Acta path. microbiol. scand. Section B. 80,292-296, 1972

STUDIES ON THE COMPLEMENT FIXATION TEST WITH

MYCOPLASMA PNEUMONIAE ANTIGEN 7 . Purification of Not Cell-Bound Antigen in Broth Culture

JAN ENO

The Bacteriological Laboratory, UllevHl Hospital, Oslo, Norway

Methods for purifying the not cell-bound complement fixing antigen (NCBA) of M . pneurnoniae, present in cellfree filtrates from broth cultures of the organism, have been examined. Precipitations with ammonium sulphate (AS) and isopycnic CsCl gradient centrifugation have both proved effective methods by which to separate NCBA from broth medium proteins. Using 31 per cent AS, ab. 40 per cent of the NCBA measured in native filtrates was regained in the precipitates, the protein content per CF-unit of which was ab. 5 per cent of that in the native filtrates. With increasing salt concentrations, the protein figure of the precipitates rose far more steeply than the NCBA yield, resulting in an inverse ratio between the yield and the degree of purification. The AS precipitation method also facilitated a substantial concentration of NCBA. In the CsCl gradient centrifugation, NCBA was located in the buoyant density range 1.084-1.105 g/ml, indicating a lipid nature of NCBA. By a combined application of 31 per cent AS precipitation and CsCl gradient centrifugation, a concentrated preparation of NCBA was produced with a yield on the 30 per cent level and a protein figure below 1 per cent of that in native filtrates.

The not cell-bound complement fixing anti- isopycnic gradient centrifugation employing gen (NCBA) of M. pneumoniae in broth cesium chloride was found to be effective in culture has been described in earlier papers attaining a concentration and a separation of referred to in (3 ,4) . NCBA is readily fil- NCBA from broth medium proteins. The re- terable through membrane filters which ef- sults of the application of these methods are fectively retain colony forming units. In reported. native filtrates, NCBA is present in a highly impure state. The purpose of the present examination has been to determine methods

M A T E R I A L S A N D M E T H O D S

by which the antigen might be purified; the protein figures and the complement fixing (CF) titres of the materials have been used as parameters. The combined use of preci- pitation with ammonium sulphate (AS) and

Received 26.x.7 1 Requests for reprints should be addressed to:

Dr. ]an Eng, UllevHl Hospital, The Bacteriological Laboratory, Oslo, Norway.

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The BHrd strain of M . pneumoniae was cultivated in PPLO broth medium; M.pneurnoniae cells washed free from NCBA were used as inoculum. The development of NCBA in the broth cultures was examined by the direct measurement technique. Within 2 days of NCBA being first demonstrable in filtered samples of a culture, the whole broth culture was passed through Millipore @ filter discs, type GS. The native filtrate thus obtained w a s the starting material of the purification experiments. All methods employed in the production of native

filtrates along the above lines are referred to in (3). The CF technique employed is described in (2) . After inactivation at 56” C for 30 min, the mate- rials were tested against a diluted anti-M. pneu- moniae rabbit hyperimmune serum containing 4 CF-units for linear measurements of their CF- titres. The CF-titre was read as the dilution giving 50 per cent inhibition of haemolysis. For estima- tion of the protein contents of the materials, two methods were applied: 1. the Folin-Ciocalteu re- agent was used as described and referred to in (2), 2. a modified Biuret method was employed as described in (7). The results of the Biuret tests were read in a Perkin Elmer Spectrophotometer a t wavelengths of 310 mp and 390 mp and the pro- tein figures read from a standard curve based upon crystalline egg albumin. Precipitation of native filtrate with AS was performed with (NH,),SO, pro analysis “Merck” at p H 7. The p H of the filtrate was adjusted to 7 with 1 M Tris-maleate buffer pH 8.1 (5). The AS precipitation technique employed was as described in (3). After centri- fugation, the precipitate was resuspended in veronal buffered saline p H 7.2 (“diluent”), and then dia- lysed at + 4°C against several changes of diluent un- til the addition of a saturated solution of BaCl to a sample of the dialysate no longer resulted in a visible precipitation, thus indicating a high degree of freedom from AS. The volume of the AS pre- cipitate after dialysis was measured. For isopycnic gradient centrifugations, a 60 per cent w/w stock preparation of CsCl “BDH’ was used for prepar- ing aqueous solutions with buoyant densities of 1.4, 1.3, 1.2 and 1.1 g/ml. One ml of each solution was deposited in layers in the foregoing sequence in 5 ml cellulose nitrate centrifuge tubes, with 1 ml of the material to be purified at the top. The tubes were centrifuged in a Spinco ultracentrifuge, model L, rotor SW 50, a t 45,000 rpm for 19 hours. The tubes were harvested from the bottom by puncture, 18 fractions of equal volume being collected by means of a Buchler polystaltic pump. The buoyant density of individual fractions was determined by direct weighing in tared 0.1 ml micropipettes.

E X P E R I M E N T A L

The effect of the AS concentration upon the yield and degree of purification of NCBA was examined by precipitation experiments with three different salt concentrations; these are defined as follows:

“31%AS”: to 100 ml filtrate was added 18.13 g AS “36%AS”: to 100 ml filtrate was added 21.75 g AS “47%AS”: to 100 ml filtrate was added 29.00 g AS

“ % A S ’ corresponds to per cent saturation at 25°C (6).

Each concentration of AS was added to a sepa-

rate volume of 24 ml filtrate; the amount of AS to be used was calculated from the total volume obtained after addition of the amount of 1M Tris- maleate buffer needed to bring the p H to 7.0. The precipitations were performed as described in “Methods”. The precipitates were resuspended in 1.5 ml diluent and the actual volumes of the dia- lysed precipitates gauged at the end of the dialysis. The CF-titres of the three precipitates and that of the native filtrate were measured in the same CF set-up. Two different native filtrates, produced in separate cultivation experiments, were precipitated with AS as described. All measurements of the protein figures of the filtrate and the AS pre- cipitates in Exp. 1 were performed by the Biuret method, in Exp. 2 the Folin test was used through- out. A separate preparation of 31 per cent AS precipitate was further purified by CsCl gradient centrifugation. One ml AS precipitate was de- posited on the top of each of two identical tubes as described in “Methods”. After centrifugation and fractionation of the gradients, the CF-titres of the fractions and that of the 31 per cent AS pre- cipitate were measured in the same CF set-up. Attempts to estimate the protein figures of the fractions from Gradient 1 by the Folin test were partially unsuccessful, as precipitates were formed following addition of the Folin-Ciocalteu reagent. In fractions rich in proteins, the blue colour formed was bound to the precipitate. The protein figures of the fractions from Gradient 2 were estimated by the Biuret method.

R E S U L T S

The two separate filtrates employed in the AS precipitation experiments had CF-titres of 12 and 10, and their protein figures were 22,000 and 14,750 pg/ml respectively. Table 1 shows the yields and the protein figures of the resulting precipitates. The yields are given as the number of CF-units (CF-titrexvo- lume) measured in the dialysed precipitates expressed as a percentage of the number measured in 24 ml native filtrate. The protein figures in pg/ml per CF-unit were calculated by dividing the protein figures of the preci- pitates in pg/ml by their CF-titres. The values found in the native filtrates are also given at the foot of the table. The table shows that, by using 31 per cent AS, 42/35 per cent of the NCBA in the native filtrate was recovered in the precipitate, the protein figure of which in pg/ml per CF-unit was

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Fig. 1 . Purification of a 31 per cent ammonium sulphate precipitate (see text) of not cell-bound com- plement fixing antigen (NCBA) of Mycoplasma pneumoniae by means of isopycnic CsCl gradient cen- trifugation. The CF-titres, protein figures, and buoyant densities of individual fractions are shown.

4/7 per cent of that in the native filtrate. With increasing salt concentration, the pro- tein figure of the precipitates rose far more steeply than the NCBA yield.

CF examination of the fractions from the CsCl gradient centrifugations yielded very similar results in both gradients, the highest titre being found in the top fraction (fr. 18); this fraction had a higher CF-titre than the 31 per cent AS precipitate to be purified. The following three fractions (17-15) showed decreasing titres, and no NCBA could be

traced below fraction 15. The bottom frac- tions, 6-1, displayed an anticomplementary effect when tested in diluent. The CF-results of Gradient 2 are shown in Fig. 1, which also gives the protein figures and the buoyant density values of the fractions. The NCBA- containing fractions had a buoyant density of from 1.084 g/ml in fr. 18 to 1.105 g/ml in fr. 15. Protein was measured in all fractions (except fr. 2) , but the distribution of pro- teins was characterized by a marked concen- tration in the fractions 7-3, corresponding to

TABLE 1. Precipitation with Ammonium Sulphate ( A S ) of Not Cell-Bound Complement Fixing Antigen of Mycoplasma pneumoniae from Native Broth Culture Filtrates-Effect of the AS Concentration upon the Yield and the Protein Content of the Precipitates. The results of T w o Experiments Are Shown

No. of CF-units precipitated

abs. fig.

Protein content of precipitates in % of in &ml in % of

filtrate fig. per CF-unit filtrate fig. % AS

31 120/84 42/35 73/104 4/7 36 191/116 66/48 234/386 13/26 47 234/221 81/92 580/621 32/42

Native filtrate 288/240 1833/1475

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TABLE 2. Purification of a 31 Per Cent Ammonium Sulphate (AS) Precipitate (See Tex t ) of Not Cell- Bound Complement Fixing Antigen (NCBA) of Mycoplasma pneumoniae by Means of Zsopycnic CcCl Gradient Centrifugation. The Yield and the Protein Figure of the NCBA-Containing Fractions Com-

pared with That of the AS Precipitate Are shown

1 ml 31 Yo AS precipitate 48 12,850 268 Fractions 18-15" 39 1,023 26 Fractions 18-1 39 13,066

buoyant density range 1.084-1.105 g/ml.

TABLE 3. p H 5.3-Treatment of a 31 Per Cent Ammonium Sulphate (AS) Precipitate of Not Cell-Bound Complement Fixing Antigen of Mycoplasma pneumoniae (See Tex t ) . The Effect upon the CF-Titre and

the Protein Figure in T w o Experiments Are Shown

CF-titre Protein in pg/ml

Protein in pg/ml

per CF-unit

51 96 AS precipitate untreated 140/128 12,050/9,270 86/72 31 96 AS precipitate treated at pH 5.3 100/64 8,850/8,000 89/125

the buoyant density range 1.253-1.382 g/ml. Table 2 shows the NCBA yield and the pro- tein figure of the combined fractions 18-15. Out of 48 CF-units measured in the 31 per cent AS precipitate, 39 CF-units (81 per cent) were recovered in the combined top fractions, the protein figure of which in pg/ml per CF-unit was of that in the AS precipitate.

Purification of AS precipitate of NCBA at PH 5.3. This method has been applied to 31 per cent AS precipitate by a procedure de- scribed in (4) ; it consists of removal by cen- trifugation of material which is easily pre- cipitable a t pH 5.3. The procedure removes the slight bluish-white opaqueness present in the untreated 31 per cent AS precipitate (3). The procedure was found essential as a pre- treatment when 31 per cent AS precipitate was used as antigen in immune electron mi- croscopy of NCBA (4). The effect upon the CF-titre and protein figure is illustrated in Table 3, which shows that the procedure causes a stronger reduction of the NCBA

content than of the protein content of the 31 per cent AS precipitate, resulting in an increase in the protein figure in pg/ml per CF unit.

D I S C U S S I O N

In the present work, both AS precipitations and isopycnic CsCl gradient centrifugation have proved effective in separating NCBA of M. pneumoniae from broth medium proteins. The problem of separating NCBA from non- protein medium constituents, e. g. broth me- dium lipids, has not been investigated. The precipitability of NCBA of M . pneumoniae with AS has been reported earlier (1 ), and has been examined in more detail in the pre- sent work. With this method, an inverse ratio between the yield and the degree of purifica- tion has been demonstrated (Table 1 ) . How- ever, even the AS precipitates with yields on the 80-90 per cent level showed significantly lowered protein figures. From the relatively high yields of NCBA obtained with low salt concentrations (31 per cent AS), NCBA of

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M . pneumoniae can be characterized as easily precipitable with AS. The AS precipita- tion method facilitates a substantial concen- tration of the antigen. In the CsCl gradient centrifugation, a further purification of the NCBA in the AS precipitates from broth medium proteins was achieved, and in the top fraction a concentration occured as well. The distribution of antigen between the four top fractions found in the present experiments may, at least partially, have been affected by the technique employed for harvesting the gradient. Antigen was found in the buoyant density range 1.084-1.105 g/ml, indicating a lipid nature of NCBA of M.pneumoniae. By the combined application of 31 per cent AS precipitation and CsCl gradient centri- fugation to native filtrates from broth cul- tures of M . pneumoniae, a concentrated pre- paration of NCBA was produced with a yield on the 30 per cent level and a protein figure below 1 per cent of that in the native filtrate.

R E F E R E N C E S

1. Eng,].: Studies on the complement fixation test with Mycoplasma pneumoniae antigen. 5. Time pattern of not cell-bound antigen in broth culture. Acta path. microbiol. scandinav. Sec- tion B 78: 101-105, 1970.

2 . Eng, ] . : ibid. 6. Preparation of cell antigens with low contents of medium constituents. ibid. Section B 79: 107-116, 1971.

3. Eng, J.: Immune precipitation of not cell-bound antigen of Mycoplasma pneumoniae. ibid. S e c - tion B 79: 754-758, 1971.

4. Eng, ]. & Froholm, L. 0.: Immune electron microscopy of not cell-bound antigen of Myco- plasma pneumoniae. ibid. Section B 79: 759- 763, 1971.

5. Gomori, G.: Preparation of buffers for use in enzyme studies. In: Colowick, S. P. 8: Kaplan, N . 0 . (Ed.): Methods in enzymology vol. I. Academic Press Inc., New York 1955. pp. 138- 146.

6. Green, A . A . & Hughes, W . L.: Protein frac- tionation on the basis of solubility in aqueous solutions of salts and organic solvents. ibid. vol. I. pp. 67-90.

7. Zamenhof, S.: Preparation and assay of deoxyri- bonucleic acid from animal tissue. ibid. vol. 111. pp. 696-704.

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