A radioimmunoassay for antibody to pneumococcal polysaccharides

Journal o f Immunological Methods, 49 (1982) 83--88 Elsevier Biomedical Press

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A RADIOIMMUNOASSAY FOR ANTIBODY TO PNEUMOCOCCAL POLYSACCHARIDES

A.A.A. LATIF and A.D.B. WEBSTER Division o f Immunological Medicine, Clinical Research Centre, Watford Road, Harrow, Middx., U.K. (Received 5 June 1981, accepted 1 September 1981)

A radioimmunoassay for type specific pneumococcal polysaccharides is described. This involves obtaining specific polysaccharides from culture supernatants by precipitation with ethanol saturated with potassium acetate, and then after further purification labelling the tyraminated polysaccharide with 12s iodine. The radioimmunoassay itself is a standard a m m o n i u m sulphate precipitation technique (Farr assay) using IgG myeloma protein as a carrier. We have found that measuring antibodies after test immunization with a polyvalent pneumococcal vaccine is a useful aid in diagnosing functional antibody deficiency.

Key words: anti-pneumococcal polysaccharide antibodies -- immunodeficiency -radioimmunoassay

INTRODUCTION

Interest in the immune response of humans to pneumococcal polysaccharides has recently been stimulated b y the availability of commercial polyvalent vaccines. These vaccines consist of polysaccharides from up to 14 different strains of pneumococci and have been used clinically to protect susceptible patients against pneumococcal infection (Smit et al., 1977). Our interest centres on the possibility that such vaccines can be used to test the functional integrity o f the immune response in patients with suspected immunodeficiency. This paper described a radioimmunoassay which is suitable for measuring the antibody response to a wide range of pneumococcal polysaccharides. MATERIALS AND METHODS

Fourteen strains of pneumococci (contained in Pneumovax) were obtained from the Central Public Health Laboratory in Colindale, U.K. These were the following types: 1, 2, 3, 4, 6A (6), 7F (51), 8, 9N (9), 12F (12), 14, 18C (56), 19F (19), 23F (23) and 25 (the American nomenclature is in parentheses). 0022-1759/82/0000--0000/$02.75 © 1982 Elsevier Biomedical Press

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Preparation of polysaccharides (PnPs) The m e t h o d used essentially followed that described by Mitchell et ai. (1972). Each strain was cultured in 100 ml of Todd-Hewitt broth to which was added, after autoclaving, 2.5 g of glucose and 2.5 g of glycerophosphate sterilised by millipore filtering. The cultures were incubated at 32°C for 12-16 h, following which 1 ml was removed and inoculated into 500 ml of fresh broth. After further incubation at 32°C for 16 h, 5 ml were removed and inoculated into 2000 ml of broth in a 5 litre flask to provide good aeration. The cultures were incubated at 32°C for 24 h, after which a sample was taken and cultured on a blood agar plate to check the purity and strain of the pneumococcus. 40% formalin was added to the culture to a final concentration of 2%. The cultures were then centrifuged at 1500 X g for 20 min to separate the organisms. The supernatants were collected and concentrated using a Diaflo ultrafilter (Micon PM 30 > 30,000 MW). 1.5 vol of ethanol saturated with potassium acetate was added to the concentrated material and left to stand for 2 h at 4°C. Precipitated polysaccharides were then centrifuged at 1000 X g for 20 min, re~lissolved in water and re-precipitated with ethanol saturated with potassium acetate. After further centrifugation, the precipitate was washed twice with 70% alcohol followed by absolute alcohol, then treated with ether and dried in a rotary evaporator desiccator. The final p r o d u c t was a powder. 1 g of each polysaccharide was dissolved in 1/1000 M MgC12, to which 500 pg of RNase and 50 pg of DNase were added and the whole incubated for 24 h at 37°C with constant agitation. 1 mg of pronase was then added and the whole incubated for a further 24 h at 37°C. The treatment with RNase, DNase and pronase was repeated and then the polysaccharide precipitated with 1.5 vol of saturated ethanol containing potassium acetate. Further purification was carried o u t by precipitating with ethanol saturated with potassium acetate, washing with 70% alcohol followed b y absolute alcohol, and finally drying in ether as described above.

Purity of polysaccharides The material was strongly positive in the Molisch test b u t negative in the ninhydrin test. Ultraviolet spectrometry showed t w o major peaks at approx. 330 and 245 nM, with no absorption at 260 nM. Three of the type specific polysaccharides w e r e tested b y immunodiffusion against C reactive protein for the presence of C polysaccharide (Pepys et al., 1977). Variable amounts were present, t y p e III having virtually none while type 6A was significantly contaminated. However, any possibility that contaminating C polysaccharide might interfere with the assay for t y p e specific pneumococcai polysaccharide was avoided b y performing the test in 10 mM EDTA which prevents C reactive protein reacting with C polysaccharide (Gotschlich and Edelman, 1967).

Preparation of radioiodinated polysaccharide The purified polysaccharides were first tyraminated. 10 mg of purified polysaccharide were solubilized in distilled water and the pH adjusted to

85 10.5--11 b y adding 1 M NaOH. 2 mg of cyanogen bromide were added with constant stirring and the pH adjusted by further addition of 1 M NaOH. After 6 min the pH was reduced to 8.5 by adding 2 M NaHCO3 and then tyramine (13.7 mg dissolved in 1 ml of 1 M NaHCO3). The mixture was stirred at r o o m temperature for 4--5 h and then dialysed against distilled water for 48 h at 4°C with t w o changes, followed by further dialysis for 24 h against 0.3 M phosphate buffer, pH 7.5 at 4°C. The material recovered after dialysis was stored at --70°C until labelling with ~2sI. The labelling procedure was as follows: 1 mCi of [~2SI]Na was added to 100 pg of tyraminated polysaccharide and then incubated for 65 sec after the addition o f 10 pl of chloramine-T (2 mg/ml freshly prepared and used within 20 min). The reaction was terminated with 20 pl sodium metabisulphite (2 mg/ml) for 3 min, then 50 pl of 1% potassium iodide followed by 2 ml of 0.4% BSA in BBS pH 8.0. The free ~2sI was separated from the b o u n d ~2sI by dialysis against 1 litre of BBS or phosphate-buffered saline for 48 h with changes of dialysis fluid at 4, 12 and 24 h. 1 ml of dialysis fluid at each change was retained for isotope counting. After dialysis, the radiolabelled polysaccharide was centrifuged at 10,000 × gay for 20 min and the a m o u n t of 12sI b o u n d calculated b y subtracting the counts in the dialysis fluid from the counts added at the beginning of the procedure. More than 50% binding was considered Satisfactory.

Immunization schedules Five healthy adult volunteers were immunized subcutaneously with 0.5 ml of Pneumovax. Serum was collected before immunization, and again 4 days, 8 days and 14 days after vaccination. Another group of 5 adult volunteers was immunized with 0.1 ml intradermally of t y p e III pneumococcal polysaccharide (Welcome Reagents Ltd.). Serum was collected before vaccination and 1, 3 and 6 weeks afterwards.

Haemagglutination technique In some subjects, antibodies to t y p e III polysaccharide were measured by agglutination of antigen coated sheep erythrocytes using chromic chloride as a coupling agent {Gold and Fudenberg, 1967).

Radioimmunoassay for type specific antibody Serial dilutions of the test serum were made and 0.1 ml of each dilution added to 0.1 ml of a 1 : 10 dilution of human IgG m y e l o m a protein in BBS. EDTA was added at this stage at a final concentration o f 10 mM. ~2SI-labelled t y p e specific pneumococcal polysaccharide (0.1 ml) was diluted 1/125 in 0.3% BSA/BBS. All the assays were done in duplicate and left for 16 h at r o o m temperature. 0.3 ml of saturated ammonium sulphate solution was then added to each tube and after mixing left at 4°C for 3 h. The precipitate was then washed with an equal volume of a m m o n i u m sulphate/BBS b y centrifugation at 1500 × g for 20 min at 4°C. This was repeated 3 times, the

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washed precipitate finally being counted in a scintillation counter. Serial dilutions of a standard positive serum were measured each time to demonstrate linearity between counts and increasing serum concentrations (Fig. 1).

Expression of results The results are expressed as unit binding activity (UBA), the figure being obtained from a standard curve relating cpm to dilutions of serum from a patient with a high titre of antibody to the particular PnPs being assayed (Fig. 1). One UBA is equivalent to the counts obtained from the 'blank' tube which contains all the reagents except the test serum. The counts obtained with serum from a patient with agammaglobulinaemia was always very close to those in the 'blank' tube. RESULTS

The radioimmunoassay proved to be suitable for measuring antibodies to all the 14 types of pneumococcal polysaccharide. A standard curve relating counts to increasing dilutions of test serum is shown for type III polysaccha-

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Fig. 1. Standard curve relating c p m to units of binding activity for type III PnPs. The point at the origin represents the lowest c p m obtained with the highest dilution o f serum (i.e. 1 : 10,240), the highest c p m corresponding to the lowest dilution. Fig. 2. Increase over 14 days in units of binding activity for type III PnPs in serum from 5 healthy subjects i m m u n i z e d with P n e u m o v a x .

87 TABLE 1 Relationship between radioimmunoassay and haemagglutination titres to type III PnPs. The UBA obtained for 5 subjects immunized with type III PnPs are shown, and compared with haemagglutination titres (in parenthesis). Vaccination

Subjects 1

2

3

4

5

Pre

15.0(16)

5.0(16)

7.5 (16)

39.0 (32)

21.5 (32)

Post 1 week 3 weeks 6 weeks

15.0 (16) 16.0 (16) 17.0 (16)

5.5 (16) 46.0 (64) 92.5 (64)

80.0 (128) 430.0 (512) 300.0 (256)

90.0 (128) 330.0 (256) 330.0 (256)

190.0 (256) 510.0 (512) 460.0 (512)

ride (Fig. 1). Fig. 2 shows the counts obtained from pre- and post-immunization sera when assayed for antibodies to type III. As expected, there were considerable variations in the pre-vaccination levels presumably reflecting previous exposure to this specific polysaccharide. Table 1 compares the results of the radioimmunoassay with the haemagglutinatiom titres of sera obtained from 5 healthy subjects at various times after vaccination with type III specific pneumococcal polysaccharide. Although there is no close agreement between the number of counts obtained and the haemagglutination titre, the results show parallelism between the two methods in individual subjects. The lack of correlation between UBA and titres, is probably due to the preponderance of either IgG or IgM antibodies in the individual sera. DISCUSSION

Immunization against a wide range of pneumococci is recommended in asplenic or splenectomised patients (Ammann et al., 1977). Its potential use in preventing pneumococcal pneumonia in patients with chronic chest disease is also under investigation. For these reasons it is important to have a rapid and convenient method for measuring the antibody responses in such patients. Schiffman et al. (1980) have already published a method for the measurement of antibodies to pneumococcal polysaccharide which involves incorporating [ 14C] glucose into the capsular polysaccharide while the organism is growing in culture. Our method offers no particular labour-saving advantage although it does avoid the handling and purification of large quantities of radioactive material. Pneumococcal polysaccharides may be contaminated by C reactive substance (C polysaccharide) unless further purification procedures are undertaken. The level of contamination seems to vary between polysaccharides.

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C polysaccharide is likely to be radiolabelled along with the type specific polysaccharides and cause spuriously high counts by reacting with C reactive protein in the test serum. However, this problem can be overcome by adding EDTA to the assay mixture. Nevertheless, although this may be an important issue when testing for cross-reactivity between different polysaccharide types, it is unlikely to significantly affect the many-fold increases in counts after immunization. We have shown that our method is suitable for measuring antibody responses following immunization with either polyvalent or type specific polysaccharides. Seventeen normal subjects have so far been immunised with Pneumovax, and when their serum has been assayed for both type III and 6A antibodies, all have shown a response at 8 days to one or both of these types. We are currently using this test in the routine assessment of suspected immunodeficiency and have found it useful in diagnosing patients with functional antibody deficiency who have normal or slightly low levels of serum immunoglobulins. We have also found it useful as a guide to the severity of the antibody deficiency in patients with unequivocal hypogammaglobulinaemia. ACKNOWLEDGEMENTS

We are grateful to Professor J.H. Humphrey for advising us on the purification of pneumococcal polysaccharide and to Dr. G. Colman of the Central Public Health Laboratory, Colindale, for providing the type specific pneumococci and for checking the purity of our cultures. We also thank Dr. F. Sheffield and Dr. G.L. Asherson for helpful advice. REFERENCES Ammann, A.J, J. Addiego, D.B. Wara, B. Lubin, W.B. Smith and W.C. Mentzer, 1977, New Engl. J. Med. 297,897. Gold, E.R. and H.H. Fudenberg, 1967, J. Immunol. 99,859. Gotschlich, E.C. and G.M. Edelman, 1967, Proc. Natl. Acad. Sci. U.S.A. 57, 706. Mitchell, G.F, J.H. Humphrey and A.R. Williamson, 1972, Eur. J. Immunol. 2, 460. Pepys, M.B, A.C. Dash and M.J. Ashley, 1977, Clin. Exp. Immunol. 30, 32. Schiffman, G., R.M. Douglas, M.J. Bonner, M. Robbins and R. Austrian, 1980, J. Immunol. Methods 33, 133. Smit, P., D. Oberholzer, S. Hayden-Smith, H.J. Koornhof and M.R. Hilleman, 1977, J. Am. Med. Ass. 238, 2613.