Journal o f Immunological Methods, 31 (1979) 71--81 © Elsevier/North-Holland Biomedical Press
71
A MODIFIED CYTOPLASMIC ANTIGEN OF C A N D I D A A L B I C A N S F O R SERODIAGNOSIS OF SYSTEMIC CANDIDIASIS
MALCOLM G. HOLLIDAY
Department o f Microbiology, St. George's Hospital, Stafford, Great Britain (Received 9 April 1979, accepted 28 July 1979)
A modified cytoplasmic antigen, prepared by Natamycin degradation of Candida albicans cells is described. The reactivity of this antigen in detecting precipitins to Candida albicans proved to be very similar qualitatively and quantitatively to a standardised reference antigen, prepared by X-press disruption when both were compared by immunological techniques; the majority of antigenic components in each proving to be identical. When tested against 127 human sera of u n k n o w n antibody content the two antigens showed 100% correlation by counterimmunoelectrophoresis and 87.5% correlation by double diffusion. The modified antigen proved to be reproducible and reliable in use and is easily prepared in the routine laboratory.
INTRODUCTION
Systemic candidiasis is emerging as a major life-threatening obstacle to the successful management of high-risk patients such as those with leukaemia or other conditions requiring extensive surgery or immunosuppressive, steroid and antibiotic therapy. Despite an awareness of the marked increase in such infections however, many workers believe that in 60--80% of these cases the infection is rarely diagnosed in time for effective treatment (Gaines and Remington, 1973), frequently diagnosis only being made at post-mortem (Andersen and Stenderup, 1974). Deep Candida infections do n o t present an unequivocal clinical picture and results of cultures are difficult to interpret (Goldstein and Hoeprich, 1972); therefore an urgent need arose for alternative diagnostic parameters to augment clinical and mycological criteria (Andersen and Stenderup, 1974}. This need appears to have been partly filled b y the introduction of several serological procedures, of which the detection of precipitating antibodies to Candida albicans cytoplasmic antigens is presently held to be the most satisfactory (Rosner et al., 1970; Taschdjian et al., 1972; Odds et al., 1975). Taschdjian et al. (1969a and b) have suggested that precipitin reactions in agar gel against cytoplasmic Candida antigens permit diagnosis of systemic candidiasis with 85--90% reliability. Widespread diagnostic use of this m e t h o d in many laboratories has hitherto been precluded by the difficulties in making a relatively stable and
72 reproducible cytoplasmic antigen. The antigen described here was made by a simple m e t h o d suitable for routine laboratories, without the use of specialised equipment for cell breakage, separation of fractions, cooling and centrifugation. The purpose of the present investigation was to compare the reactivity of the modified antigen with a reference cytoplasmic antigen of Candida albicans tested against a standard antiserum to Candida albicans and serum from a patient with systemic candidiasis and to investigate the performance of the modified antigen in routine use for screening sera. Batches of the modified antigen were investigated for reproducibility and stability over a period of 12 months. MATERIALS AND METHODS Preparation o f an tigens Candida albicans group A (Hasenclever, 1961) (kindly provided by D.W. Warnock, Microbiology Department, Bristol Royal Infirmary) was grown on 6 Sabouraud's agar plates for 48 h at 37°C. The growth was then washed off the plates in 25 ml of 0.154 M NaC1 (Axelson, 1973) and centrifuged for 10 min at 3,000 rpm in an MSE Minor centrifuge. The supernatant was discarded and the cell deposit was re-suspended in 0.154 M NaC1 and centrifuged twice more using the same procedure. The final cell deposit, which averaged 2--3 g wet weight of cells, was then suspended in 10 ml of 10,000 pg/ml Natamycin (Pimafucin) in 0.2 M sodium phosphate buffer, pH 8.6, containing 0.1% sodium azide. This was left in the dark at room temperature for 14 days with frequent mixing, after which it was again centrifuged at 3,000 rpm for 10 min. The supernatant, a pale, straw-coloured fluid was carefully pipetted off and re-centrifuged. The final supernatant proved to be cell-free upon microscopy and was dialysed against distilled water at 4°C for 48 h. After dialysis, the extract was vacuum-desiccated at room temperature and stored at --12°C until needed, when it was dissolved in sterile physiological saline to give a concentration of 20 mg/ml, and sodium azide was added to give a final concentration of 0.1% (referred to in the text as NAT antigen). Purified mannan was prepared using the m e t h o d of Faux (1968) and used at a concentration of 1 mg/ml. A standardised cytoplasmic extract of Candida albicans, designated Cytoplasmic Proteins (Candida albicans) Whole Extract 76/513, was obtained from the National Institute for Biological Standards and Control, Holly Hill, Hampstead, and was used at a concentration of 13 mg/ml (referred to in the text as NIBSC antigen). This antigen was prepared by X-press disruption and differential centrifugation. Se ra A standardised polyvalent antiserum to Candida albicans was kindly
73 provided by Dr. Nils Axelson, The Protein L a b o r a t o r y , Copenhagen. {This antiserum is n o w available f r om DAKO Ltd., Denmark, and is referred to in the t e x t as STAB.) 125 sera were obtained f r om patients having rout i ne l aborat ory investigations p e r f o r m e d . These sera were of u n k n o w n a n t i b o d y cont ent . Positive serum was obtained f r o m a patient with systemic candidiasis. All sera were stored at --12°C until needed. All sera were tested against the NIBSC antigen, 13 mg/ml, the NAT antigen, 20 mg/ml, and the purified mannan antigen, 1 mg/ml, by counterimmunoelectrophoresis and double diffusion m e t h o d s to compare the results obtained with each antigen in normal use. The NIBSC and N A T antigens were com pared by double diffusion, immunoelectrophoresis, c o u n t e r i m m u n o e l e c t r o p h o r e s i s , two-dimensional immunoelectrophoresis, t a n d e m two-dimensional immunoelectrophoresis, two-dimensional immunoelectrophoresis (absorption o f antigen in situ), and line immunoelectrophoresis (direct comparison of antigens), against the standardised a n t i b o d y and against the positive hum an serum in order to assess their respective reactivities.
Double diffusion (DD) (Taschdjian et al., 1972) Tests were carried o u t in 1% purified agar (oxoid) in physiological saline, containing 0.4% sodium citrate and 0.1% sodium azide, layered to a depth of 2 m m in 90-mm petri dishes. The central serum well was 12 m m in diameter and the peripheral wells were 4 m m in diameter and 6 m m from the central well. Diffusion was carried o u t at r o o m t e m p e r a t u r e for up to one week, after which the plates were washed overnight in distilled water, dried and stained in 0.05% Naphthalene Black. Double diffusion absorption of antiserum was p e r f o r m e d using the above procedure e x c e p t t hat two 12-mm wells were cut side by side, 10 m m apart, with peripheral 4-mm wells 6 mm f r o m each. Positive hum an serum was placed in one large well, and the same positive h u man serum absorbed by the NAT antigen was placed in the other. NAT antigen was placed in one peripheral well, and NIBSC antigen in the other. Absorption of antiserum was carried o u t as follows: 6 drops of serum were placed in a test tube, and 3 drops o f NAT antigen were added to it. After leaving at r o o m t e m p e r a t u r e for 1 h, the serum was centrifuged and the supernatant placed in the appropriate well.
Immunoelectrophoresis methods All immunoelectrophoresis tests were carried o u t in 1% agarose (BDH) in barbitone buffer pH 8.6 (ionic strenght p = 0.02) containing 0.1% sodium azide. Counterimmunoelectrophoresis. Agarose gel was layered to a depth of 1 m m on 7.5 X 5 cm glass slides, with antigen and serum wells 2 m m in diameter and separated by 2 m m edge to edge. Serum was placed in the
74 anode well and antigen in the c a t hode well, and electrophoresis was perf o r m e d for 1 h at 2.5 mA/slide. Immunoelectrophoresis. Tests were carried o u t using a modification of the m i c r o - m e t h o d of Faux (1968). Agarose gel was layered to a depth of 1 m m on glass slides as before. T w o wells were cut 2 m m in diameter, at a distance o f 2 m m from the central trough which was 2 m m wide. Antigen was placed in the wells and electrophoresis was p e r f o r m e d at 10 mA/slide for 1 h, after which serum was placed in the central trough and the precipitin reactions were allowed to develop for 24 h in a moist atmosphere. Two-dimensional immunoelectrophoresis (Axelson, 1971). Tests were carried o u t in 1-mm thick agarose gel on 5 X 5 cm glass plates. A 2-mm well in one corner was filled with antigen and first dimension electrophoresis was p e r f o r m e d at 10 V/cm for 1 h, with this well at the cathode, to separate the antigen horizontally. The unw ant ed gel was then removed, leaving a narrow strip containing the separated antigen, and replaced with gel containing the antiserum to a final c o n c e n t r a t i o n of 12 pl/cm 2 to a depth of 1 mm. The plate was r o tate d through 90 ° and second-dimension electrophoresis perf o r m e d at 2 V/cm for 20 h with the antigen strip at the cathode. For antigen absorption in situ the first dimension electrophoresis was carried out as above, with NIBSC antigen in the well, but before removal of the unw ant ed gel, a ditch 2 m m wide was cut parallel to the separated antigen and filled with a large a m o u n t of the NAT antigen, which was allowed to diffuse into the gel for 1 h. The edges of the ditch were then pushed together, the unwanted gel removed, and antiserum containing gel (12 t~l/cm 2) poured on the rest of the plate to a depth of 1 mm, and second-dimension electrophoresis p e r f o r m e d as before. Tandem two-dimensional immunoelectrophoresis (Axelson and Bock, 1972). The p r o cedur e here was the same as for ordinary two-dimensional immunoelectrophoresis except that two wells were cut 2 m m in diameter and 4 mm apart in the plane of horizontal separation. NAT antigen was placed in the first well and NIBSC antigen in the second, before first-dimension electrophoresis. The antigens were allowed to diffuse into the gel for 1 h and the wells were then sealed with m ol t en agarose gel after which first- and second-dimension electrophoresis was carried out as before. Line immunoelectrophoresis, direct comparison o f antigens (Axelson and Bock, 1972). Tests were carried out on 5 X 5 cm glass plates o n t o which antiserum containing gel (12 pl/cm 2) was layered to a depth of 1 mm. When this had set, a strip 5 m m wide was removed from the cathode end and replaced by 2 equal gels, side by side, containing equal amounts of NAT and NIBSC antigens, respectively. Electrophoresis was then p e r f o r m e d at 3 V/cm for 20 h. All immunoelectrophoresis slides and plates were washed in distilled water for 24 h, dried and stained in 0.5% Naphthalene Black after devel opm ent of precipitation patterns. 'Cambrelle', a 100% nyl on 'melded' fabric, made by ICI and marketed by
75 Plant Protection Ltd., Farnham, Surrey, was used as wick material in all immunoelectrophoresis techniques (Strong and Lea, 1977).
Nature of precipitin reactions Two types of precipitin reactions have been observed in agar gel precipitations with C. albicans antigens (Faux, 1968; Warnock et al., 1976): (a) A broad, diffuse 'H' type reaction attributable to a heat-stable polysaccharide antigen on the cell wall -- the Mannan antigen. (b) A sharp, well defined 'R' t y p e reaction attributable to heat-labile internal protein antigens -- the cytoplasmic antigens. Seven different batches of NAT antigen were prepared over a period of 12 months and each was compared shortly after preparation by immunoelectrophoresis against a positive patient's serum, to investigate the reproducibility of the preparation method. To investigate the stability of the NAT antigen during both normal use and prolonged storage, small aliquots from each of 7 batches were stored at --12°C and tested as follows: One sample from each batch, stored for varying times up to 12 months was tested by immunoelectrophoresis against positive human serum. The results of these were compared with the results of each batch tested shortly after preparation against the same serum. A sample from the first batch made was tested several times, being thawed and then refrozen each time. The results were compared with the results of the above tests. The sample was tested once a month for 9 months. RESULTS
Comparison of antigens by precipitation techniques (Table 1) using positive human serum and a reference antiserum Preliminary double diffusion investigation with 3 different concentrations of the NAT antigen (100 mg/ml, 40 mg/ml and 20 mg/ml) tested against STAB showed 12, 11 and 10 precipitin lines, respectively. The NIBSC antigen tested in the same way gave 10 lines (Fig. 1). Reactions of identity can be seen for most of the lines although the complexity of the reactions partially obscures this. The NAT 20 mg/ml antigen seemed the concentration most closely resembling the NIBSC antigen and this concentration was used in the other experiments and designated NAT antigen. When tested against positive human serum both the NAT and NIBSC antigens gave 8 lines. The NAT antigen and the NIBSC antigen each showed 8 lines against the unabsorbed positive serum, most showing reactions of identity (Fig. 2) without reactions with the absorbed serum. Immunoelectrophoresis of the NAT and NIBSC antigens showed 11 and 9 lines, respectively, against positive human serum, and 11 and 13 lines, respectively, against STAB, the NAT extract lines being slightly weaker in both cases. Counterimmunoelectrophoresis of the NAT and NIBSC antigens
76 TABLE 1 COMPARISON OF PRECIPITIN PATTERNS OF NAT AND NIBSC ANTIGENS TESTED AGAINST ST.AB. AND POSITIVE HUMAN SERUM Method
Double diffusion Immu noelectrophoresis Two-dimensional IEP Two-dimensional IEP ; Absorption of antigen in situ
Tandem two-dimensional IEP Line IEP; direct comparison of antigens CIE
Number of precipitin lines seen Standard antibody
Positive human serum
NIBSC antigen (13 mg/ml)
NAT antigen (20 mg/ml)
NIBSC antigen (13 mg/ml)
NAT antigen (20 mg/ml)
]0 13 43
10 11 44
8 9 22
8 11 21
NT NT 30
NT NT 28
14 13 14
12 15 16
4
3
2
2
NT = not tested due to lack of ST.AB.
ii~iiiiii~ii!
Fig. 1. Double diffusion of NAT antigens and NIBSC antigen against ST.AB. (central well). I, NAT 100 mg/ml, II, NIBSC 13 mg/ml; III, NAT 40 mg/ml; IV, NAT 20 mg/ml. Reactions of identity can be observed between the NAT and NIBSC antigens. It can be seen that NAT 20 mg/ml most closely resembles the NIBSC antigen. Fig. 2. Double diffusion of (well A) and positive human 1 mg/ml; 2, NAT 20 mg/ml; NIBSC 13 mg/ml; 7, Mannan
NAT and NIBSC antigens against positive human serum serum absorbed by NAT antigen (well B). Well: 1, Mannan 3 Mannan 1 mg/ml; 4, NAT 20 mg/ml; 5, NAT 20 mg/ml; 6, 1 mg/ml; 8, NAT 20 mg/ml.
77 each showed 2 lines against positive hum a n serum and 3 and 4 lines, respectively, when tested against the STAB. In all cases general similarity was observed between the precipitation patterns of b o t h antigens. Two-dimensional immunoelectrophoresis. The NAT antigen showed 21 peaks and the NIBSC antigen showed 22 peaks when tested against positive h u man serum, and against STAB, 44 and 43 peaks, respectively (Figs. 3 and 4). In all these cases the general precipitation patterns of both antigens were similar. When NIBSC was electrophoresed and absorbed by NAT in the trough, against a positive hum an serum, only 2 very minor c o m p o n e n t s were unabsorbed, remaining as peaks, while faint reactions of identity, continuous lines, were observed with 12 o t h e r c o m p o n e n t s . The NAT antigen showed 13 reactions o f identity with the NIBSC antigen when tested against positive hum a n serum in tandem. The NAT antigen apparently contained 2 antigenic c o m p o n e n t s n o t evident in the NIBSC antigen. By line immunoelectrophoresis 14 reactions of identity were observed (Fig. 5). One or possibly two minor c o m p o n e n t s in the N A T antigen failed to show reactions of identity with the NIBSC antigen. When NAT and NIBSC were tested together against STAB, 28 reactions of identity were observed. Only one, or possibly two, c o m p o n e n t s in the NIBSC antigen failed to show reactions of identity with the NAT antigen.
Reproducibility of the modified antigen Seven batches of NAT antigen prepared at different times were tested shortly after preparation by immunoelectrophoresis against positive h u m a n serum. In all cases the n u m b e r of precipitin lines observed was the same, i.e.
Fig. 3. Two-dimensional immunoeleetrophoresis of NAT antigen against ST.AB; 44 peaks were seen, although many are not visible on the figure. Origin on the right. Fig. 4. Two-dimensional immunoelectrophoresis of NIBSC antigen against ST.AB; 43 peaks were seen, although many are not visible on the figure. Origin on the right. Note similarity in pattern to Fig. 3.
78
Fig. 5. Line immunoelectrophoresis; direct comparison of antigens, a, NAT antigen and b, NIBSC antigen against ST.AB. Reactions of identity can be seen for the majority of lines formed by each antigen, indicating that the major components of each are identical. 28 reactions of identity were seen, although not all are revealed on the figure.
11, a l t h o u g h slight variation o c c u r r e d in the strength o f s o m e lines. T h e r e f o r e it appears t h a t the m e t h o d s h o w e d suitable reliability in preparing the antigen, and 7 d i f f e r e n t b a t c h e s s h o w e d a c c e p t a b l e reproducibility.
Stability of the modified antigen All e x t r a c t s w h e n t a k e n directly f r o m - - 1 2 ° C and tested b y i m m u n o e l e c t r o p h o r e s i s against positive h u m a n s e r u m gave 11 lines, regardless o f the length o f storage time, u p to 12 m o n t h s . H o w e v e r , the e x t r a c t t h a t was t h a w e d and r e f r o z e n s h o w e d progressive loss o f a n t i g e n i c i t y , d r o p p i n g to 5 lines after 3 m o n t h s and 4 after 9 m o n t h s .
TABLE 2 SCREENING OF SERA WITH MANNAN, NAT AND NIBSC ANTIGENS BY CIE AND DD Method
Counterimmunoelectrophoresis Double diffusion
Total No. of sera tested
127 127
Number of positive reactions "H" type precipitins
"R" type precipitins
Mannan
NAT
NIBSC
NAT
NIBSC
46 13
47 8
47 7
2 2
2 2
79 Screening o f sera with mannan, N A T and N I B S C antigens by CIE and DD It can be seen from Table 2 that the results obtained b y NAT and NIBSC antigens show good correlation. By CIE both gave 47/127 positive 'H' t y p e reactions and 2/127 positive 'R' type reactions, i.e. 100% correlation. By DD, the NAT antigen gave 8/127 positive 'H' type reactions and the NIBSC gave 7/127. Both gave 2/127 positive 'R' t y p e reactions, i.e. 87.5% correlation. DISCUSSION Natamycin is a polyene antibiotic closely related to Nystatin and its primary antifungal action is based on its ability to alter the permeability of the cytoplasmic membrane, allowing the loss of inorganic phosphate ions, triose and sugar phosphates, proteins, nucleotides and other essential cellular constituents from the cell (Raab, 1972). This action provides the basis for the collection of antigenic intracellular components relatively unchanged and uncontaminated by cell wall material, hopefully in the same condition as they may be released from phagocytosed yeast cells in the body. It has been shown (Venezia and Robertson, 1974) that extracts made from protoplasts of yeast-phase Candida albicans cells are very similar to the cytoplasmic extract made by Taschdjian et al. (1967) ('S' antigen produced by a sonication method). Preliminary testing showed that pH 8.6, with a concentration of 10,000 pg/ml of Natamycin, and an extraction time of 2 weeks provided the best antigenic material. The loss of antigenicity due to the heat-labile character of many of the cytoplasmic components, did n o t become evident until after 2 weeks extraction at room temperature. The reaction was allowed to progress in the dark, as Natamycin is susceptible to direct sunlight, and at room temperature, as at 4°C no antifungal effect is observed (Raab, 1972). One of the main problems in assessing the diagnostic significance of precipitins to Candida antigens is the use of many different antigenic preparations (Faux et al., 1975) and if cytoplasmic antigens differ markedly in their cell wall antigen content, widely discrepant results could occur (Venezia and Robertson, 1974). It is shown in these investigations that the a m o u n t of cell wall mannan present in the NAT antigen must be very similar to that present in the NIBSC antigen, the results showing 87.5% correlation, by DD and 100% correlation by CIE and also must be very similar in a m o u n t to the pure mannan extract, as the results show 61.5% correlation by DD and 97.2% correlation by CIE. Therefore results obtained by each of the 3 antigens should show few discrepancies. The detection of 'R' t y p e precipitins to the cytoplasmic protein components by NAT and NIBSC antigens showed 100% correlation b y DD and 100% correlation by CIE b u t due to the small number of sera tested, this figure may be suspect.
80 H o w e v e r , as t h e m a j o r i t y o f antigenic c o m p o n e n t s in each e x t r a c t s h o w e d r e a c t i o n s o f i d e n t i t y it can be a s s u m e d t h a t t h e N A T antigen will give similar results to t h e N I B S C antigen with o t h e r positive sera. On t h e evidence here t h e N A T antigen is r e p r o d u c i b l e a n d c o m p a r e s f a v o u r a b l y w i t h t h e N I B S C r e f e r e n c e antigen. T h e stability o f t h e N A T antigen was n o t investigated t h o r o u g h l y , b u t a p p e a r s to be o f a similar o r d e r to t h a t o f the N I B S C antigen. B o t h antigens s h o w e d r a p i d d e g e n e r a t i o n o n r e p e a t e d t h a w i n g a n d re-freezing, b u t w h e n k e p t c o n s t a n t l y at - - 1 2 ° C n o o b v i o u s d e t e r i o r a t i o n was n o t e d a f t e r 12 m o n t h s . A x e l s o n states t h a t his c y t o p l a s m i c antigen r e m a i n s stable f o r u p to 18~ m o n t h s at - - 2 0 ° C . ( A x e l s o n and B o c k , 1972). T h e N a t a m y c i n antigen can be p r e p a r e d easily in t h e r o u t i n e l a b o r a t o r y w i t h o u t great e x p e n s e or e f f o r t . ACKNOWLEDGEMENTS M y t h a n k s to B r o c a d e s , G r e a t Britain L t d . , W e y b r i d g e , S u r r e y , w h o k i n d l y s u p p l i e d t h e N a t a m y c i n ; Dr. E. N n o c h i r i , C o n s u l t a n t Microbiologist, Staff o r d a n d B u r t o n o n T r e n t ; Mr. I.L. M c C a r t n e y , C h i e f T e c h n i c i a n , Microbiolo g y , S t a f f o r d ; Mr. M. Mitchell, C h i e f T e c h n i c i a n , Clinical C h e m i s t r y , Staff o r d ; T h e D e p a r t m e n t o f Medical P h o t o g r a p h y , B u r t o n District H o s p i t a l ; P.H.L., Carlisle and the P a t h o l o g y D e p a r t m e n t , S t a f f o r d , f o r t e c h n i c a l assistance a n d use o f e q u i p m e n t ; Mrs. D. J o h n s o n f o r t y p i n g t h e m a n u s c r i p t ; and the staff of the Microbiology Department, Stafford, for encouragement a n d advice, w i t h o u t w h o m m u c h o f this w o r k w o u l d have b e e n impossible. REFERENCES Andersen, P.L. and A. Stenderup, 1974, Scand. J. Infect. Dis. 6, 69. Axelson, N.H., 1971, Inf. Immunity 4,252. Axelson, N.H., 1973, Inf. Immunity 7,946. Axelson, N.H., 1976, Scand. J. Immunol. 5, 177. Axelson, N.H. and E. Bock, 1972, J. Immunol. Methods 1, 109. Evans, E.G.V. (ed.), 1976, in: The Serology of Fungal Infections and Farmer's Lung Diseases (University Printing Office, Leeds) p. 3. Faux, J.A., 1968, Immunological Studies of the Antigens of Candida albicans in man, PhD Thesis, University of London. Faux, J.A., V.C. Stanley, H.R. Buckley and B.M. Partridge, 1975, J. Immunol. Methods 6,235. Gaines, J.D. and J.S. Remington, 1973, Arch. Intern. Med. 132, 699. Goldstein, E. and P.D. Hoeprich, 1972, J. Infect. Dis. 125, 190. Hasenclever, H.F. and W.O. Mitchell, 1961, J. Bact. 82,570. Odds, F.C., E.G.V. Evans and K.T. Holland, 1975, J. Immunol. Methods 7,211. Raab, W.P., 1972, in: Natamycin (Pimaricin) -- its Properties and Possibilities in Medicine (Thieme, Stuttgart) p. 16. R0sner , F., F.D. Gabriel, C.L. Taschdjian, M.B. Cuesta and P.J. Kozinn, 1970, Amer. J. Med. 51, 54. Sargent, J.R. and S.G. George, 1975, Methods in Zone Electrophoresis, B.D.H. Chemicals, Ltd.
81 Strong, W.M. and D.J. Lea, 1977, J. Med. Lab. Sci. 34,355. Taschdjian, C.L., P.J. Kozinn, A. Okas, L. Caroline and M.T. Halle, 1967, J. Infect. Dis. 117, 180. Taschdjian, C.L., M.B. Cuesta, P.J. Kozinn and L. Caroline, 1969a, Amer. J. Clin. Path. 52, 468. Taschdjian, C.L., P.J. Kozinn, H. Fink, M.B. Cuesta, L. Caroline and A.B. Kantrowitz, 1969b, Sabouraudia 7, 110. Taschdjian, C.L., P.J. Kozinn, M.B. Cuesta and E.F. Toni, 1972, Amer. J. Clin. Path. 57, 195. Venezia, R.A. and R.G. Robertson, 1974, Amer. J. Clin. Path. 61,849. Warnock, D.W., D.C.E. Speller, J.A. Morris and P.H. Mackie, 1976, J. Clin. Path. 29,836.
71
A MODIFIED CYTOPLASMIC ANTIGEN OF C A N D I D A A L B I C A N S F O R SERODIAGNOSIS OF SYSTEMIC CANDIDIASIS
MALCOLM G. HOLLIDAY
Department o f Microbiology, St. George's Hospital, Stafford, Great Britain (Received 9 April 1979, accepted 28 July 1979)
A modified cytoplasmic antigen, prepared by Natamycin degradation of Candida albicans cells is described. The reactivity of this antigen in detecting precipitins to Candida albicans proved to be very similar qualitatively and quantitatively to a standardised reference antigen, prepared by X-press disruption when both were compared by immunological techniques; the majority of antigenic components in each proving to be identical. When tested against 127 human sera of u n k n o w n antibody content the two antigens showed 100% correlation by counterimmunoelectrophoresis and 87.5% correlation by double diffusion. The modified antigen proved to be reproducible and reliable in use and is easily prepared in the routine laboratory.
INTRODUCTION
Systemic candidiasis is emerging as a major life-threatening obstacle to the successful management of high-risk patients such as those with leukaemia or other conditions requiring extensive surgery or immunosuppressive, steroid and antibiotic therapy. Despite an awareness of the marked increase in such infections however, many workers believe that in 60--80% of these cases the infection is rarely diagnosed in time for effective treatment (Gaines and Remington, 1973), frequently diagnosis only being made at post-mortem (Andersen and Stenderup, 1974). Deep Candida infections do n o t present an unequivocal clinical picture and results of cultures are difficult to interpret (Goldstein and Hoeprich, 1972); therefore an urgent need arose for alternative diagnostic parameters to augment clinical and mycological criteria (Andersen and Stenderup, 1974}. This need appears to have been partly filled b y the introduction of several serological procedures, of which the detection of precipitating antibodies to Candida albicans cytoplasmic antigens is presently held to be the most satisfactory (Rosner et al., 1970; Taschdjian et al., 1972; Odds et al., 1975). Taschdjian et al. (1969a and b) have suggested that precipitin reactions in agar gel against cytoplasmic Candida antigens permit diagnosis of systemic candidiasis with 85--90% reliability. Widespread diagnostic use of this m e t h o d in many laboratories has hitherto been precluded by the difficulties in making a relatively stable and
72 reproducible cytoplasmic antigen. The antigen described here was made by a simple m e t h o d suitable for routine laboratories, without the use of specialised equipment for cell breakage, separation of fractions, cooling and centrifugation. The purpose of the present investigation was to compare the reactivity of the modified antigen with a reference cytoplasmic antigen of Candida albicans tested against a standard antiserum to Candida albicans and serum from a patient with systemic candidiasis and to investigate the performance of the modified antigen in routine use for screening sera. Batches of the modified antigen were investigated for reproducibility and stability over a period of 12 months. MATERIALS AND METHODS Preparation o f an tigens Candida albicans group A (Hasenclever, 1961) (kindly provided by D.W. Warnock, Microbiology Department, Bristol Royal Infirmary) was grown on 6 Sabouraud's agar plates for 48 h at 37°C. The growth was then washed off the plates in 25 ml of 0.154 M NaC1 (Axelson, 1973) and centrifuged for 10 min at 3,000 rpm in an MSE Minor centrifuge. The supernatant was discarded and the cell deposit was re-suspended in 0.154 M NaC1 and centrifuged twice more using the same procedure. The final cell deposit, which averaged 2--3 g wet weight of cells, was then suspended in 10 ml of 10,000 pg/ml Natamycin (Pimafucin) in 0.2 M sodium phosphate buffer, pH 8.6, containing 0.1% sodium azide. This was left in the dark at room temperature for 14 days with frequent mixing, after which it was again centrifuged at 3,000 rpm for 10 min. The supernatant, a pale, straw-coloured fluid was carefully pipetted off and re-centrifuged. The final supernatant proved to be cell-free upon microscopy and was dialysed against distilled water at 4°C for 48 h. After dialysis, the extract was vacuum-desiccated at room temperature and stored at --12°C until needed, when it was dissolved in sterile physiological saline to give a concentration of 20 mg/ml, and sodium azide was added to give a final concentration of 0.1% (referred to in the text as NAT antigen). Purified mannan was prepared using the m e t h o d of Faux (1968) and used at a concentration of 1 mg/ml. A standardised cytoplasmic extract of Candida albicans, designated Cytoplasmic Proteins (Candida albicans) Whole Extract 76/513, was obtained from the National Institute for Biological Standards and Control, Holly Hill, Hampstead, and was used at a concentration of 13 mg/ml (referred to in the text as NIBSC antigen). This antigen was prepared by X-press disruption and differential centrifugation. Se ra A standardised polyvalent antiserum to Candida albicans was kindly
73 provided by Dr. Nils Axelson, The Protein L a b o r a t o r y , Copenhagen. {This antiserum is n o w available f r om DAKO Ltd., Denmark, and is referred to in the t e x t as STAB.) 125 sera were obtained f r om patients having rout i ne l aborat ory investigations p e r f o r m e d . These sera were of u n k n o w n a n t i b o d y cont ent . Positive serum was obtained f r o m a patient with systemic candidiasis. All sera were stored at --12°C until needed. All sera were tested against the NIBSC antigen, 13 mg/ml, the NAT antigen, 20 mg/ml, and the purified mannan antigen, 1 mg/ml, by counterimmunoelectrophoresis and double diffusion m e t h o d s to compare the results obtained with each antigen in normal use. The NIBSC and N A T antigens were com pared by double diffusion, immunoelectrophoresis, c o u n t e r i m m u n o e l e c t r o p h o r e s i s , two-dimensional immunoelectrophoresis, t a n d e m two-dimensional immunoelectrophoresis, two-dimensional immunoelectrophoresis (absorption o f antigen in situ), and line immunoelectrophoresis (direct comparison of antigens), against the standardised a n t i b o d y and against the positive hum an serum in order to assess their respective reactivities.
Double diffusion (DD) (Taschdjian et al., 1972) Tests were carried o u t in 1% purified agar (oxoid) in physiological saline, containing 0.4% sodium citrate and 0.1% sodium azide, layered to a depth of 2 m m in 90-mm petri dishes. The central serum well was 12 m m in diameter and the peripheral wells were 4 m m in diameter and 6 m m from the central well. Diffusion was carried o u t at r o o m t e m p e r a t u r e for up to one week, after which the plates were washed overnight in distilled water, dried and stained in 0.05% Naphthalene Black. Double diffusion absorption of antiserum was p e r f o r m e d using the above procedure e x c e p t t hat two 12-mm wells were cut side by side, 10 m m apart, with peripheral 4-mm wells 6 mm f r o m each. Positive hum an serum was placed in one large well, and the same positive h u man serum absorbed by the NAT antigen was placed in the other. NAT antigen was placed in one peripheral well, and NIBSC antigen in the other. Absorption of antiserum was carried o u t as follows: 6 drops of serum were placed in a test tube, and 3 drops o f NAT antigen were added to it. After leaving at r o o m t e m p e r a t u r e for 1 h, the serum was centrifuged and the supernatant placed in the appropriate well.
Immunoelectrophoresis methods All immunoelectrophoresis tests were carried o u t in 1% agarose (BDH) in barbitone buffer pH 8.6 (ionic strenght p = 0.02) containing 0.1% sodium azide. Counterimmunoelectrophoresis. Agarose gel was layered to a depth of 1 m m on 7.5 X 5 cm glass slides, with antigen and serum wells 2 m m in diameter and separated by 2 m m edge to edge. Serum was placed in the
74 anode well and antigen in the c a t hode well, and electrophoresis was perf o r m e d for 1 h at 2.5 mA/slide. Immunoelectrophoresis. Tests were carried o u t using a modification of the m i c r o - m e t h o d of Faux (1968). Agarose gel was layered to a depth of 1 m m on glass slides as before. T w o wells were cut 2 m m in diameter, at a distance o f 2 m m from the central trough which was 2 m m wide. Antigen was placed in the wells and electrophoresis was p e r f o r m e d at 10 mA/slide for 1 h, after which serum was placed in the central trough and the precipitin reactions were allowed to develop for 24 h in a moist atmosphere. Two-dimensional immunoelectrophoresis (Axelson, 1971). Tests were carried o u t in 1-mm thick agarose gel on 5 X 5 cm glass plates. A 2-mm well in one corner was filled with antigen and first dimension electrophoresis was p e r f o r m e d at 10 V/cm for 1 h, with this well at the cathode, to separate the antigen horizontally. The unw ant ed gel was then removed, leaving a narrow strip containing the separated antigen, and replaced with gel containing the antiserum to a final c o n c e n t r a t i o n of 12 pl/cm 2 to a depth of 1 mm. The plate was r o tate d through 90 ° and second-dimension electrophoresis perf o r m e d at 2 V/cm for 20 h with the antigen strip at the cathode. For antigen absorption in situ the first dimension electrophoresis was carried out as above, with NIBSC antigen in the well, but before removal of the unw ant ed gel, a ditch 2 m m wide was cut parallel to the separated antigen and filled with a large a m o u n t of the NAT antigen, which was allowed to diffuse into the gel for 1 h. The edges of the ditch were then pushed together, the unwanted gel removed, and antiserum containing gel (12 t~l/cm 2) poured on the rest of the plate to a depth of 1 mm, and second-dimension electrophoresis p e r f o r m e d as before. Tandem two-dimensional immunoelectrophoresis (Axelson and Bock, 1972). The p r o cedur e here was the same as for ordinary two-dimensional immunoelectrophoresis except that two wells were cut 2 m m in diameter and 4 mm apart in the plane of horizontal separation. NAT antigen was placed in the first well and NIBSC antigen in the second, before first-dimension electrophoresis. The antigens were allowed to diffuse into the gel for 1 h and the wells were then sealed with m ol t en agarose gel after which first- and second-dimension electrophoresis was carried out as before. Line immunoelectrophoresis, direct comparison o f antigens (Axelson and Bock, 1972). Tests were carried out on 5 X 5 cm glass plates o n t o which antiserum containing gel (12 pl/cm 2) was layered to a depth of 1 mm. When this had set, a strip 5 m m wide was removed from the cathode end and replaced by 2 equal gels, side by side, containing equal amounts of NAT and NIBSC antigens, respectively. Electrophoresis was then p e r f o r m e d at 3 V/cm for 20 h. All immunoelectrophoresis slides and plates were washed in distilled water for 24 h, dried and stained in 0.5% Naphthalene Black after devel opm ent of precipitation patterns. 'Cambrelle', a 100% nyl on 'melded' fabric, made by ICI and marketed by
75 Plant Protection Ltd., Farnham, Surrey, was used as wick material in all immunoelectrophoresis techniques (Strong and Lea, 1977).
Nature of precipitin reactions Two types of precipitin reactions have been observed in agar gel precipitations with C. albicans antigens (Faux, 1968; Warnock et al., 1976): (a) A broad, diffuse 'H' type reaction attributable to a heat-stable polysaccharide antigen on the cell wall -- the Mannan antigen. (b) A sharp, well defined 'R' t y p e reaction attributable to heat-labile internal protein antigens -- the cytoplasmic antigens. Seven different batches of NAT antigen were prepared over a period of 12 months and each was compared shortly after preparation by immunoelectrophoresis against a positive patient's serum, to investigate the reproducibility of the preparation method. To investigate the stability of the NAT antigen during both normal use and prolonged storage, small aliquots from each of 7 batches were stored at --12°C and tested as follows: One sample from each batch, stored for varying times up to 12 months was tested by immunoelectrophoresis against positive human serum. The results of these were compared with the results of each batch tested shortly after preparation against the same serum. A sample from the first batch made was tested several times, being thawed and then refrozen each time. The results were compared with the results of the above tests. The sample was tested once a month for 9 months. RESULTS
Comparison of antigens by precipitation techniques (Table 1) using positive human serum and a reference antiserum Preliminary double diffusion investigation with 3 different concentrations of the NAT antigen (100 mg/ml, 40 mg/ml and 20 mg/ml) tested against STAB showed 12, 11 and 10 precipitin lines, respectively. The NIBSC antigen tested in the same way gave 10 lines (Fig. 1). Reactions of identity can be seen for most of the lines although the complexity of the reactions partially obscures this. The NAT 20 mg/ml antigen seemed the concentration most closely resembling the NIBSC antigen and this concentration was used in the other experiments and designated NAT antigen. When tested against positive human serum both the NAT and NIBSC antigens gave 8 lines. The NAT antigen and the NIBSC antigen each showed 8 lines against the unabsorbed positive serum, most showing reactions of identity (Fig. 2) without reactions with the absorbed serum. Immunoelectrophoresis of the NAT and NIBSC antigens showed 11 and 9 lines, respectively, against positive human serum, and 11 and 13 lines, respectively, against STAB, the NAT extract lines being slightly weaker in both cases. Counterimmunoelectrophoresis of the NAT and NIBSC antigens
76 TABLE 1 COMPARISON OF PRECIPITIN PATTERNS OF NAT AND NIBSC ANTIGENS TESTED AGAINST ST.AB. AND POSITIVE HUMAN SERUM Method
Double diffusion Immu noelectrophoresis Two-dimensional IEP Two-dimensional IEP ; Absorption of antigen in situ
Tandem two-dimensional IEP Line IEP; direct comparison of antigens CIE
Number of precipitin lines seen Standard antibody
Positive human serum
NIBSC antigen (13 mg/ml)
NAT antigen (20 mg/ml)
NIBSC antigen (13 mg/ml)
NAT antigen (20 mg/ml)
]0 13 43
10 11 44
8 9 22
8 11 21
NT NT 30
NT NT 28
14 13 14
12 15 16
4
3
2
2
NT = not tested due to lack of ST.AB.
ii~iiiiii~ii!
Fig. 1. Double diffusion of NAT antigens and NIBSC antigen against ST.AB. (central well). I, NAT 100 mg/ml, II, NIBSC 13 mg/ml; III, NAT 40 mg/ml; IV, NAT 20 mg/ml. Reactions of identity can be observed between the NAT and NIBSC antigens. It can be seen that NAT 20 mg/ml most closely resembles the NIBSC antigen. Fig. 2. Double diffusion of (well A) and positive human 1 mg/ml; 2, NAT 20 mg/ml; NIBSC 13 mg/ml; 7, Mannan
NAT and NIBSC antigens against positive human serum serum absorbed by NAT antigen (well B). Well: 1, Mannan 3 Mannan 1 mg/ml; 4, NAT 20 mg/ml; 5, NAT 20 mg/ml; 6, 1 mg/ml; 8, NAT 20 mg/ml.
77 each showed 2 lines against positive hum a n serum and 3 and 4 lines, respectively, when tested against the STAB. In all cases general similarity was observed between the precipitation patterns of b o t h antigens. Two-dimensional immunoelectrophoresis. The NAT antigen showed 21 peaks and the NIBSC antigen showed 22 peaks when tested against positive h u man serum, and against STAB, 44 and 43 peaks, respectively (Figs. 3 and 4). In all these cases the general precipitation patterns of both antigens were similar. When NIBSC was electrophoresed and absorbed by NAT in the trough, against a positive hum an serum, only 2 very minor c o m p o n e n t s were unabsorbed, remaining as peaks, while faint reactions of identity, continuous lines, were observed with 12 o t h e r c o m p o n e n t s . The NAT antigen showed 13 reactions o f identity with the NIBSC antigen when tested against positive hum a n serum in tandem. The NAT antigen apparently contained 2 antigenic c o m p o n e n t s n o t evident in the NIBSC antigen. By line immunoelectrophoresis 14 reactions of identity were observed (Fig. 5). One or possibly two minor c o m p o n e n t s in the N A T antigen failed to show reactions of identity with the NIBSC antigen. When NAT and NIBSC were tested together against STAB, 28 reactions of identity were observed. Only one, or possibly two, c o m p o n e n t s in the NIBSC antigen failed to show reactions of identity with the NAT antigen.
Reproducibility of the modified antigen Seven batches of NAT antigen prepared at different times were tested shortly after preparation by immunoelectrophoresis against positive h u m a n serum. In all cases the n u m b e r of precipitin lines observed was the same, i.e.
Fig. 3. Two-dimensional immunoeleetrophoresis of NAT antigen against ST.AB; 44 peaks were seen, although many are not visible on the figure. Origin on the right. Fig. 4. Two-dimensional immunoelectrophoresis of NIBSC antigen against ST.AB; 43 peaks were seen, although many are not visible on the figure. Origin on the right. Note similarity in pattern to Fig. 3.
78
Fig. 5. Line immunoelectrophoresis; direct comparison of antigens, a, NAT antigen and b, NIBSC antigen against ST.AB. Reactions of identity can be seen for the majority of lines formed by each antigen, indicating that the major components of each are identical. 28 reactions of identity were seen, although not all are revealed on the figure.
11, a l t h o u g h slight variation o c c u r r e d in the strength o f s o m e lines. T h e r e f o r e it appears t h a t the m e t h o d s h o w e d suitable reliability in preparing the antigen, and 7 d i f f e r e n t b a t c h e s s h o w e d a c c e p t a b l e reproducibility.
Stability of the modified antigen All e x t r a c t s w h e n t a k e n directly f r o m - - 1 2 ° C and tested b y i m m u n o e l e c t r o p h o r e s i s against positive h u m a n s e r u m gave 11 lines, regardless o f the length o f storage time, u p to 12 m o n t h s . H o w e v e r , the e x t r a c t t h a t was t h a w e d and r e f r o z e n s h o w e d progressive loss o f a n t i g e n i c i t y , d r o p p i n g to 5 lines after 3 m o n t h s and 4 after 9 m o n t h s .
TABLE 2 SCREENING OF SERA WITH MANNAN, NAT AND NIBSC ANTIGENS BY CIE AND DD Method
Counterimmunoelectrophoresis Double diffusion
Total No. of sera tested
127 127
Number of positive reactions "H" type precipitins
"R" type precipitins
Mannan
NAT
NIBSC
NAT
NIBSC
46 13
47 8
47 7
2 2
2 2
79 Screening o f sera with mannan, N A T and N I B S C antigens by CIE and DD It can be seen from Table 2 that the results obtained b y NAT and NIBSC antigens show good correlation. By CIE both gave 47/127 positive 'H' t y p e reactions and 2/127 positive 'R' type reactions, i.e. 100% correlation. By DD, the NAT antigen gave 8/127 positive 'H' type reactions and the NIBSC gave 7/127. Both gave 2/127 positive 'R' t y p e reactions, i.e. 87.5% correlation. DISCUSSION Natamycin is a polyene antibiotic closely related to Nystatin and its primary antifungal action is based on its ability to alter the permeability of the cytoplasmic membrane, allowing the loss of inorganic phosphate ions, triose and sugar phosphates, proteins, nucleotides and other essential cellular constituents from the cell (Raab, 1972). This action provides the basis for the collection of antigenic intracellular components relatively unchanged and uncontaminated by cell wall material, hopefully in the same condition as they may be released from phagocytosed yeast cells in the body. It has been shown (Venezia and Robertson, 1974) that extracts made from protoplasts of yeast-phase Candida albicans cells are very similar to the cytoplasmic extract made by Taschdjian et al. (1967) ('S' antigen produced by a sonication method). Preliminary testing showed that pH 8.6, with a concentration of 10,000 pg/ml of Natamycin, and an extraction time of 2 weeks provided the best antigenic material. The loss of antigenicity due to the heat-labile character of many of the cytoplasmic components, did n o t become evident until after 2 weeks extraction at room temperature. The reaction was allowed to progress in the dark, as Natamycin is susceptible to direct sunlight, and at room temperature, as at 4°C no antifungal effect is observed (Raab, 1972). One of the main problems in assessing the diagnostic significance of precipitins to Candida antigens is the use of many different antigenic preparations (Faux et al., 1975) and if cytoplasmic antigens differ markedly in their cell wall antigen content, widely discrepant results could occur (Venezia and Robertson, 1974). It is shown in these investigations that the a m o u n t of cell wall mannan present in the NAT antigen must be very similar to that present in the NIBSC antigen, the results showing 87.5% correlation, by DD and 100% correlation by CIE and also must be very similar in a m o u n t to the pure mannan extract, as the results show 61.5% correlation by DD and 97.2% correlation by CIE. Therefore results obtained by each of the 3 antigens should show few discrepancies. The detection of 'R' t y p e precipitins to the cytoplasmic protein components by NAT and NIBSC antigens showed 100% correlation b y DD and 100% correlation by CIE b u t due to the small number of sera tested, this figure may be suspect.
80 H o w e v e r , as t h e m a j o r i t y o f antigenic c o m p o n e n t s in each e x t r a c t s h o w e d r e a c t i o n s o f i d e n t i t y it can be a s s u m e d t h a t t h e N A T antigen will give similar results to t h e N I B S C antigen with o t h e r positive sera. On t h e evidence here t h e N A T antigen is r e p r o d u c i b l e a n d c o m p a r e s f a v o u r a b l y w i t h t h e N I B S C r e f e r e n c e antigen. T h e stability o f t h e N A T antigen was n o t investigated t h o r o u g h l y , b u t a p p e a r s to be o f a similar o r d e r to t h a t o f the N I B S C antigen. B o t h antigens s h o w e d r a p i d d e g e n e r a t i o n o n r e p e a t e d t h a w i n g a n d re-freezing, b u t w h e n k e p t c o n s t a n t l y at - - 1 2 ° C n o o b v i o u s d e t e r i o r a t i o n was n o t e d a f t e r 12 m o n t h s . A x e l s o n states t h a t his c y t o p l a s m i c antigen r e m a i n s stable f o r u p to 18~ m o n t h s at - - 2 0 ° C . ( A x e l s o n and B o c k , 1972). T h e N a t a m y c i n antigen can be p r e p a r e d easily in t h e r o u t i n e l a b o r a t o r y w i t h o u t great e x p e n s e or e f f o r t . ACKNOWLEDGEMENTS M y t h a n k s to B r o c a d e s , G r e a t Britain L t d . , W e y b r i d g e , S u r r e y , w h o k i n d l y s u p p l i e d t h e N a t a m y c i n ; Dr. E. N n o c h i r i , C o n s u l t a n t Microbiologist, Staff o r d a n d B u r t o n o n T r e n t ; Mr. I.L. M c C a r t n e y , C h i e f T e c h n i c i a n , Microbiolo g y , S t a f f o r d ; Mr. M. Mitchell, C h i e f T e c h n i c i a n , Clinical C h e m i s t r y , Staff o r d ; T h e D e p a r t m e n t o f Medical P h o t o g r a p h y , B u r t o n District H o s p i t a l ; P.H.L., Carlisle and the P a t h o l o g y D e p a r t m e n t , S t a f f o r d , f o r t e c h n i c a l assistance a n d use o f e q u i p m e n t ; Mrs. D. J o h n s o n f o r t y p i n g t h e m a n u s c r i p t ; and the staff of the Microbiology Department, Stafford, for encouragement a n d advice, w i t h o u t w h o m m u c h o f this w o r k w o u l d have b e e n impossible. REFERENCES Andersen, P.L. and A. Stenderup, 1974, Scand. J. Infect. Dis. 6, 69. Axelson, N.H., 1971, Inf. Immunity 4,252. Axelson, N.H., 1973, Inf. Immunity 7,946. Axelson, N.H., 1976, Scand. J. Immunol. 5, 177. Axelson, N.H. and E. Bock, 1972, J. Immunol. Methods 1, 109. Evans, E.G.V. (ed.), 1976, in: The Serology of Fungal Infections and Farmer's Lung Diseases (University Printing Office, Leeds) p. 3. Faux, J.A., 1968, Immunological Studies of the Antigens of Candida albicans in man, PhD Thesis, University of London. Faux, J.A., V.C. Stanley, H.R. Buckley and B.M. Partridge, 1975, J. Immunol. Methods 6,235. Gaines, J.D. and J.S. Remington, 1973, Arch. Intern. Med. 132, 699. Goldstein, E. and P.D. Hoeprich, 1972, J. Infect. Dis. 125, 190. Hasenclever, H.F. and W.O. Mitchell, 1961, J. Bact. 82,570. Odds, F.C., E.G.V. Evans and K.T. Holland, 1975, J. Immunol. Methods 7,211. Raab, W.P., 1972, in: Natamycin (Pimaricin) -- its Properties and Possibilities in Medicine (Thieme, Stuttgart) p. 16. R0sner , F., F.D. Gabriel, C.L. Taschdjian, M.B. Cuesta and P.J. Kozinn, 1970, Amer. J. Med. 51, 54. Sargent, J.R. and S.G. George, 1975, Methods in Zone Electrophoresis, B.D.H. Chemicals, Ltd.
81 Strong, W.M. and D.J. Lea, 1977, J. Med. Lab. Sci. 34,355. Taschdjian, C.L., P.J. Kozinn, A. Okas, L. Caroline and M.T. Halle, 1967, J. Infect. Dis. 117, 180. Taschdjian, C.L., M.B. Cuesta, P.J. Kozinn and L. Caroline, 1969a, Amer. J. Clin. Path. 52, 468. Taschdjian, C.L., P.J. Kozinn, H. Fink, M.B. Cuesta, L. Caroline and A.B. Kantrowitz, 1969b, Sabouraudia 7, 110. Taschdjian, C.L., P.J. Kozinn, M.B. Cuesta and E.F. Toni, 1972, Amer. J. Clin. Path. 57, 195. Venezia, R.A. and R.G. Robertson, 1974, Amer. J. Clin. Path. 61,849. Warnock, D.W., D.C.E. Speller, J.A. Morris and P.H. Mackie, 1976, J. Clin. Path. 29,836.
