Specificity of prostaglandin A1 antiserum Against prostaglandins A1 and B1

Volume 30, numbe~ 1

[;EBS LETTERS

SPECIFICITY AGAINST

OF PROSTAGLANDIN PROSTAGLANDINS

February 1973

A t ANTISERUM A t AND B t *

A m i r a m R A Z * * and William A. S T Y L E S 7~:e Worcexter F o u n d ~ tio. ~fiJr E x p e r m w n t a l Biology. Shrew.+burt. :. ]~fa~.. U S A

Received 1 January 1973

l. I n t r o d u c t i o n Recent a t t e m p t s b y J a f f e e t al. [ | I to p r o d u c e antibodies t o w a r d s P G A I have resulted in an antise,-ur.:twh/ch c r o s s - r c a c l e d significantly w i t h P G E ! a n d PGE 2. Levine a n d c o - w o r k e r s [2] h a v e a t t e m p t e d to obtain a n t i b o d i e s t o P G E t in; r a b b i t s b y i m m u n i z i n g with PGE t - b o v i n e s e r u m a l b u m i n c o n j u g a t e s , b u t 0brained a n t i b o d i e s d i r e c t e d m a i n l y against P G B 1 . Smlitar a t t e m p t s b y Y u a n d Burke [3} have resulted in the p r o d u c t i o n o f P G E t antisera w h i c h cross :eacted t o an e q u a l e x t e n t o r m o r e with the A and B pr0staglandins, a n d o f P G A 1 antis~ra w h i c h cross reacted to a higher e x t e n t w,~tix P G B t. Z u s m a n et al. [4] have a t t e m p t e d t o p r o d u c e a n t i b o d i e s to F G E 2 but o b t a i n e d a n t i s e r a d i r e c t e d m a i n l y against P G A 2 and with cross reactivities t o w a r d s P ( ; A 1 ( 5 3 % ) a n d PGE2 ( 2 6 ~ ) . As a result o f t h e ~ studies [ 1 - 4 ~ , several irxvestigatots [2, 3] have suggested t h a t it w o u l d be very d i f f i c u l t , i f n o t i m p o s s i b l e , to o b t a i n an*Abodies d i r e c t e d specifically against the P G E a n d F G A pr0staglandins. The first r e p o r t o n t h e successful p r o d u c t i o n o f ami-PGA 1 s e r u m w h i c h is highly specific f o r ..i~A l

* ~fis work was supported by AID contract csd/2837. "* VisR~ng sc~entis[ on leave from the Department o f Bidchemistry, Tel-Aviv University, Ramat-Aviv, Tel-Arty, Isa=.~:I.

Abbrc~,farions:

PGE 1• prosta~landin E ! ; PG E 2 , prostaglandin F 2; PGA I prostaglandin A l ; PGB!, prostaglandin Bt ; PGF tc~;prostagtandin Vt~; PGFs~,, pmstaglandin Vs0t. Xortf:-Hoiland l~lblishit~g C o m l m r t y - A m x t e r d ~ o t

was r e p o r t e d b y S t y l e s and Rivetz [ 5 ] . These a u t h o r s i m m u n i z e d rabbits with p o l y L : l y s i n e - - P G A 1 c o n j u gate a b s o r b e d on P n e u m o c o c c u s R 3 6 A strains cells, and o b t a i n e d PGA 1 antiserum w h i c h cross reacted s o m e w h a t with P G A 2 (9.7%), but o n l y very m i n i m a l iy with PGE t (2.9%). Yhis re por~ describes the relative specificity o f this P G A I a n t i s e r u m t o w a r d s P G A t and PGB I . Also included is a disct:ssion o f the pro= rein carrier a n d m e t h o d o f i m m u n i z a t i o n as t h e y affeet the st~ccificity o f an tisera raised against P G A I and against PGE i .

2. Materials a n d m e t h o d s Prestaglandin E 1 was g e n e r o u s l y supplied b y Dr. J o h n E. Pike o f the U p j o h n Co. a n d Prostaglandin A l was a gift o f the O n e C o m p a n y , T o k y o , J a p a n . P G B ! was prcpaced f r o m P G E t b y heating in 0.5 N K O H in m e t h a n o l - w a t e r (1 : I ) at 5 5 - 5 8 ° for 3 0 rain. The reaction m i x t u r e was then e v a p o r a t e d to d r y n e s s , dissolved in water, acidified to p H = 3.O with I N HCI and e x t r a c t e d 3 times with 5 m! diethyl e t h e r . The pooled e t h e r e x t r a c t s were washed with w a t e r to p H = 6 . 0 - 6 . 5 , d, ied o v e r N a 2 S O 4 and e v a p o r a t e d to dryness. A recent report b y Z u s m a n [6] has indic a t e d that tile m e t h o d o f c o n v e r l m g PGE 1 to FGB i b y heating in 1 N methanol':c K O H for 2 - 5 rain at l O0 ° ntay yield p r o d u c t s o t h e r than PGB I . I1~.e p u r i t y o f the p r e p a r e d P G B t was therefore c h e c k e d s p e c ~ r o p h o t o metrically. The p r o d u c t was f o u n d to possess the characteristic a b s o r p t i o n s p e c t r u m o f P G B I wi~h an a b s o r p t i o n tnaximuln at 278 n m .

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r_-~ ,-~-. I- l ~ t i b i t [ o n o f [3HIPGA~-anti-PGA~ btndivg by PGA t and PG, B~. Assay c o n d i t i o n s are d e s c r i b e d in M e t h o d s . T h e an',isernm d i l a t i o n e m p l o y e d was 1:800 (final dilution in the assay m e d i u m was 1:40130).

PGA l antiserum was o b t a i n e d f r o m rabbits immunized with p o l y - L q y s i n e - P G A l conjugate [ 5 ] . The procedure e m p l o y e d in the rad,~oimmunoassay has been described elsewhere [ 5 ] .

3. Results and discussion The PGA 1 antiseru~.n p r o d u c e d in rabbits immunized with a p o l y - L - l y s i n e - P G A 1 conjugate b o u n d 50,~ o f the added [3H]PGA 1 ( 2 0 0 0 cpm, 10 p g ) a t a dilution o f 1:800 (final dilution o f the antiserum in the assay medium was I _'4000). The standard curve o f this ant,:serum with unlabeled PGA, and the cross reaction with unlabeled PGB t are shown in fig. 1. PGA l and PGB 1 appear to be equally effective in displacing antibody b o u n d I3H]PGA. These results indicate that the antibod,.'e.~ we obtained possess very similar binding characleristit's towards PGA: and PGB 1. Support t'or this tentative conclusion wa, o b t a i n e d from studies in which [3tlI'*GBI was first added to the PGA 1 antiserum. :rod then displaced by varying amounts o f unlabeled PGA 1 or PGB 1 . The results o f these experiments are given in fig. 2 and again indicate b o t h PGA I and P G B 1 to be a.pprt;y':nmte.Iy equal in displacing the a n t i b o d y - b o u n d [311]PGBI. it should be emphasized that despite the apparent inability o f the antibodies p r o d u c e d to distinguish between PGA 1 and P G B I , they are nevertheless specific for the 22

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Fig. 2. lnlhibition o f [ 3[-[]PGI$ I - a n t i - p G A t b i n d i n g b y l~3A and PGB ! . Assay c o n d i t i o n s ate d e s c r i b e d in M e t h o d s . "I'he a n t i s e r u m d i l u t i o n e m p l o y e d was ! :400 (final dilution in the assay m e d i u m was 1:2000).

c y t o p e n t e n e ring structure, showing only small cross r e a c t i ~ t y with PGE l , P ~ E 2 , P ~ F 2 ~ , and PG[ZIa [51 . T w o conclusions can_ be drawn f r o m these results. The first is that the affinity o f tile antibodies for initial binding o f PGA ! is stronger t h a n towards PG[I I since in order t o achieve the same e x t e n t o f binding (50%) o f [3H]prostaglandin, it was necessa;)" to use a more c o n c e n t r a t e d antiseium for PGB I (dilution of antiserum 1 : 4 0 0 ) than for I G A 1 (dilution o f antiserum ! :800). The second cc.nctusion is that the abilities of unlabeled FGA 1 and I~GB I t o displace [311]prostaglandin ( P G A I o~ P G B I ) are very similar. Taken together, these cor.clus,ons in6icate t o us lhat the antibodies we o b t a i , e d were p r o d u c e d in response to the presence o f PGA l-C°ntaining i m m u n o g e n o n l y and are directed against PGA 1 moieties, b u t are not capable o f distinguishing between the PGAj and I ~ B 1 structures. Previous studie:: [2, 3, 7] have indicated that the presence or absent e o f the h y d r o x y l groups at C 9 and C ! 1 arid the k e t o group al C 9 are the m:-j0r factors in producing the specific i m m u n o g e n i c response against the par:icular prostaglandin molecule. The c y c l o p e n t e n e ring in b o t h PGA l and PGB I is planar although structural differences b e t w e e n these two prostaglandins d o exist (e.g. the o r i e n t a t i o n s o f the C 8 and CI2 side drains). These differences appear howe~er t o be to~) small to affect significantly the overall binding nff'mities o f anti-PGA t antibodies towards P G A 1 as c o m p a r e d to F-GB I . The for,~,mtion o f anti,bodies directed against PGA 1 foliowiog , m m u n i z a t i o n with PGA l-containing

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Fig. 3. I n h i b i t i o n o f [ 3 1 [ ] g G E t - a n t i - P G E t b i n d i n g b y P G E 1 , PGA! a n d P G B t . A s s a y a s d e s c r i b e d in M e O t o d s . T h e a n t i s e ;am d i l u t i o n e m p l o y e d w n s l: 1 2 0 0 .

immunogen is in c o n t r a d i s t i n c t i o n to the results o f Jafl~ et al. [11, Levine e t ai. 121 and Yu and Burke [31. levine and c o - w o r k e r s [2] have a t t e m p t e d to prodttce antibodies to PGE I b y i m m u n i z i n g rabbits with PGE lp01y-L-iysine-succinylated h e m o c y a n i n . T h e antibodies they o b t a i n e d were h o w e v e r d i r e c t e d mainly against PGBt with w e a k e r binding o f P G A I a n d negligible binding o f PGE 1 _ T o a c c o u n t for these results, t h e y proposed that PGE I was c o n v e r t e d t o PGA 1 d i n i n g the earbodiimide c o n j u g a t i o n r e a c t i o n , so that the conjugate used for i m m u n i z a t i o n c o n t a i n e d F G A ! rather than P G E l_ T h e y f u r t h e r p r o p o s e that prostaglandin isomerase activity: originally d e m o n s t r a t e d b y Jones in cat plasma [81 and m o r e r e c e n t l y also in plasma o f rabbif, dog and rat [9, IO[ catalyzes the m vh,o conversion o f p r o t e i n - b o u n d P G A I to P G B ! yielding an i m m u n o g e n c o n t a i n i n g P G B t moieties. Support for this suggested m e c h a n i s m was r e p o r t e d by Yu and Bttrke [3] w h o o b t a i n e d P G E I a n t i s e r u m which cross r e a c t e d t o an equal or m o r e e x t e n t with PGA 1 and PGB t. T h e y also o b t a i n e d PGA 1 antiserunl ~itich cross r e a c t e d to a higher e x t e n t with PGB t . The data p r e s e n t e d h e r e , while n o t e x c l u d i n g the possibility that such a m e c h a n i s m is o p e r a t i v e , indicates tha~ tt~e n~ethod o f i m n l u n i z a t i o n as r e p o r t e d b y Stytos and Rivetz [5l in which the poly-.l_-lysinePGA I conjugate was a b s o r b e d to the P n e u m o c o c c u s cells, prevented a possible conversion o f FGA l to PGB 1 . In addition it was r e c e n t l y possible to obtain

February 1973

a PGE 1 antiserum with high specificity towards PGE~. Since b o t h Levine et al. [2~ and Yu and Burke [3J have suggested that p r o d u c t i o n o f antibodies against E type prostaglandins w i t h o u t considerable cr~ss-reactivity with tile A and B prostaghmdins would be very difficult if r,.ot impossible, we were interested in determining the cross reactivities o f the PGE t antiserum against PGA 1 and PGB 1 . l l m s e results are given in fig. 3, and indicate that neither PGA 1 nor P(.;B 1 could displace 50% o f tire [3H J PG E 1 initially b o u n d . Accurate evaluation o f the cross reactivittes o f these pro,~a~andins were t h e r e f o r e not possible ; the cross reactivities are however less than !%. O f special signifi~.~ancc is the fact that the binding affinities o f P G A l and PGB I towards the PGE [ antibodies, how,±ver small, are nevertheless very similar. These results are in agreement with those o b t a i n e d earlier (fig. 1 and 2) with regard to the relative binding aflinities o f P G A ! and PGB I towards PGA I anti.~erum. A radioimmunoassay for prostaglandins provides a simple technique for the quantitalive detern;_i~]ation o f these c o m p o u n d s in biological fluids and tissues. An absolute necessity for the d e v e l o p m e n t o f a specific radioimmunoassay is the p r o d u c t i o n o f antibodies capable o f distinguishing between the various structurally related prostaglandins. Styles and Rivetz 15 J, b y choosing a suitable protein conjugate in c o m b i n a t i o n with a ~ c o n d carrier ( P n e u m o c o c c u s cell~)were able to prevent the possible conversion o f PGA 1 ~o PGB I and thus olGtain a P G A I antiserum directed mainl3 against PGA l . Furtttermore, the data pre~ented here on the cross reactivities o f the PGE ! anti~erum with PGA t and PGB t (fig. 3) indicates tha~ the chemical conversion o f PGE~ to PGA t at~d the ~ . z y m a t i c conversion o f PGA 1 to PGB 1 as suggested by Levine et al. [21 and Yn and Burke [ 3 ] , was b l o c k e d . It ttlercfere appears that the type o f protein carrier used for conjugation and the m e t h o d o f immtmigation are the major d e t e r m i n a n t s which affect the resulting speeificities o f the antibodies p r o d u c e d , and that by proper selection o f the.~ determinants, it is possible to obtain specific antibodies against prostaglandins E or prostaglandin A c o m p o u n d s . * T h e P G E t autis~rum was p r e p a r e d at t h e W o r c e s t e r F , m u d a t i o n . Manuscript dealing w i t h the m e t h o d o f prepara. tion and d c i a i l e d immunologic',d specificity is in prepztration.

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Volume 30, number I

FEBS L E T I ' E R S

Acknowledgements We a c k n o w l e d g e t h e v a l u a b l e t e c h n i c a l a s s i s t a n c e o f D i a n e H. B r o w n m i d J o h n M a n g i a r d i .

References [ 11 B,M. Jaft'e, J.W. Sraith, W.T. Newton ang C.W. Parker, Science 17I ( ! 9 7 1 ) 494. [21 L. Lcvinc, R.M. Guticrrez C c m o ~ k and H. Van Vunakis, J. Biol. Chem. 240 (1971) 6782.

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: F e b r u a r y 1973

131 5-C. Yu and G. Burke, Prostaggandin~ 2 (1972) ! [. [4l R.M. Zusman, B.V. C a l d ~ e l l and L. Speroft, Prost ~,glaadins 2 ( 1 9 7 2 ) 41. [51 W.A. SWlo~ and B. Rivetz, Prostagl~-'tdin$, in press: [6] R-M. Zusman, Prostaglar.dins 1 (1972) 168. (7] B.V. Caldwell, S. Burstein. W.A. Brock a n d L. Spe:off, J. Clin. Endoc. 33 (1971) 171. [8[ R.L. Jones, Biochem. J . ! 19 (1970) 64P. [91 E. l t o r t o n . R. Jones, C. T h o m p s o n and N. Poysec, Ann. N.Y. Acad. Sci. 1 8 0 ( 1 9 7 1 ) 351. 110l !1. Polct a n d L. Le'Ane, Hiochem. Biophys. Res. C o m m u n . 45 ( 1 9 7 | ) ! 169.