Immunoelectrophoretic characterization of the molecular weight polydispersion of polysaccharides in multivalent bacterial capsular polysaccharide vaccines

Immunoelectrophoretic characterization of the molecular weight polydispersion of polysaccharides in multivalent bacterial capsular polysaccharide vaccines

Journal of Biological StandarL.~aation (1985) 11, 6 5 - 7 4 Immunoelectrophoretic characterization o f the molecular w e i g h t polydispersion o f p...

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Journal of Biological StandarL.~aation (1985) 11, 6 5 - 7 4

Immunoelectrophoretic characterization o f the molecular w e i g h t polydispersion o f polysaccharides in multivalent bacterial capsular polysaccharide vaccines* M. Porro, ~f3; S. Fabbiani, t 1. Marsili, Y S. V i t i t and M. SaIettit

The molecular weight polydispersion of single antigens present in multivalent bacterial capsular polysaccharide vaccines has been characterized by an immunoelectrophoretic methcx/. C h r o m a tographic effluents from Sepharose 8el of bacterial capsular polysaccharide vaccines v.'ere tes~ed by fused-rocket immunoelectrophoresis and the distribution coefficient (Kd) of each polysaccharide present in the m i x t u r e was calculated. The m e t h o d appeared to be efficient and reproducible. How-ever, different Kd s-alues were obtained by immunoelectrophoretic and chemical or physical analysis of the chromatographic effluents o f each single polysaccharide component. The use o f this immunoelectrophoretic procedure w ~ extended to the potency control o f m u l t i v a l e n t meningococcal and pneumococcal polysaccharide vaccinc~ in order to detect changes in the molecular weight polydispersion o f each antigen with time.

INTRODUCTION Polysaccharides are k n o w n to be very poor a n t i g e n s in a n i m a l m o d e l s , b u t their a n t i g e n i c i r y in h u m a n s , i n c l u d i n g that o f bacterial capsular polysaccharide v a c c i n e s , has b e e n s h o w n to be directly related to their m o l e c u l a r size. " • T h u s , r e q u i r e m e n t s for t h e control o f m o l e c u l a r w e i g h t p o l y d i s p e r s i o n ( p o t e n c y control) o f t h e bacterial -

-

1 . 2 . 3

-

* Received for publication 18 August 1982. t I S V T Sclavo, Research and D e v e l o p m e n t , Biological.s, Via Fiorentin~ 1. 53 i 0 0 Siena, Italy. :~ Reprint requests to Dr. Massimo Porto. 0092-115718~1001065+ I0 SO3.O0/O

198_~ "The lm~r~liorat~ Axso*:ialion of Blo~ogitat Staru~ardization

65

M. PORRO

ET AL.

capsular polysaccharides present in meningococcal and pneumococcal vaccines for h u m a n use were introduced by the W . H . O . 4"5'6 on the basis of gel chromatographyand Sepharose 4B as molecular sieve. These procedures are accurate and suitable for single polysaccharide antigens or for a mixture of antigens which do n o t interfere one with another when tested by chemical m e t h o d s in the chromatographic effluents. However, several problems arise when a m i x t u r e of antigens with similar chemical moieties in their structures m u s t be tested after molecular sieving. A similar problem occurs with the pneumococcal polyvalent vaccine, which at the present time includes I4 polysaccharide antigens, w h e n potency control of the single components is performed over a period of time. This paper describes the use of an immunoelectrophoretic m e t h o d to d e t e r m i n e the molecular w e i g h t polydispersion of each antigen c o m p o n e n t of the polyvalent pneumococcal vaccine, there being no significant immunochemical crossreactions between the type specific poIysaccharides present in the vaccine when these are tested with the corresponding homologous rabbit anrisera. 7 The use of this procedure was extended to the groups A and C meningococcal bivalent vaccine, although in this case a chemical differentiation of two antigens in the chromatographic effluents was possible. From these experiments, different values of distribution coefficients (Kd) were found with physical and chemical analyses o f chromatographic effluents of single polysaccharides after separation on Sepharose gel. Experiments to explain these differences were carried out. MATERIALS AND METHODS

PoIysaccharides Pneumococcal capsular polysaccharides serotypes 1, 2, 3, 4, 6A, 7F, 8, 9 N , 12F, 14, 18C, 19F, 23F and 25 (Danish nomenclature) were prepared in our laboratories and were present in an experimental lot o f vaccine for h u m a n use (ISVT Sclavo, Siena, Italy). The characteristics of the polysaccharides complied with the proposed requirem e n t s of W . H . O . 6 for pneumococcal polysaccharide vaccine. Meningococcal capsular polysaccharides of serogroups A and C and meningococcal vaccines were commercial products (ISVT Sclavo, Siena, Italy).

A ntisera Rabbit pneumococcal typing antisera were obtained from the Pneumococcal Departm e n t of the \ V . H . O . Centre for Reference and Research, Starens Seruminstitut, Copenhagen, D e n m a r k . Horse meningococcal group A and C antisera were kindly supplied by Dr C. E. Frasch (Bureau o f Biologics, Bethesda, Maryland).

Chromatographic media and gel chromatography Sepharose 2B (lot 7594), Sepharose 4B (lot 3325) and Sepharose 6B (lot 10457) were obtained from Pharmacia (Uppsala, Sweden). All chromatographic media were equilibrated in 0-2 ~,l s o d i u m chloride solution with p H adjusted to 7-0 + 0" t w i t h base as required. C o l u m n s o f 1-6 × 100 c m were emplo~-ed. Gel chromatography of single polysaccharide antigens were performed by loading w i t h about 3 m g ofpolysaccharide. C o l u m n effluents were monitored at 206 n m with 66

ESTIMATION OF POLYDISPERSION OF POLYSACCHARIDES BY IF.P Uvi kord S s p e c t r o p h o t o m e t e r (LKB, B r o m m a , Sweden). Fractions of approximately 4" 5 ml were collected and chemically assayed for uronic acid (types 1, 3 , 8, 9 N ) by the Gregory m e t h o d , 8 m e t h y l pentose (types 2, 6 A , 7F, 18C, 19F, 23F) by the Kabat m e t h o d , 9 hexosamines (types 4, 12F, 14, 25) by the Ashwell m e r h o d , m p h o s p h o r u s (group A) by the m e t h o d of Chen, Toribara and W a r n e r II and sialic acid (group C) according to the m e t h o d of Svennerholm. .2 These fractions were also tested by fused-rocket immunoetectroplaoresis as later described. Gel c h r o m a t o g r a p h y oflyolyvalent pneumococcal vaccine and bivalent meningococcal vaccine was p e r f o r m e d with a loading of the equivalent of ten h u m a n doses of these vaccines, corresponding to a total of about 5 0 0 / a g for each antigen. T h e collected chromatographic fractions were tested by fused-rocket immunoelectrophoresis.

Immunoelectrophoresis Rocket i m m u n o e l e c t r o p h o r e s i s of the pneumococcal polysaccharide type 19F was performed according t o a modified W e e k e procedure: 13 agarose gels (Agarose M=LKB, B r o m m a , S w e d e n ) a t concentrations o f 0-5, l and 2% w/v in 0-02 M Tris barbiturate buffer p H -- 8-6 were e m p l o y e d on plastic plate supports. Each gel contained 0- 59~. v/v of h o m o l o g o u s rabbit a n t , s e r u m c o r r e s p o n d m g to 0-75 ~1 c m ~ gel area. In each a n t i b o d y c o n t a i n i n g agarose gel, 5 /al a l i q u o t s of solutions of polysaccharide (0- I m g ml - ~) at three different K d values (0-10, 0-47, 0-71 on Sepharose ZiB), were loaded. Electrophoresis was performed at; 70 v/cm for 2 h in a M u l t i p h o r electrophoresis apparatus ( ' L K B ~ B r o m m a , Sweden) at constant t e m p e r a t u r e ( 15 4- IoC). Following electrophoresis, gels on plastic plate supports were moistened w i t h distilled water, pressed w i t h filter paper and d r i e d at 37°(2 for 30 rain. Rocket immunoprecipi= tates were then detected by Coomassie Blue-R 2 5 0 stain. Fused-rocket i m m u n o e l e c t r o p h o r e s i s of polyvalent vaccines and of the single polysaccharide antigens, following the molecular sieving on different Sepharose gels, was performed according to a modified Svendsen metl*od: 14 10 /tl aliquots of the c o l u m n fractions were transferred to a row of wells in an antibody-free agarose gel 1% w/v. Samples were allowed to diffuse into the gel for 60 rain. Electrophoresis was performed in agarose 1% w/v c o n t a i n i n g 0" 5% v/v of h o m o l o g o u s a n t i s e r u m to a specific polysaccharide a n t i g e n u n d e r the same _conditions described for rocker i m m u n o e l e c trophoresis.

Quantitatiz~e precipiti~ procedure for nitrogen antibody estimation T h e m i c r o p r e c i p i t i n t e c h n i q u e of Schiffman, Kabat and T h o m p s o n was used as modified by Etzeler.*3 Briefly, aliquots of solutions of type 6A pneumococcal polysaccharide at t w o different K d values (0-.17 and 0-51) and type 19F pneumococcal polysaccharide at three different K d values(O- 10, 0-47 and 0-7 I) containing a m o u n t s ofpolysaccharide r a n g i n g b e t w e e n 1 - 1 0 / * g , were incubated for 1 h at 37°(2 in conical tubes w i t h a constant v o l u m e ( 1 0 / a l ) of h o m o l o g o u s rabbit antiserum. T h e final salt concentration was 0-95~ s o d i u m chloride and th e final v o l u m e in each tube was 100 ~*l Tube s were k e p t at 4°(2 for 18 h, then th e precipitates were c o l l e c t e d b y centrifugation at 10 0 0 0 r rain -1 and waghed in cold saline. Nitro~g,en was d e t e r m i n e d in the washed precipitates by the n i n h y d r i n m e t h o d of Etzeler, 15 using a m m o n i u m sulphate as standard. T h e calculated q u a n t i t i e s o f a n t i b o d y n i t r o g e n in t h e precipitates collected were p l o t t e d a g a i n s t t h e respective a m o u n t s o f polysaccharide used to check the

67

M. P O R R O E T A L .

behaviour of the typical i m m u n o p r e c i p i t a t i o n curve. From this curve, the m a x i m u m and average value of the nitrogen antibody quantity precipitated by I ktg of polysaccharide (at different Kd's) were calculated.

Determination of the distribution coefficient (Kd) of polysaccharides Calibration of the chromatographic columns was performed by d etermin atio n of interstitial volume (Vi) using s o d i u m azide and void volume (Vo) using Blue Dextran 2000 (Pharmacia-Uppsala, Sweden). In each chromatographic run, Vi was d e t e r m i n e d from the position of the elution peak for s o d i u m azide. The elution volume (Ve) of the Polysaccharide antigens was calculated as follows: the chemical a m o u n t or the height of the i m m u n o p r e c i p i t a t e rockets d e t e r m i n e d in each of the chromatographic fractions collected were plotted against the c h r o m a t o g r a p h i c effluent volumes. On the m a x i m u m peak of the curve obtained, two tangents were drawn and their intersection point represented the value of the elution c o l u m n . The Kd value corresponding to the Ve value was calculated according to Acker's formula Kd ---- (Ve -- Vo)/(Vi -- Vo). t¢' RESULTS Table I summarizes the distribution coefficient values of the single polysaccharides investigated, calculated according to Acker's formula. Comparisons of the results of chemical or i m m u n o e l e c t r o p h o r e t i c analyses for single pneumococcal and meningococcal antigens are shown. Similar values were also found w h e n the multivalent vaccines were subjected to i m m unoelectrophoret ic invesrigat ion for each polysaccharide component after rfiolecular sieving on Sepharose 4B. Type 7F and Type 14 pneumococcal polysaccharides were not included because of the absence of anodal migration of these antigens at p H 8-6, when subjected to an electric field. 7 Experiments under similar conditions using a borate-derivative buffer I7 for these polysaccharides are planned in our laboratory. Plate I shows some examples of fused-rocket immunoelectrophoresis for pneumococcal antigens after gel c h r o m a t o g r a p h y of the m u l t i v a l e n t vaccine on Sepharuse 4B and Plate 2 shows a comparison between physico-chemical and immu n o electrophoretic analysis of the molecular w e i g h t polydispersion of two polysaccharide antigens. I m m u n o e l e c t r o p h o r e t i c analysis gave good reproducibility of results w i t h a m a x i m u m discrepancy corresponding to 0"02 for a typical Kd of several polysaccharides. Some single polysaccharides were also tested on different gel matrices to compare the i m m u n o e l e c t r o p h o r e t i c behaviour o f the molecular weight polydispersion of polysaccharides unde r conditions of different molecular sieving. Plate 3 shows a comparison of molecular polydispersion detected by immunoelectrophoresis, obtained from Sepharose 4 B and 6B for the single pneumococcal type 8 polysaccharide. A similar comparison for pneumococcal type zi polysaccharide from Sepharose 2B and 4B is shown in Plate 4. As recorded in Table 1, differences existed in all Kd values d e t e r m i n e d by chemical or i m m u n o e l e c t r o p h o r e t i c procedures and were more m a r k e d w h e n the molecular sieving properties of the gels were increased. T h e reasons for these differences were attributed as follows: the possible molecular sieve effect afforded by agamse gel support in i mmunoe le c t r ophor e s i s on different ch ro mato g rap h ic polydispersion, and the possibility that a t y p i n g a n t i s e r u m could form different amourtts of i m m u n o p r e c i p i t a t e s w h e n reacting w i t h different polysaccharide polydispersions. R o c k e r ]mmunoelectrophoresis 68

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tography of multivalent vaccines (set- Mrtho,J,'~) on Seph,lrost- ,tB. I)neumoco~(al/~)1).s.1¢¢ harzdc's: (.=) tylx" I, (b) type 6A, (c) t)'p¢ 18C, (d) tylx" 25, re) type 9 N , if) typc 191:, (~:) tyix" t21". Arn)~.~'~ llkllt,~tc the chromau)graphic fraction corresponding to the ~l~id volumc.

t/,~a,lg pu~qt 6,~ ~

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Pla~e 2. A direct comparison between physico-chcmica! and immunociectrophoreric analysis of ~he molecular weight polydispersion of type 1 (a) and type 12F (b) pneumo~occal polysaccharides after gel chromatography on Sepharose 4B. ( A ) physical analysis (reading at 206 am); (O) chemical analysis (uronic acid for type 1 potysaccharide and hexosamines for type t2F polysaccharide). Arrow's indicate the c h r o m a t o g r a p h i c fraction corresponding to the void volume.

(b)

Plate 3. lmmunoelectrophoredc comparison of molecular polydispersion of"pneumoct~ccal polysaccharide type 8 after molecular sieving on different gel matrices. (a) from Sepharose 4B, (b) from Srpharose 6B. Arrows indicate the chromatographic fraction corresponding to the void volume.

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Plate 4. Immunoelectrophoretic comparison of molecular polydispt.rsion ofpneumococcal polysaccharide type 4, after molecular sieving on different gel mairic(~. (a) from Sephatose 2B, (b) from Sepharose 4B. Arrows indicate the chromatographic fraction correspondin~ to the void volume.

(a)

(b)

(c)

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2

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Plate 5. Rocker immunoelcctrophorcsis on agarose gel suplx)rts of three different molecular distributions of the type l°,F pneumococcal l',olysaccharide, l--polysacchar~de with Kd = O" IO (O. 1 mg m l - l). 2--polysaccharide with Kd = 0.47 (0-1 mg m l - t ) , 3--polysaccharide with Kd = 0"71 (0.1 mg ml - I). Agarost. gel supports were at the concentration of: 0"St;; w/v (a), 19; w/v (bL and 2r/ w./v (c).

XO

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Kd on Sepharose 6B

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Pneumococcal type 1 2 3 4 6A 8 0"01 9N 0.01 12F 18C 19F 2~)F 25 Meningococcal group A C 0'05 0'09

0'04 0.08

lmmunoelectroPolysaccharide Chemical phoretic antigen procedure procedure A Kd

-

.

0'48 0'37

0'15 0'11 0,12 0'05 0.14 0'08 0'09 0'30 0'37 0'26

0'01

0"13 0.20 0'09 0"20 0'19 0'~,2 0.25 0'22 0,18 O' 12 O. 14 0'34

0'05 0'05 0.05

0'11 0' ! 1

0'08 0'15 0.04 0' 19 0'04 0,21 0,13 0'17 0.04 0'04 0'05 0.04

lmmunoelectro, Chemical phoretic procedure procedure A Kd

Kd on Sephamse 4B

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lmmunoelectto. Chemical phoreric procedure procedure

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TABLE 1. A comparison of the Kd values of some bacterial capsular polysaccharide preparations obtained by chemical and immunoelectrophotetic analyses of column effluents from different chromatographic media

0.24 0' 18

0.22

Kd

5tm

O t-'

O

5

t~

O

Z O

5

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,-1

M. PORRO E T AL.

TABLE 2. The quantitative precipitation o f rabbit typing sera by polysaccharities with different values of distribution coefficient Polys~ccharide type

Kd polysaccharide (chemical procedure)

6A

0- 16 0-51 0-05 0-47 0-71

19F

Hg N A b " /~g-t potysaccharide /Maximum Average 3"00 1-50 5"50 2-85 0"31

2-76 1-31 1- 15 0-70 0-15

• Nitrogen antibody quantity. The values represent the maximum and the average amount of antibody nitrogen precipitated by 1 ~g of'the indicated polysaccharide on the basis of the immuno-precipitation curves obtained as described in the Methods section. in P l a t e 5 s h o w s t h e m o l e c u l a r sieve effect o f s o m e agarose g e l s u p p o r t s , in a d d i t i o n to a d i f f e r e n t b e h a v i o u r o f t h e a n t i s e r u m , o n t y p e 19F p o l y s a c c h a r i d e w i t h d i f f e r e n t d i s t r i b u t i o n coefficient values. In fact, t h e 'rockets" s h o w e d a g o o d d e f i n i t i o n o f c o n t o u r s o n l y w h e n a p o l y d i s p e r s i o n o f h i g h m o l e c u l a r w e i g h t o f t h e p o l y s a c c h a r i d e was e m p l o y e d a n d r h e d i f f e r e n c e s in t h e h e i g h t s o f r h e r o c k e t s b e t w e e n t h e d i f f e r e n t m o l e c u l a r p o l y d i s p e r s i o n s o f t h e s a m e a m o u n t o f p o l y s a c c h a r i d e were m o r e e v i d e n t w h e n t h e c o n c e n t r a t i o n o f t h e agarose g e l s u p p o r t was increased. T h e d a t a s h o w n in T a b l e 2, o b t a i n e d w i t h t y p e s 6 A a n d 19F p n e u m o c o c c a l polysaccharides, quantitatively confirm the finding that different molecular weight polydispersions ofa polysaccharide precipitate different amounts of typing antibodies. A l t h o u g h d i s c r e p a n c i e s existed b e t w e e n t h e c h e m i c a l a n d i m m u n o e l e c t r o p h o r e t i c d e t e r m i n a t i o n s o f K d values, it was possible ro i n v e s t i g a t e t h e b e h a v i o u r o f t h e m o l e c u l a r p o l y d i s p e r s i o n o f b a c r e r / a l c a p s u l a r p o l y s a c c h a r i d e s p r e s e n t in a m u l t i v a l e n t v a c c i n e by c o m p a r i n g i m m u n o e l e c r r o p h o r e t i c analysis o f c h r o m a t o g r a p h i c effluents at t h e t i m e o f vaccine p r e p a r a t i o n a n d at later t i m e s . T a b l e s 3 a n d 4 s h o w r e s p e c t i v e l y , a p o t e n c y c o n t r o l for s o m e a n t i g e n s p r e s e n t in t h e p o l y v a l e n t p n e u m o c o c c a l vaccine a n d for t h o s e p r e s e n t in t h e m e n i n g o c o c c a l b i v a l e n t vaccine.

TABLE 3- lmmur~oelectrophoretic determination of Kd values o f pneumococcal polysaccharide at different times after manufacture using Sepharose 4B Kd values

Polysaccharide antigen

0P

12*

18*

Maximum A Kd

6A 18C 19F 23F

0-21 0-21 0- 12 0" 14

0-23 0-2L 0- 11 0- 15

0-23 0-21 0-11 0- 15

0-02

o "F~rne (months).

70

0-01

ESTIMATION

OF POLYDISPERSION

OF POLYSACCHARIDES

BY I E P

TABLE 4. Immunoelectrophoreticand chemical detection of~lroup A and C meningococcal polysaccharides present in bivalenr vaccine after separation on Sepharose 4B at different times after manufacture Kd values

Chemical procedure Polysaccharide antigen Group A Group C

] m m u n o e l e c t rophor<'t ic p r o c e d u r e

O"

18"

0"

18 s

0- 37 (75-5%) 0-26 (88-9%)

0-44 (7 1-89~) 0-26 (8R- 5~ )

0,48

0'55

0-3"I

0-37

• T i m e (months}. T h e K d ~ealues are calcu]arod o n the second m a j o r peak o f the c h r o m a t o g r a m . T h e values i n p a t e n : b e s e t mdicatecl the c h e m i c a l a m o u n t of" polysaccharide recovered at K d = 0 - 5 .

DISCUSSION Bacterial capsular l~lysaccharides are found to be polydispersion systems when purification following their isolation is performed, For bacterial capsular polysaccharides present in vaccines for h u m a n use, the characterization of rhe polysaccharide molecular weight polydispersion is expresse d as the Kd value calculated after gel chromatography o f the single polysaccharide antigens. 4"5"¢' Because the Kd value represents a significant porency rest for the i m m u n o g e n i c i r y of the meningococcal polysaccharides in man 2"18 and for poJysaccharides in general, t'l~ we examined the molecular weiBhr polydispersion of each polysaccharide anriBen present in mulrivalent vaccines, such as t h e 14 valent pneumococcal polysaccharide vaccine, in order to control the i m m u n o g e n i c activity of the vaccine with time. Fused-rocket immunoelectrophoresis using rabbit pneumococcal typing antisera appeared to be a sui table m e t h o d to visualize specifically the chromatographic behaviour of each polysaccharide antigen present in the vaccine. A similar i m m u n o e l e c t r o p h o r e t i c m e t h o d was recently adopted as a q u a n t i t a t i v e assay to verify the a m o u n t of polysaccharides in the pneumococcal vaccine in the final container.7 These authors reported in their experimental conditions, a h i g h specificity between rabbit pneumococcal typing antisera and the respective polysaccharide monovalent preparations. Similar findings were found in our experiments when both monovalent or multivaten: pneumococcal polysaccharide preparations were investigated" only one precipitin line was-observed. Meningococcal polysaccharides of group A and C were equally specific w h e n rested with their respective antisera. T h e present procedure appeared to be h i g h l y sensitive and reproducible, However, differences in t h e Kd values of single polysaccharides were found from those obtained by chemical or physical analysis'of the same chromatographic effluents, These differences were investigated and ascribed to at least two factors: the molecular sieving effect of the agarose gel support on the molecular polydispersion o f the polysaccharides, and the different q u a n t i t y o f immunoprecipirates obtained when different molecular sizes of a 7t

M. P O R R O l i T A L .

polysaccharide polydispersion were precipitated by t h e same amount of specific antiserum. The latter observation is i n g o o d agreement with that published by Lee, Pearson and Robbins, 7 which recorded a change between the molecular size of the pneumococcal capsular polysaccharides and their mass/'rocket" height ratio. However, the discrepancies of Kd values may be considered a minor disadvantage since a relative measure of the Kd values ofpolysaccharides at the time of vaccine preparation and in the following storage period may be performed. The gradual depolymerization of the molecular polydispersion of pneumococcal polysaccharides is reported to be more rapid when polysaccharides are stored in aqueous solutions than when kept at --20°C as dried powder. 7 A potency control for pneumococcal vaccine relative to time may be performed by investigation of the immunoelectrophoretic molecular weight polydispersion of all or some of the polysaccharides such as types 6A, 18C, I9F and 23F, which contain in their molecular structure the potential acid-base labile and temperature sensitive phosphodiester bond. 2° Alternatively, a method to check the Kd shifts ofpneumococcal polysaccharides could be done by storing the individual solutions of the polysaccharide components for the same time and under the same conditions as the vaccine preparation. This procedure became laborious and differences in the stability of the stored antigens in individual solutions or mixed with the other polysaccharides could exist. Using the present procedure, the absence of significant shifts in the distributioncoefficient values of types 6A, 18C, 19F and 23F p01ysaccharides present in the pneumococcal vaccine were determined 18 months from the date of the vaccine preparation (Table 3). In the case of multivalent meningococcal polysaccharide vaccine, requirements for the determination of distribution coefficients of t h e a n t i g e n s in the final container exists5"21Formeningococcal group A and C combined vaccine a chemical differentiation of the antigens is possible and easy, although a problem similar to that o f the pneumococcal polyvalent vaccine is encountered when a. tetravalent meningococcal v a c c i n e c o n t a i n i n g .groups Y and W 135 needs tO be investigated for the _distribution coefficient values of the single antigens. For this purpose it must be pointed out that, as shown in Table 4, the immunoelectrophoreti~ detection 0 f K d values for group A and C polysaccharide gave discrepancies with the chemical determination of Kdyalues which, for. a potency control with t i m e , provided unsatisfactory r e s u l t s o f stability of the molecular polydispersion of the po!ysaccharides w h e n compared wit h the polydisper, sions detected by chemical analysis. However; corrections in these values could be made by considering the difference in t h e K d ValUeS shown by the two methods of analysis o f the single polysaccharide a n t i g e n s . The problem of the detection of the amoun t of antigen recovered after gel chromatography, a l t h o u g h not .reclu!r~ for mefiingococcal

polysaccharidescombined in thevaccine, deservessome Consideration.Meningococcal polysaccharides.showed a larger .molecular ~weight polydispersion ,_.after molecular sieving on Sepharose gel, thari pnetunoc0ccal i~lysaccharides.ilTheseP~2operties 0f the polysaccharide ~:af!tigens, : as mentione d above, i ~ e responsible ?for a Change i~i n immunoelectrophoresis i n the/corresp0nding:: pO!ysacc~idel ~c0ncent~t!ord'rocket heightlrati0. Th~us, a : q ~ t i t a t / v e estimation o f a pglysacchafide may h ~ o m e very in~ i f ~ k e t im~un~ 0e!ecTcr0Phoresis i # peff.0rmed 0n differen~ : m o l e c u l ~ i s i z ~ o f a /nolecular P01ydispe~i0n u n l ~ s i ~ m ~ y selected nac/ie0~ar Sizes of t h e s a m e polysac~:ha~ide a r e a v a i l a b l e a s there a~re:=standards. The nature o f the: different r0¢~ket ~i mm, tmop~cipitares-obtained using the~same a m o u n t o f aniiserum agairisi:-a p 0 1 y s a c ~ i d e ~ w i i h . i t i f f d r e n t polydispersions w a s n o t 72

ESTIMATION OF POLYDISPERSION OF POLYSACCHARIDES BY IEP i n v e s t i g a t e d , b u t r e a s o n a b l e e x p l a n a t i o n s c o u l d be f o u n d in t h e h e t e r o g e n e i t y o f t h e a n t i b o d y p o p u l a t i o n s p r e s e n t in t h e r a b b i t a n t i s e r a w i t h respect to t h e p o l y s a c c h a r i d e d e t e r m i n a n t ( s ) r e c o g n i z e d a n d / o r in t h e h e t e r o g e n e i t y o f t h e affinity o f a n t i b o d y populations reacting with the polysaccharide determinant(s). 13ecause these bacterial c a p s u l a r p o l y s a c c h a r i d e s are c o m p o s e d o f r e p e a t i n g m o n o or o l i g o s a c c h a r i d e u n i t s , z°'22"z" t h e s e c o n d h y p o t h e s i s c o u l d be m o r e c o n v i n c i n g in t h e l i g h t o f t h e o b s e r v a t i o n s o f K r a u s e 24 a n d H a b e r a n d S t r o s b e r g 2-s on t h e h o m o g e n e i t y o f t h e a n t i b o d i e s to c e r t a i n p o l y s a c c h a r i d e a , t i g e n s in several a n i m a l m o d e l s s u c h as r a b b i t s a n d m i c e , a n d t h e views o f Berzofsky a n d S c h e c h t e r 26 o n t h e c o n c e p t s o f c r o s s - r e a c t i v i t y a n d specificity o f t h e a n t i g e n s . F r o m t h e a b o v e m e n t i o n e d f i n d i n g s , a n o t h e r p o i n t can be raised. T h e i m m u n o c h e m i c a l m e t h o d s e m p l o y e d in t h e a n t i b o d y t i t r a t i o n o f h u m a n sera to m e n i n g o c o c c a I a n d p n e u m o c o c c a l p o l y s a c c h a r i d e vaccine, s u c h as R a d i o i m m u n o a s s a y 27"2~ a n d ELISA, 2~'3° n e e d p u r i f i e d m e n i n g o c o c c a l a n d p n e u m o c o c c a l p o l y s a c c h a r i d e s h o m o l o g o u s to t h o s e p r e s e n t in t h e vaccines. A l t h o u g h in ~ach t i t r a t i o n test p e r f o r m e d , t h e p o l y s a c c h a r i d e a n t i g e n e m p l o y e d is t h e s a m e for 'pre' a n d ' p o s t ' i m m u n i z a t i o n sera, differences in t h e sera titres u s i n g p o l y s a c c h a r i d e s w i t h d i f f e r e n t K d values can exist, a n d t h i s a p p e a r s to c o n s t i t u t e a f u n d a m e n t a l variable w h e n e s t i m a t i o n s o f h u m a n sera t i t r e s are m a d e in several l a b o r a t o r i e s , even w h e n t h e s a m e i m m u n o c h e m i c a l p r o c e d u r e is u s e d . T o avoid s i g n i f i c a n t d i s c r e p a n c i e s t h e t i t r a t i o n o f h u m a n a n t i b o d i e s to p n e u m o c o c c u s or m e n i n g o c o c c u s by i m m u n o c h e m i c a l m e t h o d s s h o u l d m a k e use o f p u r i f i e d bacterial c a p s u l a r p o l y s a c c h a r i d e s w i t h K d values c o r r e s p o n d i n g to t h o s e r e q u i r e d by W . H . O . for t h e p o l y s a c c h a r i d e s p r e s e n t in vaccines for h u m a n use.

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