Non-specific inhibition of the precipitin reaction between teichoic acids and antisera

Immumochemistry., 1973, Vol. 10, pp. 93-99.

Peqlalaoa Press. Printed ia Great Britain

NON-SPECIFIC INHIBITION OF THE PRECIPITIN REACTION BETWEEN TEICHOIC ACIDS AND ANTISERA K. W. K N O X The Institute of Dental Research, United Dental Hospital, Sydney, N.S.W. 2010, Australia and A. J. W I C K E N School of Microbiology, University of New South Wales, P.O. Box 1, Kensington, N.S.W. 2033, Australia (Firstreceived 7 February 1972; in revisedform I I Ju/y 1972) A l m r m - A i n n i n e ester hydrochlorides, which have been used in studying the specificity of antibedim to teichok: a~ds, were shown to cause non-speoSc inhibition of the precipitin rexmiou. Nonspecific inhibition was also cJumed by lysine and glucosamine hydrochiorides and inoqlm~ snits. The marked effect of divalent cations would be consistent with their forming complexes with teichok: acids. It is concluded that the effect is due to an increase in the ion concenU'afion in 8eneral rather tlum to a decrease in pH.

In studying the specificityof the antigen-antibody reaction, the contribution of a component of the

Preparation of cell wall Suspensions of washed oripmisnm were ¢fisrupted with. Ballotini beads (No. 13) and the wall obtained by centrifugalion and waahh~_ with 0"85% NaCI and distilled water (Heptinstall el ai., 1970). A part of the prepm'ation from strata NCIB 7220 was treated with hydroxylamine to remove D-aianine ester residues from teichoic acid (Heptinsudl et aL, 1970; Kelemeumand Bnddiley, 1961).

antisen to the reaction is usually examined by determining the ability o f the isolated component or a derivative to inhibit the reaction. D-Aianine is a charaeteristic component of teichoic acids and, in d e t m m i n i ~ its contribution to serological specificity, frequent use is made o f the methyl ester hydrochl0ride (Matsuno and Slade, 1970; McCarty, 1964; Karakawa and K a n e , 1971). During studies being carried out in these laboratories on the serological proper,.ies of teichoic acids, (Knox and Wicken, 1970, 1972a) it was noted that the alanine esters would inhibit the precipitin reaction even if the teichoic acid lacked alan/he. T h e observations reported in this paper show that the precipitation of teichoic acid by antiserum is particularly sensitive to the ionic concentration, and indicate that alanine esters and other ionized compounds can cause non-specific inhibition o f the precipitin reaction.

Preparalfon of teichoicacid Teichoic acid was extracted from cell wall with cold 10% trichloracefic acid, prec/pitated with 5 vol 96%

ethan~ midpuria~ furtherby ctemmomaphyou Sephadex G75 (Knox and Wickan, 1972a; Wicke~ and Knox, 1970). Po/ysacchar~les Dex~an from Leuconostoc mesenteroides B1415 was kindly provided by Dr. A. Jeanes, Northern Regioaal Laboratory. P e o r ~ IIL, U.S.A., and a prepm'ation of type III pneumococcal polymcchm'ide by Dr. O. Bateman, Agricultural Protection Board of Western Australia. InCh/tots of the precipitinreaction All the inorllank salts examined were analytical grade. Other compounds tested were D-alanine and the methyl ester hydrochiorides of D,L-alanine and L-ainnine (Sisma Chemical Co., SL Louis, Mo., U.S.A.), the bydrochiorides of D,L-lysim~ and D-Siacesamine (British Drug Houses, Poole, England), and N-acetyl~ucosamine (Calbinchzm, Los Anples, Calif., U.S.A.). The methyl ester hydrochloride of l>.siaai~, available as a syrup from Cycio Chemical Co., Los Anples, Calif.. U.S.A., was unstable under the conditions of transport to Australia.

MATDUAI~ AND lm~saOl~ Organ~nu Lactobacillus plantarum NCIB 7220 was kindly suppfied by Dr. M. E. Sharpe, National Institute for Research in Dairying, Reading, Enginnd, and the RI mutant of L. plantarum ATCC 10241 by Professor W. J. Wolin, College of Allrioulture, Univera/ty of Illinois, Urban, III., U.S.A. Strain N C I B 7220 beloulls to serological group D (Sharpe, 1955), the group anti&~n being D-glucosyi-substituted ribitoi teichoic acid (Sharpe, Davison and Baddiley, 1964; Knox and Wicken, 1972a); the RI mutant forms a cell wall teichoic acid virtually lacking the D-glucose substituent found in the parent strain (Douglas and Wolin. 1971). Orlpmisms were grown for 18hr at 37°C in the medium described by Sharpe et al. (1964).

Preparation of antisera The procedures employed were essentially those described previously (Knox et aL. 1970). For the preparation of antiserum to cell wall, suspensions (10 ms/mi 93

94

K.W. KNOX and A. J. WICKEN

RESULTS Precipitin reactions in O"1 M sodium chloride Precipitin reactions were carried out with the antigen and inhibitor dissolved in 0.85% (0.145 M) NaCI resulting in a final NaC! concentration of approximately 0.1M. U n d e r these conditions o-alanine (100 ~mole/0-2 ml) did not inhibit the precipitin reaction between wall teichoic acid from strain N C I B 7220 (10~g/0.2ml) and antiserum 215 (0.2m l) whereas O,L-alanine methyl ester HCI and L-alanine methyl e s t e r - H C l markedly decreased the amount of antibody precipitated Precipitin reaction All precip/tin reactions were carried out in duplicate (Table 1). Removal of ~ a l a n i n e from the wall teichoic acid with 0. I ml or 0.2 ml antiserum and a final volume of 0,6 ml (Knox et al., 1970). Precipitin reactions were initially by treatment with ammonia (Knox et al., 1970) carried out with the antigen and potential inhibitor dis- gave a product retaining 95 per cent of its reacsolved in 0-85% (0-145M) NaCI; thus the final NaCI tivity with antiserum 215, indicating that D-alanine concentration on adding 0.2 ml of each solutior to 0.2 ml was not a major antigenic determinant. The reacserum approximated 0.1 M. To compare the effects of tion of this alanine-free teichoic acid with antidifferent ionized compounds, including NaCI, on the pre- serum 215 was also inhibited by the alanine cipitin reaction, experiments were also carried out in esters (Table 1). Evidence that the inhibition by which 0.1 mi-0-4 ml of a 0.6 M solution of the potential inhibitor in water was added to 0.1 mi serum, volumes these esters was probably due to non-specific made up to 0-5 mi with water, and teichoic acid added effects was provided by the observation (Table 1) that l y s i n e - H C ! and g l u c o s a m i n e - H C l were also (5 ~tg/0-1 mi water). effective inhibitors. However, the specificity of To examine the effect of inhibitors on the precipitation of antibody from buffered serum, antiserum 216 (1 or antiserum to dbitol teichoic acid from strain 1.5 ml) was dialysed for four days at 2-.4"C against 7220 depends on the ~,-D-glucosyl substituents and three changes of 50-60voi of each of 10 buffers of the inhibition by D-glucosamine-HCl could be constant ionic strength, ! I 0- I, between pH 4-6 (acetate) partly due to its specific binding with antibody; and 8.4 (Tris-hydmxymethyl-aminomethane-HCl)with this was further indicated by the observation that phosphate buffers providing intermediate values (Long, 1961); sera dialysed at p H 4,6=6,0 were centrifuged N-acetylglucosamiue inhibited the precipitation of before use. Dialysed antiserum (0-Imi) was diluted teichoic acid, though it was less effective than to 0.3 ml with the dialysis buffer of the same p H and glucosamiue hydrochloride. Evidence that glucosamine-HC1 could cause 5/~g, 10/~g and 15/~g ribitolteichoic acid (strain N C I B 7220) added in 0.3 mi water, The effect of Dd.-aianine non-specific inhibition was shown by its effect ester-HCl and lysine-HCl on the amount of antibody (Table I) on the reaction between R1 teichoic precipitated was examined by adding 100~tmole/0.1 ml acid (10~tg) and homologous antiserum (0-2ml water to representative buffered s:,"a (0-1 mi diluted to serum 213). This teichoic acid lacks glucose 0.3mi) followed by 10/~g teichoic acid/0.2mi water. substituents and N-acetylglucosamine did not Precipitin reactions were set up at 2-40C and all subsequent steps carried out at this temperature to minimize inhibit. In this system also, the alanine ester the effect of temperature changes on pH. The pH values and lysine hydrochlorides were inhibitory though the effect was less than with antiserum 215 of the superuatant solutions from the precipitin reactions (Table 1). were subsequently read at 6"C.

in 0.85% NaCI) were injected intravenously into rabbits twice a week for 3 weeks, the doses being 0. I mi, 0-2 ml, 0.5 mi and three of 1.0 mi. Sera obtained by the injection of whole organisms or cell wall reacted strongly with the homologous wall teichoic acid and cross-reacted with dextran (Knox and Wicken, 1972a,b). Results are given for antisera 215 and 216 (obtained by injecting whole organisms of strain NCIB 7220). antiserum 226 (hyd~xylamine-treated wall from strain NCIB 7220) and antiserum 213 (whole organisms of the RI mutant). Antiserum to pneumoonccus type III was purchased from Burroughs Welkome & Co., London.

Table 1. Non-specific inhibition of the precipitation of ribiml teichoic acids by antibodies. Antiserum (0.2 mi) was mixed with I00/zmole of each substance (in 0-2 ml 0-85% NaCI) and 10 ~41teichoic acid (in 0-2 ml 0.85% NaCI) added. Results are given for L. plantarum NCIB 7220 and L. plantarum R1 mutant wall teichoic acids and for NCIB 7220 teichoic acid treated with ammonia to remove alanine esters. For each system the amount of antibody precipitated in the presence of the potential inhibitors is expressed as a percentage of the antibody precipitated from 0.2 mi serum by 10/~8 teichoic acid (in 0.4 ml 0.85% NaCI) Percent antibody precipitatedwith inhibitorpresent

Antiserum 215 215 213 226

Teichoic acid NCIB 7220 Ammon/atreated RI mutant NCIB 7220

D,L-alanine L-alanine ester-HCl ester-HCl

L-lysine- D-glucosamineHCI HCI

N-acetylglucosamine

Sodium chloride

51 68

47 65

38 51

31 36

65 76

61 67

76 68

76 64

68 51

56 43

100 78

64 67

Inhibitiou of Pr~ip/tin Rm~tion Antiserum to cell waft that had been treated with hydmxylaminc to remove D-alantne substiments (rabbit 226) was also tested for non-specific inhibitiot~ The results (Table 1) are generally comparable with those o b e y e d for antiserum 215, though the extent of inhibition is less. In addition to the hydrochlorides an increase in the NaC! concentration also inhibited the precipitin reaction (Table 1). To allow comparison with the other results the solution added contained 100~anole NaCI dissolved in 0.2ml 0-85% (0.145 M) NaCI and thus increased the final molarity of added NaCI from 0.1 M to 0-27 M.

95

tOO

<

0'!

Precipitation of polysaccharides in O.I M N aCl To determine whether the effect of the inh~itors was primarily on the teichoic acid component of the antigen-untibody complex, their effect on the precipitation of dextran (30 ttg) by antiu~rum 226 (0.1 ml) was also examined. The inhibitions were comidm'abiy less thaa for the homologous reJtction, the value for lO0~u,mole o,t.-alanine methyl ester-HCI, for example, being 16~. In the reaction between type I I I Imeumococcus polysaccharide (10#40 and homologous antiserum (O.Iml), 100tunole lysine-HC! save 15 per cent inhibitiun whereas D , L ~ e methyl ester-HCl did not 8ive detectable inlu'bition. Effect of ion concentration on the precipitin reaction The observation that an increase in NaCI concentration inhibited the precipitin reaction prompted a comparison of the effect of carrying out precipitin tests in which both the teichoic acid and potential inhibitor were dissolved in water and reaction mixtures were diluted to 0.6 ml with water. The results in Fig. 1 compare the amount of antibody precipitated from 0-1 ml antiserum 226 by 5 ~g N C I B 7220 wall teichoic acid (expressed as 100 per cent) with the amounts precipitated in the presence of 0-I ml-0.4ml of 0"6 M NaCI, D,L-alaniue methyl ester-HCl and lysineHCI, the final concentration thus being 0-1 M-0-4 M. Lysine-HCl was the most effextive inhibitor with NaCI bein8 as effective as the alanlne ester. In contrast, the precipitation of dextran (30 ~g) by antiserum 226 (0-1 ml) was inhibited to only a slight extent by the increase in NaCI concentration. Salts representative of the lyotrophic series were tested at a final concentration of 0.1 M for their ability to inhibit the precipitin reaction between 0-1 mi serum 226 and 5 ~4~ NCIB 7220 wall teichoic acid. the conditions being the same as employed in the previous experiment. In 0.1 M NaF the amount of antibody precipitated was 95 per cent of that precipitated in water, compared with 90 per cent for NaCl. Greater inhibition was achieved with KCI, KI and KCNS where the

O-2

0,3

04

F ~ 1. Effectof~he moJmiW of sodium ~ mNIMm~ ine eater and lysine hydrochlork~ ou the precipitation of anu'body by ~¢ho~ ackl. Antit~'um 226 (0. I ml) w u mixed with 0-I-0-4 ml of 0-6M solutions of each slibsgln~ in watt, dihlt~! to 0.5 ~ with water and t,~.la~ acid (5 ~j/O.! ml watt) 8dded. The amount of antibody wec@imted at each mncenU~ tion of inhibitor (0- i-0-4 M) is exptmsmi as a larcantalge of thin precipitated from ~Jml uaum by teichei¢ reid (5 ~I/0.5ml water): 0, D,L-alanineestm'-HCl; &, lys~.HCl; I"3,sodium chlm-ide. For ¢ o R , the d'ect of increas/~ the raohn'ity of NaCI on the precipitatiou of clesu'an(30 ~ by antiserum 226 is alto shown (0); these results are expretu~ as a percenta~ of the amount of antibody precipitated by dexu.an (30 ~l/0.SmJ water) from serum (0.1 ml).

respective values [or antibody precipitation were 75 percent, 55 per cent and 41 percent. An inhibitory effect b y divalent cations was indicated by experiments showing that in 0.1 M MgCls and CaCIs the respective values for antibody precipitated were 48 per cent and 43 per cent, decreasing in each instance to 15 per cent in 0.4 M salt.

Inhibition reactions in buffered serum Owing to the differences in pH of the solutions of the various hydrochlorides, the final pH at which precipitin reaction were carried out also varied. Under the conditions employed in the experiments with N C I B 7220 wall teichoic acid and homologous antiserum (Table 1), the pH (at room temperature) remained unchanged at 8.5 on adding the NaCI solution (cf. McCarty, 1964), but decreased to 7.7 with lysiue-HCl, 6.8 with V,L-alanine methyl ester-HCl and 6.7 with glucosamine HCI. For reactions carried out in water (Fig. 1), the pH values with 0.1 Mand 0.4 MlysineHCI were 7-8 and 7.5 respectively, whereas with D,L-ainnine methyl ester-HCl the corresponding values were 7-1 and 6-5. To determine whether the observed inhibition by lysine-HCl and D,L-alunine methyl ester-HCl could be accounted for by the decrease in pH,

96

K.W. KNOX and A. J. WICKEN

the effect of pH on the amount of antibody precipitable from antiserum 216 (0.1 ml) by strain NCIB 7220 ribitol teichoic acid was examined. The pH of the precipitin reactions containing 0.1ml dialysed serum (see Methods), 0.2ml dialysis buffer and teichoic acid (5-15/~g/0.3 ml water) in a final ionic strength (1) of 0-05 were as follows, with the pH of the dialysis buffers being given in brackets: acetate buffer 4.55(4-55), 5.15(5-15) and 5-9(6.1); phosphate buffer 5.9 (5.85), 6.55(6.6), 7-1(7.1), 7.6(7.5); Tris-HC1 buffer 7-0(6-9), 7.9(7.7) and 8.4(8-35). The results in Fig. 2 express the maximum amount of antibody precipitated at each pH value as a percentage of the amount precipitable from serum (0.1 nil) in the absence of buffer, but without allowance being made for the increase in volume of serum on dialysis (15-20 per cent). An inhibition of precipitation at low pH is indicated (Fig. 2). but this is not achieved until the pH falls below 5. The maximum amount of antibody was precipitated at pH 5-9, 93-97 per cent compared with 82 per cent at pH 7.6. A similar effect of pH was observed for the precipitin reaction between buffered serum (67/~l) and 20-30/~g of membrane teichoic acid from strain NCIB 7220 (Knox and Wicken, 1972a), whereas in the cross-reaction with dextran (50/~g to 0.5ml buffered serum) the amount of antibody precipitated at pH 5.9 was only 80 per cent of that precipitated at pH 7.9. Table 2 shows the effect of adding 100/~moie lysine-HCl and D,L-alanine methyl ester-HCl on the pH and precipitation of strain NCIB 7220 ribitoi teichoic acid (10/~g) from buffered serum at pH 7.9 (Tris-HCl), 7-6 (phosphate) and 5-9 (acetate). In each case. particularly ~t pH 5-9, inhibition was achieved. Only in the case of adding alanine ester to serum at pH 5-9 would the decrease in pH have contributed to the inhibition, and then by only about 5 per cent (Fig. 2). With the other sera, the decrease in pH on adding inhibitor would have increased the amount of antibody precipitable-by 15 per cent with alanine ester added to serum at pH 7.9.

100~

7s

<

25

I

°a

5

I

I

6

7

Fig. 2. Effect of pH on the precipitation of antibody by ribitoi teichoicacid. Antiserum 216 was dialysed against buffers at different p H values but constant ionic strength (I = 0. I). To tubes containing dialysed serum (0-I ml) diluted to 0-3 ml with dialysis buffer were added 5. I0 and 15 ~ ribitolteichoic acidl0.3 ml water. The maximum amount of antibody precipitated at each pH is expressed as a percentage of the amount precipitatedfrom undialysed serum by adding teichoic acid in water (0-5 ml). The p H values of the reaction mixture were determined by testingthe supernatant

solutions from the precipitin reaction: A, acetate buffer; 0, phosphate buffer: I'1,Tris--HCl buffer. Adding 50/,¢mole phosphate buffer p H 7.6 (0. I rni 0.5 M ) to serum dialysed against phosphate buffer p H 7-6 decreased the amount of antibody precipitated by teichoic acid (10/,¢g) to 60 per cent, showing that phosphate is an effective inhibitor of the precipitin reaction. DISCUSSION

Evidence that D-alanlne could contribute to the specificity of teichoic acid was first obtained by McCarty (1964) who showed that removal of aianine from the glycerol teichoic acid of group A streptococci markedly decreased its reactivity with antiserum; these results were confirmed and extended by Matsuno and Slade (1970). Karakawa and Kane ( 1971), using the same procedures, con-

of antibody precipitated as a percent of that precipitated at each p H in the absence of inhibitor pH Lysine

Antibody (%) Alanine

Control

Lysine

ester

7-9 7.6 5-9

7-6 7-4 5-8

6"5 6-55 5-55

I 9

pH

Table 2. Inhibition of the prccipitin reaction in buffered sera. To dialysed serum (0.I m D diluted to 0.3 ml with dialysis buffer was added 100/=mole inhibitor/0.1ml water and 10/4 ribitolteichoic acid/0-2ml water. The results compare the p H values before (Control) and after adding inhibitors, and express the amounts

Control

l

8

Alanine ester

I00 I00 I00

71 74 50

72 76 60

Inhibition of Precipitin Reaction cluded that v-alanine was also an antigenic determinant of the ribitoi teichoic acid from a Staphylococcus aureus strain. In none of these cases was D-alanine an inhibitor of the precipitin reaction, though D-alanine methyl ester HCI was a more effective inl~ibitor than L-alanine methyl ester HCI. For the two preparations of ribitol teichoic acid from L. plantarum strains, removal of D-alanine residues did not significantly decrease serological reactivity, and both D,L- and L-alanine methyl ester hydrochiorides were equally effective as inhibitors. As shown in Table 1, giucosamine-HCl and iysine-HCl are also effective inhibitors of the precipitin reaction, indicating that the precipitation of teichoic acid by antibodies is sensitive to the presence of the hydrochiorides in general. The alanine esters are thus giving a non-specific inhibition of the precipitation of L. plantarum teichoic acid, an effect independent of the presence of D-alanine as a substituenL Where Dalanine is an antigenic determinant these results would suggest that the reported inhibition by the t,-alanine ester is non-specific and the specific effect by the D-alanine ester less than the results would indicate. Non-specific inhibition may also account for observations that giucosamine-HCl is an effective inhibitor of the precipitation of group A streptococcal glycerol teichoic acid (Matsuno and Siade, 1970) and of the group F streptococcal antigen (Willers et al., 1964). Studies on antigen specificity by the use of inhibitors are routinely carried out in 0.85-0-9% NaC1, though McCarty (1964) also employed serum dialysed against 0.2M acetate buffer pH 6.0. The resultant decrease in pH when hydrochlorides of alanine esters, lysine or glucosamine are added raises the possibility that the inhibitory effect derives solely from the effect of such a decreased pH on the formation of the antigenantibody precipitate (Kabat and Mayer, 1961). It was not possible to divorce inhibition by the hydrochlorides from a decrease in pH. but it was possible to show with buffered solutions that the observed decrease in pH would not be sufficient to cause a significant inhibition of the precipitin reaction, The inhibitory effect of the hydrochlorides would therefore seem to derive from the effects of an ionized compound rather than from a decrease in pH. Confirmatory evidence for a non-specific inhibition by ionized compounds was provided by the results with sodium chloride and other salts as well as phosphate buffer. The concentration of various salts is known to affect the antigenantibody reaction (Kabat and Mayer, 1961; Kleinschmidt and Boyer, 1952), and the observed effect of the different anions, where the inhibitions followed their positions in the lyotrophic series, has also been reported for the reaction of I M M - V o l . 10No. 2 - C

97

egg albumin with specific antibodies (Kleinschmidt and Boyer, 1952). However, the reaction of teichoic acid with antibody is particularly sensitive to increases in ion concentration as is shown by comparison with the slight effects on dextran and pneumococcal type III polysaccharide. The present studies have also shown that the divalent cations give a greater inhibition of the precipitin reaction than movalent cations, the amount of antibody precipitated in 0.1 M CaCI2 and MgCI~ being approximately half of that precipitated in 0.1 M NaCI. This could be accounted for by their ready formation of complexes with phosphate groupings of a teichoic acid molecule. Evidence for the formation of such complexes by ribitoi teichoic acid has been presented by Heptinstall et al. (1970), and it is proposed that their formation by membrane glycerol teichoic acid would provide a means of maintaining a high concentration of divalent cations in the region of the membrane (Heptinstall et al., 1970; Hughes et al., 1971). Non-specific inhibition of the precipitation of teichoic acid by phosphates was also observed by Morse (1963) who studied the poss~ie role of sugar phosphates in the specificity of Staph. epidermidis glycerol teichoic acid. However. other investigators who have proposed a role for phosphorylated components as antigenic determinants in group N streptococcal glycerol teichoic acid (Elliot't, 1963), Bacillus subtilis W23 teichoic acid (Chin et al., 1966) and yeast phosphomannan (Raschke and Ballou, 1971) did not allow for the possibility of non-spocific inhibition. Recent studies with the plant iectin concanavalin A have shown that its reaction with telchoic acids, but not neutral polysaccharides, is particularty sensitive to the concentration of inorganic phosphate (Reeder and Ekstedt, 1971) and other salts (Doyle and Birdsell, 1972). Doyle and Birdsell (1972) propose that the teichoic acid molecule changes from a rigid rod conformation in low ionic concentration to a random coil which masks antigenic determinants at high ionic concentration; their observation of a decrease in intrinsic viscosity of teichoic acid solutions on increasing the ionic concentration supported the suggestion of a change in shape. A change in shape could also account for the present observation that the maximum amount of antibody is precipitated by teichoic acid (but not dextran) below pH 6. Evidence for an effect of pH values below 6 on the configuration of teichoic acid occuring as a surface component of Staph. aureus has been provided by James and Brewer (1968) who studied the electrophoretic mobility of cells as a function of pH. Acknowledgement-This work was supported by a grant from the National Health and Medical Council of Australia.

98

K. W. K N O X and A. J. W I C K E N REFEIIF..NCF..S

Chin T., Burger M. M. and Glaser L. (1966) Archs Biochem. Biophys. 116, 358. Douglas L. J. and Wolin M. J. (1971) Biochemistry 10, 1551. Doyle R. J. and Birdsell D. C. (1972)J. Bacteriol. 109, 652. Elliott S. D. (1963) Nature, Lond. 200. 1184. Heptinstall S.. Archibald A. R. and Baddiley J. (1970) Nature, Lond. 225, 519. Hughes A. H., Stow M., Hancock I. C. and Baddiley J. (1971) Nature, Lond. 229, 53. James A. M. and Brewer J. E. (1968) Biochem. J. 107, 817. Kabat E. A. and Mayer M. M. (1961) Experimental lmmunochemistry, p. 42. Thomas, Springfield, Ill. Karakawa W. W. and Kane J. A. (1971) J. Immun. 106. 900. Kelemen M. V. and Baddiley J. (1961) Biochem. J. 80. 246. Kleinsehmidt W. J. and Boyer P. D. (1952) J. lmmun. 69, 247. Knox K. W., Hewett M. J. and Wicken A. J. (1970) J. gen. Microbiol. 60, 303.

Knox K. W. and Wicken A. J. (1970) J. gen. Microbiol. 63,237. Knox K. W. and Wicken A. J. (1972a) Infect. Immun. 6, 43. Knox K. W. and Wicken A. J. (1972b)Archs. oral Biol. 17, 1491. Long C. (1961) Biochemists' Handbook. pp. 31-33. Spon, London. Matsuno T. and Slade H. D. (I 970) J. Bacteriol. 102. 747. McCarty M. (1964) Proc. natn. Acad. Sci. U.S.A. 52. 259. Morse S. I. (1963)J. exp. Med. 117, 19. Raschke W. C. and Ballou C. E. (1971) Biochemistry. 10, 4130. Reeder W. J. and Ekstedt R. D. (1971)J. Immun. 106. 334. Sharpe M. E. ( 1955)J. gen. Microbiol. 12. 107. Sharpe M. E., Davison A. L. and Baddiley J. (1964) J. gen Microbiol. 34, 333. Wicken A. J. and Knox K. W. (I 970)J. gen. Microbiol. 60, 293. Willers J. M. N., Michel M. F., Sysma M. J. and Winkler K. C. (1964) J. gen. MicrobioL 36. 95.