- Email: [email protected]
Development and characterization of a homologous radioimmunoassay for deer mouse (peromyscus maniculatus bairdii ) prolactin
Life Sciences, Vol. 33, pp. Printed in the U.S.A.
2305-2309
Pergamon
Press
D E V E L O P M E N T A N D C H A R A C T E R I Z A T I O N OF A H O M O L O G O U S R A D I O I M M U N O A S S A Y F O R DEER MOUSE (PEROMYSCUS M A N I C U I ~ T U S BAIRDII) P R O L A C T I N 1 Gregory A. Mawr, I, Peter Colosi, ' - 3 Claude Desjardins- and Frank Talamantes I' 1 Thimann Laboratories, , U n i v e r s i t y of California, Santa Cruz, C a l i f o r n l a 95064 and Department of Zoology, Institute of R e p r o d u c t i v e Biology, The U n i v e r s i t y of Texas, Austin, Texas 78712
(Received
in final
form September
19,
1983)
Summary A h i g h l y specific and sensitive homologous radioimmunoassay has been d e v e l o p e d for the secreted form of p r o l a c t i n from the deer mouse Peromyscus maniculatus bairdii. Peromyscus serum and pituitary h o m o g e n a t e s d i s p l a y e d p a r a l l e l dilution response curves, and no cross reaction was seen with either mouse prolactin, mouse growth hormone or rat prolactin. The assay was sensitive %0 25 p i c o g r a m s per tube and the intra- and inter-assay coefficients of variation were 5 and 3.6%, respectively. In addition, we have d e m o n s t r a t e d that Perom~scus prolactin does not show p a r a l l e l displacement in a homologous radioimmunoassay utilized for m e a s u r i n g p r o l a c t i n in the common l a b o r a t o r y mouse.
Homologous r a d i o i m m u n o a s s a y s (R/A) for both the stored (1) and secreted forms (2) of p r o l a c t i n (PRL) have been developed for the p u r p o s e of correlating PRL levels in the p i t u i t a r y and serum with reproductive p r o c e s s e s in the l a b o r a t o r y mouse. Mice of the genus Perom~Tscus have b e e n utilized in studies c o n c e r n i n g the photo p e r i o d i c regulation of r e p r o d u c t i v e development (9, 4, 5) and the c i r c a d i a n rhythm of serum PRL levels (6). The u n a v a i l a b i l i t y of Peromyscus PRL (pmPRL) and its corresponding antibody has necessitated the use of a h e t e r o l o g o u s system involving the rat p r o l a c t i n (rPRL) R I A for the measurement of PRL levels in P e r o m y s c u s (6). In this study we report on the development and c h a r a c t e r i z a t i o n of a h o m o l o g o u s R I A for deer mouse (Peromyscus m a n i c u i a % u s b a i r d i i ) PRL. Additionally, we have d e m o n s t r a t e d that p m P R L does not show p a r a l l e l d i s p l a c e m e n t in a h o m o l o g o u s R I A utilized for m e a s u r i n g PRL in the common laboratory mouse.
3Reprint requests~ Dr. F r a n k Talamantes, of California, Santa Cruz, C a l i f o r n i a 95064.
Thimann
Laboratories,
0 0 2 4 - 3 2 0 5 / 8 3 $3.00 + .00 Copyright (c) 1983 Pergamon Press Ltd.
University
2306
Deer M o u s e
Prolactin
Radioimmunoassay
Materials Hormone
Vol.
33, No.
23,
1983
and M e t h o d s
Preparations
Mouse prolactln (mPRL), and pmPRL were isolated, purified and c h a r a c t e r i z e d a c c o r d i n g to p r o c e d u r e s d e v e l o p e d in thls l a b o r a t o r y ~7, 8, 9, i0, 11). rPRL (B-3) was obtained from the NIADDK. Mouse growth h o r m o n e (mOH) was o b t a l n e d from Dr. Y. N. Slnha ( W h i t t i e r Institute, La Jolla, California). Antiserum Five h u n d r e d m i c r o g r a m s of h i g h l y p u r i f l e d pmPRL, d i s s o l v e d in 333 ~l of 50 mM sodium phosphate, pH 7.0, were mixed with 667 ~I of F r e u n d ' s complete adjuvant, and in3ected s u b c u t a n e o u s l y (sc) into 5 sites around the neck region in each of 3 New Zealand w h i t e rabbits. Two weeks later, each rabbit was b o o s t e d with 500 ~g of p m P R L in the same m a n n e r but e m p l o y i n g F r e u n d ' s i n c o m p l e t e ad3uvant. A f t e r two a d d i t i o n a l weeks, the rabbits were injected with 200 ~g of pmP~L. Two weeks after the second booster injection a p p r o x i m a t e l y 40 ml of blood were c o l l e c t e d from each rabbit by cardiac p u n c t u r e under k e t a m i n e anesthesia. Iodination
of p m P R L
Highly purlfled pmPRL was iodinated by a modiflcation of the l a c t o p e r o x l d a s e method (12). Five ~ g of prePRL d i s s o l v e d in i0 ~I of 0.01 M Na/~CO3, pH 8.75, 0.5 ~g of l a c t o p e r o x i d a s e in 20 ~i of 0.5 M sodium phosphate, pH 7.5, and 20 ~i of a 1:60,000 d i l u t i o n of 30% H202 distilled d e i o n i z e d water w e r e added to 1.0 mCi of c a r r i e r free N a 1 2 5 I ( A m e r s h a m Searle) and allowed to react for 3 m i n u t e s at room temperature, after which the reaction was t e r m i n a t e d by the a d d i t i o n of 300 ~i of R I A assay b u f f e r (0.01 M sodium phosphate, 0.01 M EDTA, 0.15 M NaCI, 0.1% sodium azide and 1% RIA grade bovine serum albumin, pH 7.6). The reaction m i x t u r e was i m m e d i a t e l y placed on a 1.3 i0 cm S e p h a d e x G-50 column p r e - e q u i l i b r a t e d with RIA buffer. The p r o t e i n a s s o c i a t e d peak of r a d i o a c t l v i t y was c o l l e c t e d and pooled. Five h u n d r e d m i c r o l l t e r s of the pooled G-50 eluate were then p l a c e d on a 1.3 w 20 cm S e p h a d e x G-IO0 column e q u l l i b r a t e d with RIA buffer. O n e - h a l f ml aliquots were collected. The peak fraction and the first three p o s t - p e a k fractions were used in the RIA. The specific a c t l v i t y as d e t e r m i n e d by T C A - p r e c i p i t a t e counts of the iodinated p m P R L was i00 ~ C i / ~ g of protein. Assay Procedure A double a n t i b o d y P/A was d e v e l o p e d using a n t i s e r u m from one of the rabbits a c c o r d l n g to similar P/A p r o c e d u r e s p r e v i o u s l y d e v e l o p e d in this l a b o r a t o r y for m P R L (2) and mouse p l a c e n t a l lactogen (13). Working dilutions of a n t i s e r u m were p r e p a r e d in 0.01 M sodium p h o s p h a t e - b u f f e r e d saline, pH 7.6, c o n t a l n i n g 0 . 0 1 M EDTA, 0.15 M NaCI, 0.1% sodium azide and 3% (v/v) normal rabbit serum. All s t a n d a r d s and unknown samples were diluted in RIA buffer. In the assay, I00 ~l of standard or unknown were incubated with i00 ~i of 1:30,000 d i l u t i o n of prePl~L a n t i s e r u m for 24 hours at room temperature. Ten thousand cpm of 1 2 5 1 p,IP]~L was then d e l i v e r e d in i00 ~I of a s s a y b u f f e r and allowed to incubate for an a d d i t i o n a l 24 hours. S e p a r a t i o n of bound and free h o r m o n e s was a c c o m p l i s h e d by i n c u b a t i n g the reaction m i x t u r e with 100 ~i of goat a n t i - r a b b i t gamma g l o b u l i n s (Antibodies, Inc., Davis, C a l i f o r n i a ) diluted 1:16 with 0.01 M PBS, pH 7.6. A f t e r one h a l f hour, 100 ~I of 30% (w/v) p o l y e t h y l e n e glycol (MW 6000) in d i s t i l l e d water was added (14). All tubes were then i m m e d i a t e l y v o r t e x e d and spun at 7000 rpm for 20 m i n u t e s in a Sorvall R C 2 - B c e n t r i f u g e using an HS4 rotor. The s u p e r n a t a n t s were a s p i r a t e d and the p e l l e t s counted in a B e c k m a n 8000 g a m m a spectrometer. The dilution of a n t i s e r u m e m p l o y e d p r o v i d e d total b i n d i n g ranging b e t w e e n 50 and 55% with n o n s p e c i f i c b i n d l n g b e t w e e n 4 and 5%. R e f e r e n c e standards ranged from 10 to
Vol.
33, No.
23,
1983
Deer Mouse
i000 pg of h l g h l y p u r l f i e d
Prolactin
Radioimmunoassay
2307
m P R L per tube.
m P R L RIA The c r o s s - r e a c t i v i t y of p m P R L was e v a l u a t e d in the m P R L RIA which was p r e v i o u s l y d e v e l o p e d and c h a r a c t e r i z e d in this l a b o r a t o r y (2). validation
of p m P R L RIA
The p m P R L R I A was e v a l u a t e d b y g e n e r a t i n g d i s p l a c e m e n t curves for pmPRL, a n t e r l o r p i t u i t a r y h o m o g e n a t e and serum from P e r o m y s c u s m a n i c u l a t u s bairdii. P i t u i t a r i e s w e r e o b t a i n e d from adult females. S e r u m was o b t a i n e d from intact males, lactating females and 2 - B r - ~ - e r g o c r y p t i n e t r e a t e d males (200 ~ g / a n i m a l a d m l n l s t e r e d s.c. in oii for 4 days and s a c r i f i c e d 24 h o u r s after the last i n 3 e c t i o n ) . In addition, d i s p l a c e m e n t curves w e r e g e n e r a t e d for mPRL, m G H and FPRL. Results As is shown in Figure
i, p m P R L was able to d i s p l a c e
labelled mPRL,
I00 90 80 70
60 5o
~
4O 2O I0 I .01
I .I
I''~' I
I I0
I I00
I 1,000
I0 000
ng/tube
PIG.
1
D i s p l a c e m e n t curve for p m P R L in the m P R L RIA. of five d e t e r m i n a t i o n s .
Each point
represents
the m e a n
2308
Deer Mouse Prolactin Radioimmunoassay
Vol.
33, No.
23,
1983
but the d l l u t l o n - r e s p o n s e c u r v e s o b t a i n e d did not p a r a l l e l the m P R L standard, F i g u r e 2 s h o w s the s p e c i f i c i t y and s e n s i t i v i t y of the p m P R L RIA. The
Sample Dilution ( Sera, Pituitary Homogenote ) 1:4000
I:1000
I
i
I:100
I: I0
i
i
mGH,rPRL
I00 9O 8O 70
X~
"~
~'~~N.orrnol MolSer e um
6O
BIBoso
\
4O
,
3O 2O I0
Ib
2s
so
60
25o s6o ,ooo
io,ooo
Peromyscus PRL pg/tube FIG. 2 D i s p l a c e m e n t c u r v e s for pmPRL, P e r o m y s c u s p i t u i t a r y h o m o g e n a t e , l a c t a t i n g female s e r a in the pmRIA.
and m a l e and
s e n s i t i v i t y of the a s s a y w a s 25 p g p e r t u b e and the d i s p l a c e m e n t c u r v e w a s linear b e t w e e n 25 and 500 pg. The i n t r a - ( d e t e r m i n e d f r o m 20 r e p l i c a t e s ) and i n t e r - a s s a y ( d e t e r m i n e d from l0 a s s a y s ) c o e f f i c i e n t s of v a r i a t i o n w e r e 5 and 3.6%, r e s p e c t i v e l y . M o u s e GH and r P R L s h o w e d little if any c r o s s - r e a c t i v i t y in t h i s a s s a y w h e n e v a l u a t e d for d i s p l a c e m e n t in a m o u n t s up to 1 ~ g p e r tube. m P R L w a s found to d i s p l a c e some l a b e l l e d p m P R L w h e n the c o n c e n t r a t i o n of m P R L e x c e e d e d iO ng p e r t u b e but the d i l u t i o n r e s p o n s e c u r v e d i d not p a r a l l e l the p m P R L s t a n d a r d curve. The s e r u m v a l u e s of p m P R L d e t e r m i n e d b y this R I A r a n g e d from 12 n g / m l for m a l e s to 22 n g / m l for l a c t a t i n g females. Bromocryptine t r e a t e d m a l e s h a d s e r u m P R L levels of 4.5 n g / m l ( d a t a not shown). Discussion In c o n t i n u a t i o n o f o u r s t u d i e s c o n c e r n i n g the b i o c h e m i s t r y and p h y s i o l o g y of s e c r e t e d p i t u l t a r y prolactin (2, 7, 8, 9, iO, 15), w e r e p o r t on the d e v e l o p m e n t and c h a r a c t e r i z a t i o n of a s p e c i f i c and s e n s i t i v e h o m o l o g o u s R I A for pmPRL. T h e p m P R L R I A d o e s not c r o s s - r e a c t w i t h e i t h e r mGH, mPRL, or rPRL.
(6).
S e r u m P R L l e v e l s in P e r o m y s c u s h a v e b e e n m e a s u r e d w i t h an N I H r P R L - R I A However, u t i l i z i n g an N I H r P R L or m P R L RIA, it w a s not p o s s i b l e to
Vol.
33, No.
23,
1983
Deer Mouse P r o l a c t i n
Radioimmunoassay
2309
c o r r e c t l y m e a s u r e serum PRL levels in P e r o m y s c u s due to the lack of p a r a l l e l i s m (Des3ardlns, unpublished). Furthermore, in support of these latter studies we h a v e d e m o n s t r a t e d in this study that h i g h l y p u r i f i e d p m P R L w i l l not d i s p l a c e labelled mPRL in a p a r a l l e l manner. The a p p l i c a t l o n of RIA's d e v e l o p e d for h o r m o n e s from one species to be used in other species has b e e n a v a l u a b l e tool in e n d o c r l n e research, e s p e c i a l l y in u n d e r s t a n d i n g the c o m p a r a t i v e p h y s i o l o g y of the gonadotropins; however, this m e t h o d o l o g y should not be used w i t h o u t some k n o w l e d g e of species d i f f e r e n c e s in the h o r m o n e b e i n g studied and only after rigorous p h y s i o l o g l c a l validation. The a v a i l a b i l i t y of the p m P R L RIA and the h i g h l y specific a n t i s e r a to p m P R L w i l l now allow us to study the r e g u l a t i o n of s e c r e t i o n and the p h y s i o l o g i c a l role of this h o r m o n e in a h o m o l o g o u s system. Acknowledgements We thank Dr. L1nda Ogren (UCSC), Dr. M. J. Soares (UCSC), and Don Carroll (U.T.) for their e x c e l l e n t assistance. This work was s u p p o r t e d by a UCSC FRC grant and by NIH grants R R 0 8 1 3 2 and H D - 1 3 4 7 0 to Drs. F. T a l a m a n t e s and C. D e s ] a r d l n s , respectively. References i. 2. 3. 4.
5. 6. 7. 8. 9. 10. ii. 12. 13. 14. 15.
Y.N. SINHA, F.W. SELBY, U.J. LEWIS and W.P. VANDERLAAN, E n d o c r i n o l o g y 9_!i 1045-1053 (1972). E. MARKOFF, P. COLOSI and F. TALAMANTES, Life Sci. 28 203-211 (1981). L.J. P E T T E R B O R G and R.J. REITER, J. Reprod. Fertil. 60 209-212 (1980). P . G . J O H N S T O N and I. ZUCKER, Biol. Reprod. 22 9 8 3 - 9 8 9 (1980). P.G. JOHNSTCN, M. BOSHES and I. ZUCKER, Biol. Reprod. 26 597-602 (1982). M . K . T H O M P S O N and E.L. BRADLY, Gen. Comp. Endocrinol. 39 2 0 8 - 2 1 4 (1979) L.F.SHOER, N.R. SHINE and F. TALAMANTES, Biochim. Biophys. A c t a 537 936347 ( 1978 ). P. COLOSI, E. MARKOFF, A. LEVY, L. OGREN, N. SHINE and F. TALAMANTES, E n d o c r i n o l o g y 108 8 5 0 - 8 5 4 (1981). P. COLOSI and F. TALAMANTES, Arch. Biochem. Biophys. 212 759-761 (1981). T.A. BEWLEY, P. COLOSI and F . T ~ S , B i o c h e m i s t r y 21 4238-4243 (1982). P. COLOSI, T.A. B E W L E Y and F. TALAMANTES, Arch. Biochem. Biophys. 222 621-627 (1983). J . I . T H O R E L L and B.G. JOHANNSON, Biochim. Biophys. A c t a 251 363-369 (1971). M.J. SOARES, P. COLOSI and F. TALAMANTES, E n d o c r i n o l o g y ii0 6 6 8 - 6 7 0 (1982). M.A. P E T E R S O N and R.S. SWERDLOFF, Clin. Chem. 25 1239-1241 (1979). M. J I B S O N and F. TALAMANTES, Gen. Comp. Endocrinol. 3__4 402-407 (1978).
2305-2309
Pergamon
Press
D E V E L O P M E N T A N D C H A R A C T E R I Z A T I O N OF A H O M O L O G O U S R A D I O I M M U N O A S S A Y F O R DEER MOUSE (PEROMYSCUS M A N I C U I ~ T U S BAIRDII) P R O L A C T I N 1 Gregory A. Mawr, I, Peter Colosi, ' - 3 Claude Desjardins- and Frank Talamantes I' 1 Thimann Laboratories, , U n i v e r s i t y of California, Santa Cruz, C a l i f o r n l a 95064 and Department of Zoology, Institute of R e p r o d u c t i v e Biology, The U n i v e r s i t y of Texas, Austin, Texas 78712
(Received
in final
form September
19,
1983)
Summary A h i g h l y specific and sensitive homologous radioimmunoassay has been d e v e l o p e d for the secreted form of p r o l a c t i n from the deer mouse Peromyscus maniculatus bairdii. Peromyscus serum and pituitary h o m o g e n a t e s d i s p l a y e d p a r a l l e l dilution response curves, and no cross reaction was seen with either mouse prolactin, mouse growth hormone or rat prolactin. The assay was sensitive %0 25 p i c o g r a m s per tube and the intra- and inter-assay coefficients of variation were 5 and 3.6%, respectively. In addition, we have d e m o n s t r a t e d that Perom~scus prolactin does not show p a r a l l e l displacement in a homologous radioimmunoassay utilized for m e a s u r i n g p r o l a c t i n in the common l a b o r a t o r y mouse.
Homologous r a d i o i m m u n o a s s a y s (R/A) for both the stored (1) and secreted forms (2) of p r o l a c t i n (PRL) have been developed for the p u r p o s e of correlating PRL levels in the p i t u i t a r y and serum with reproductive p r o c e s s e s in the l a b o r a t o r y mouse. Mice of the genus Perom~Tscus have b e e n utilized in studies c o n c e r n i n g the photo p e r i o d i c regulation of r e p r o d u c t i v e development (9, 4, 5) and the c i r c a d i a n rhythm of serum PRL levels (6). The u n a v a i l a b i l i t y of Peromyscus PRL (pmPRL) and its corresponding antibody has necessitated the use of a h e t e r o l o g o u s system involving the rat p r o l a c t i n (rPRL) R I A for the measurement of PRL levels in P e r o m y s c u s (6). In this study we report on the development and c h a r a c t e r i z a t i o n of a h o m o l o g o u s R I A for deer mouse (Peromyscus m a n i c u i a % u s b a i r d i i ) PRL. Additionally, we have d e m o n s t r a t e d that p m P R L does not show p a r a l l e l d i s p l a c e m e n t in a h o m o l o g o u s R I A utilized for m e a s u r i n g PRL in the common laboratory mouse.
3Reprint requests~ Dr. F r a n k Talamantes, of California, Santa Cruz, C a l i f o r n i a 95064.
Thimann
Laboratories,
0 0 2 4 - 3 2 0 5 / 8 3 $3.00 + .00 Copyright (c) 1983 Pergamon Press Ltd.
University
2306
Deer M o u s e
Prolactin
Radioimmunoassay
Materials Hormone
Vol.
33, No.
23,
1983
and M e t h o d s
Preparations
Mouse prolactln (mPRL), and pmPRL were isolated, purified and c h a r a c t e r i z e d a c c o r d i n g to p r o c e d u r e s d e v e l o p e d in thls l a b o r a t o r y ~7, 8, 9, i0, 11). rPRL (B-3) was obtained from the NIADDK. Mouse growth h o r m o n e (mOH) was o b t a l n e d from Dr. Y. N. Slnha ( W h i t t i e r Institute, La Jolla, California). Antiserum Five h u n d r e d m i c r o g r a m s of h i g h l y p u r i f l e d pmPRL, d i s s o l v e d in 333 ~l of 50 mM sodium phosphate, pH 7.0, were mixed with 667 ~I of F r e u n d ' s complete adjuvant, and in3ected s u b c u t a n e o u s l y (sc) into 5 sites around the neck region in each of 3 New Zealand w h i t e rabbits. Two weeks later, each rabbit was b o o s t e d with 500 ~g of p m P R L in the same m a n n e r but e m p l o y i n g F r e u n d ' s i n c o m p l e t e ad3uvant. A f t e r two a d d i t i o n a l weeks, the rabbits were injected with 200 ~g of pmP~L. Two weeks after the second booster injection a p p r o x i m a t e l y 40 ml of blood were c o l l e c t e d from each rabbit by cardiac p u n c t u r e under k e t a m i n e anesthesia. Iodination
of p m P R L
Highly purlfled pmPRL was iodinated by a modiflcation of the l a c t o p e r o x l d a s e method (12). Five ~ g of prePRL d i s s o l v e d in i0 ~I of 0.01 M Na/~CO3, pH 8.75, 0.5 ~g of l a c t o p e r o x i d a s e in 20 ~i of 0.5 M sodium phosphate, pH 7.5, and 20 ~i of a 1:60,000 d i l u t i o n of 30% H202 distilled d e i o n i z e d water w e r e added to 1.0 mCi of c a r r i e r free N a 1 2 5 I ( A m e r s h a m Searle) and allowed to react for 3 m i n u t e s at room temperature, after which the reaction was t e r m i n a t e d by the a d d i t i o n of 300 ~i of R I A assay b u f f e r (0.01 M sodium phosphate, 0.01 M EDTA, 0.15 M NaCI, 0.1% sodium azide and 1% RIA grade bovine serum albumin, pH 7.6). The reaction m i x t u r e was i m m e d i a t e l y placed on a 1.3 i0 cm S e p h a d e x G-50 column p r e - e q u i l i b r a t e d with RIA buffer. The p r o t e i n a s s o c i a t e d peak of r a d i o a c t l v i t y was c o l l e c t e d and pooled. Five h u n d r e d m i c r o l l t e r s of the pooled G-50 eluate were then p l a c e d on a 1.3 w 20 cm S e p h a d e x G-IO0 column e q u l l i b r a t e d with RIA buffer. O n e - h a l f ml aliquots were collected. The peak fraction and the first three p o s t - p e a k fractions were used in the RIA. The specific a c t l v i t y as d e t e r m i n e d by T C A - p r e c i p i t a t e counts of the iodinated p m P R L was i00 ~ C i / ~ g of protein. Assay Procedure A double a n t i b o d y P/A was d e v e l o p e d using a n t i s e r u m from one of the rabbits a c c o r d l n g to similar P/A p r o c e d u r e s p r e v i o u s l y d e v e l o p e d in this l a b o r a t o r y for m P R L (2) and mouse p l a c e n t a l lactogen (13). Working dilutions of a n t i s e r u m were p r e p a r e d in 0.01 M sodium p h o s p h a t e - b u f f e r e d saline, pH 7.6, c o n t a l n i n g 0 . 0 1 M EDTA, 0.15 M NaCI, 0.1% sodium azide and 3% (v/v) normal rabbit serum. All s t a n d a r d s and unknown samples were diluted in RIA buffer. In the assay, I00 ~l of standard or unknown were incubated with i00 ~i of 1:30,000 d i l u t i o n of prePl~L a n t i s e r u m for 24 hours at room temperature. Ten thousand cpm of 1 2 5 1 p,IP]~L was then d e l i v e r e d in i00 ~I of a s s a y b u f f e r and allowed to incubate for an a d d i t i o n a l 24 hours. S e p a r a t i o n of bound and free h o r m o n e s was a c c o m p l i s h e d by i n c u b a t i n g the reaction m i x t u r e with 100 ~i of goat a n t i - r a b b i t gamma g l o b u l i n s (Antibodies, Inc., Davis, C a l i f o r n i a ) diluted 1:16 with 0.01 M PBS, pH 7.6. A f t e r one h a l f hour, 100 ~I of 30% (w/v) p o l y e t h y l e n e glycol (MW 6000) in d i s t i l l e d water was added (14). All tubes were then i m m e d i a t e l y v o r t e x e d and spun at 7000 rpm for 20 m i n u t e s in a Sorvall R C 2 - B c e n t r i f u g e using an HS4 rotor. The s u p e r n a t a n t s were a s p i r a t e d and the p e l l e t s counted in a B e c k m a n 8000 g a m m a spectrometer. The dilution of a n t i s e r u m e m p l o y e d p r o v i d e d total b i n d i n g ranging b e t w e e n 50 and 55% with n o n s p e c i f i c b i n d l n g b e t w e e n 4 and 5%. R e f e r e n c e standards ranged from 10 to
Vol.
33, No.
23,
1983
Deer Mouse
i000 pg of h l g h l y p u r l f i e d
Prolactin
Radioimmunoassay
2307
m P R L per tube.
m P R L RIA The c r o s s - r e a c t i v i t y of p m P R L was e v a l u a t e d in the m P R L RIA which was p r e v i o u s l y d e v e l o p e d and c h a r a c t e r i z e d in this l a b o r a t o r y (2). validation
of p m P R L RIA
The p m P R L R I A was e v a l u a t e d b y g e n e r a t i n g d i s p l a c e m e n t curves for pmPRL, a n t e r l o r p i t u i t a r y h o m o g e n a t e and serum from P e r o m y s c u s m a n i c u l a t u s bairdii. P i t u i t a r i e s w e r e o b t a i n e d from adult females. S e r u m was o b t a i n e d from intact males, lactating females and 2 - B r - ~ - e r g o c r y p t i n e t r e a t e d males (200 ~ g / a n i m a l a d m l n l s t e r e d s.c. in oii for 4 days and s a c r i f i c e d 24 h o u r s after the last i n 3 e c t i o n ) . In addition, d i s p l a c e m e n t curves w e r e g e n e r a t e d for mPRL, m G H and FPRL. Results As is shown in Figure
i, p m P R L was able to d i s p l a c e
labelled mPRL,
I00 90 80 70
60 5o
~
4O 2O I0 I .01
I .I
I''~' I
I I0
I I00
I 1,000
I0 000
ng/tube
PIG.
1
D i s p l a c e m e n t curve for p m P R L in the m P R L RIA. of five d e t e r m i n a t i o n s .
Each point
represents
the m e a n
2308
Deer Mouse Prolactin Radioimmunoassay
Vol.
33, No.
23,
1983
but the d l l u t l o n - r e s p o n s e c u r v e s o b t a i n e d did not p a r a l l e l the m P R L standard, F i g u r e 2 s h o w s the s p e c i f i c i t y and s e n s i t i v i t y of the p m P R L RIA. The
Sample Dilution ( Sera, Pituitary Homogenote ) 1:4000
I:1000
I
i
I:100
I: I0
i
i
mGH,rPRL
I00 9O 8O 70
X~
"~
~'~~N.orrnol MolSer e um
6O
BIBoso
\
4O
,
3O 2O I0
Ib
2s
so
60
25o s6o ,ooo
io,ooo
Peromyscus PRL pg/tube FIG. 2 D i s p l a c e m e n t c u r v e s for pmPRL, P e r o m y s c u s p i t u i t a r y h o m o g e n a t e , l a c t a t i n g female s e r a in the pmRIA.
and m a l e and
s e n s i t i v i t y of the a s s a y w a s 25 p g p e r t u b e and the d i s p l a c e m e n t c u r v e w a s linear b e t w e e n 25 and 500 pg. The i n t r a - ( d e t e r m i n e d f r o m 20 r e p l i c a t e s ) and i n t e r - a s s a y ( d e t e r m i n e d from l0 a s s a y s ) c o e f f i c i e n t s of v a r i a t i o n w e r e 5 and 3.6%, r e s p e c t i v e l y . M o u s e GH and r P R L s h o w e d little if any c r o s s - r e a c t i v i t y in t h i s a s s a y w h e n e v a l u a t e d for d i s p l a c e m e n t in a m o u n t s up to 1 ~ g p e r tube. m P R L w a s found to d i s p l a c e some l a b e l l e d p m P R L w h e n the c o n c e n t r a t i o n of m P R L e x c e e d e d iO ng p e r t u b e but the d i l u t i o n r e s p o n s e c u r v e d i d not p a r a l l e l the p m P R L s t a n d a r d curve. The s e r u m v a l u e s of p m P R L d e t e r m i n e d b y this R I A r a n g e d from 12 n g / m l for m a l e s to 22 n g / m l for l a c t a t i n g females. Bromocryptine t r e a t e d m a l e s h a d s e r u m P R L levels of 4.5 n g / m l ( d a t a not shown). Discussion In c o n t i n u a t i o n o f o u r s t u d i e s c o n c e r n i n g the b i o c h e m i s t r y and p h y s i o l o g y of s e c r e t e d p i t u l t a r y prolactin (2, 7, 8, 9, iO, 15), w e r e p o r t on the d e v e l o p m e n t and c h a r a c t e r i z a t i o n of a s p e c i f i c and s e n s i t i v e h o m o l o g o u s R I A for pmPRL. T h e p m P R L R I A d o e s not c r o s s - r e a c t w i t h e i t h e r mGH, mPRL, or rPRL.
(6).
S e r u m P R L l e v e l s in P e r o m y s c u s h a v e b e e n m e a s u r e d w i t h an N I H r P R L - R I A However, u t i l i z i n g an N I H r P R L or m P R L RIA, it w a s not p o s s i b l e to
Vol.
33, No.
23,
1983
Deer Mouse P r o l a c t i n
Radioimmunoassay
2309
c o r r e c t l y m e a s u r e serum PRL levels in P e r o m y s c u s due to the lack of p a r a l l e l i s m (Des3ardlns, unpublished). Furthermore, in support of these latter studies we h a v e d e m o n s t r a t e d in this study that h i g h l y p u r i f i e d p m P R L w i l l not d i s p l a c e labelled mPRL in a p a r a l l e l manner. The a p p l i c a t l o n of RIA's d e v e l o p e d for h o r m o n e s from one species to be used in other species has b e e n a v a l u a b l e tool in e n d o c r l n e research, e s p e c i a l l y in u n d e r s t a n d i n g the c o m p a r a t i v e p h y s i o l o g y of the gonadotropins; however, this m e t h o d o l o g y should not be used w i t h o u t some k n o w l e d g e of species d i f f e r e n c e s in the h o r m o n e b e i n g studied and only after rigorous p h y s i o l o g l c a l validation. The a v a i l a b i l i t y of the p m P R L RIA and the h i g h l y specific a n t i s e r a to p m P R L w i l l now allow us to study the r e g u l a t i o n of s e c r e t i o n and the p h y s i o l o g i c a l role of this h o r m o n e in a h o m o l o g o u s system. Acknowledgements We thank Dr. L1nda Ogren (UCSC), Dr. M. J. Soares (UCSC), and Don Carroll (U.T.) for their e x c e l l e n t assistance. This work was s u p p o r t e d by a UCSC FRC grant and by NIH grants R R 0 8 1 3 2 and H D - 1 3 4 7 0 to Drs. F. T a l a m a n t e s and C. D e s ] a r d l n s , respectively. References i. 2. 3. 4.
5. 6. 7. 8. 9. 10. ii. 12. 13. 14. 15.
Y.N. SINHA, F.W. SELBY, U.J. LEWIS and W.P. VANDERLAAN, E n d o c r i n o l o g y 9_!i 1045-1053 (1972). E. MARKOFF, P. COLOSI and F. TALAMANTES, Life Sci. 28 203-211 (1981). L.J. P E T T E R B O R G and R.J. REITER, J. Reprod. Fertil. 60 209-212 (1980). P . G . J O H N S T O N and I. ZUCKER, Biol. Reprod. 22 9 8 3 - 9 8 9 (1980). P.G. JOHNSTCN, M. BOSHES and I. ZUCKER, Biol. Reprod. 26 597-602 (1982). M . K . T H O M P S O N and E.L. BRADLY, Gen. Comp. Endocrinol. 39 2 0 8 - 2 1 4 (1979) L.F.SHOER, N.R. SHINE and F. TALAMANTES, Biochim. Biophys. A c t a 537 936347 ( 1978 ). P. COLOSI, E. MARKOFF, A. LEVY, L. OGREN, N. SHINE and F. TALAMANTES, E n d o c r i n o l o g y 108 8 5 0 - 8 5 4 (1981). P. COLOSI and F. TALAMANTES, Arch. Biochem. Biophys. 212 759-761 (1981). T.A. BEWLEY, P. COLOSI and F . T ~ S , B i o c h e m i s t r y 21 4238-4243 (1982). P. COLOSI, T.A. B E W L E Y and F. TALAMANTES, Arch. Biochem. Biophys. 222 621-627 (1983). J . I . T H O R E L L and B.G. JOHANNSON, Biochim. Biophys. A c t a 251 363-369 (1971). M.J. SOARES, P. COLOSI and F. TALAMANTES, E n d o c r i n o l o g y ii0 6 6 8 - 6 7 0 (1982). M.A. P E T E R S O N and R.S. SWERDLOFF, Clin. Chem. 25 1239-1241 (1979). M. J I B S O N and F. TALAMANTES, Gen. Comp. Endocrinol. 3__4 402-407 (1978).