human pituitary FSH,l and antiurinary FSH serum in hemagglutination inhibition.’ We employed antiurinary FSH serum in radioimmunoassay and compared its activity with that of the antiserum to human pituitary FSH.

Materials

and

the hemagglutination test with the use of human menopausal gonadotropin-sensitized sheep red cells. After each rabbit received 32 ampules of urinary menopausal gonadotropin, they were bled by cardiac puncture. Antimenopausal gonadotropin serum was separated by centrifuging it at 3,000 r.p.m. for 20 minutes; then it was frozen. Antiserum from one of the rabbits showed a rather high titer of 6,000 to 8,000 (the other less than 2,000). Five milliliters of the high-titer antimenopausal gonadotropin serum was absorbed for one hour at 37O C. and 24 hours hours at 4O C. with 50,000 I.U. of urinary chorionic gonadotropin,* 50 mg. of acetoneextracted children’s serum powder, and 200 mg. of children’s urine powder obtained by kaolin absorbtion and acetone precipitation. Both the crude and the absorbed antimenopausal urinary gonadotropin sera were analyzed with immunoelectrophoresis and radioimmunoassay with the use of lz51 and double antibody technique.3 The crude antiserum produced precipitation lines with urinary menopausal gonadotropin, urinary human chorionic gonadotropin, male urine, and normal human serum. Absorbed antiserum showed a precipitation line with urinary menopausal gonadotropin only. In radioimmunoassay for gonadotrpins, the crude antiurinary menopausal gonadotro-

methods

Preparation of antiserum to human urinary FSH (antiurinary FSH) : Two white albino rabbits were injected intramuscularly with 4 ampules of urinary human menopausal gonadotropint containing 75 I.U. of FSH and 75 I.U. of LH per ampule, combined with Freund’s complete adjuvant,$ every two weeks. Test bleeding from the ear was repeated every 2 to 4 weeks, and the potency of the antiserum was examined by From the Department of GynecologyObstetrics School of Medicine, State University of New York at Buffalo. Supported in part by the Dr. Henry C. Buswell and Bertha H. Buswell Research Fellowship and by United States Public Health Service Grant No. 23070. *Dr. Henry C. Buswell and Bertha H. Buswell Research Fellowshin. 1967-69. Present adress: Department of Obstetrics and Gynecology, Nagoya University, Nagoya, Japan. +Pergonal, Lot 2214, Cutter Labs., Berkeley, California. $Difco

Labs.,

Detroit,

Michigan.

'A.P.L. 1206

supplied hy Ayerrt

Labs., New York,

New York.

Vcrlumr

108

Antiurinary

Ntmber8

I:2

Fig.

14

I:16

Dilution

of

I:4

I.32

I:64

onti-urinory

1. Affinity of antiurinary

FSH

serum

l

Crude

0

Absorbed

in radioimmunoassay

1207

ontiserum ontisfxum

I:126 I:256 I512 Cl024 FSH

(~10~)

FSH to labeled gonadotropins.

‘oor7.

0’

c

antl-urmory

+

ah-pWtory

FSH (1.2000) FSH (I 4000)

I

I

I

I

I

I

1

I

I

3.12

6.25

I25

25

50

IO0

200

400

800

mIU

,

HMG/ml

Fig. 2. Dose-response curves of 2 anti-FSH sera. pin serum showed affinity for labeled human pituitary FSH (LER 869-Z)) pituitary LH gonado(LER 960) * and urinary chorionic tropin,+ but the absorbed antiurinary menopausal gonadotropin combined specifically with pituitary FSH only (Fig. 1). Thus, we “Supplied by the National Institutes Pituitary Agency, Bethesda, Maryland. tPurified ir. OUF Laboratory from New York, New York.

of Health, A.P.L.,

Ayerst

National Lab.,

considered the absorbed antiurinary menopausal gonadotropin serum to represent antiurinary FSH. Antipituitary FSH serum obtained by immunizing a rabbit with human pituitary FSH was supplied to us by the National Pituitary Agency, National Institutes of Health. The Second International Refererxe Prrparation for Human Menopausal Gonado-

1208

Kazeto

and Hreshchyshyn Amer.

December 15, 1970 J. Obstet. Gynec.

O-

. >-

2-

I I IO

I-

anti-urinary

Fig. 3. Correlation

I loo FSH

1 loo0

m IU/ml

of urinary FSH values with the use of antipituitary

and antiurinary

FSH

scra.

tropin* was used as a standard hormone. First-morning urine specimens collected by

student nurses were assayed3for FSH with the use of antiurinary FSH serum and antipituitary FSH serum simultaneously. Results curves for the antipituitary and antiurinary FSH sera are shown in Fig. 2, and the comparison of results on assaying the urines for FSH with the useof the 2 antiDose-response

*Supplied by the London, England.

National

Institute

of Medical

Research,

sera are shown in Fig. 3. The dose-response

curves are similar, and there is good correlation of resultsin assayingthe urine specimens. Comment

It is feasible to obtain antiurinary FSH serum by immunizing rabbits with commercially available urinary human menopausal gonadotropin. Antiurinary FSH and antipituitary FSH sera appear to be immunologically identical. Antiurinary FSH serum can be used in radioimmunoassay of urinary FSH.

REFERENCES

1. Odell, W. D., and Parlow, A. F.: Clin. Res. 15: 125, 1967. 2. Mori, K. F.: J. Endocr. 42: 55, 1968.

3. Kazeto, S., Sansone, A., and Hreshchyshyn, M. M.: AMER. J. OBSTET. GYNEC. In press.