Journal of immunological Methods, 154 (1992)27-35
27
© 1992 Elsevier Science Publishers B.V. All rights reserved 0022-1759/92/$05.00
JIM 06413
Monoclonal antibody based ELISAs for cryptococcal polysaccharide * A r t u r o C a s a d e v a l l a, J e a n M u k h e r j e e b a n d M a t t h e w D. S e h a r f f ~ a Dit'ision of Infectioas Diseasesof the Depatlment of Medicine, and h Department of Cell Biology of the Albert Einstein School of Medicine. 1300 Morris Park Ave., Bronx. IVY 10461, USA
(Received 20 February 1992,revised received 9 April 1992,accepted 13 April 1992)
Mouse monoclonal antibody (MAb)-based enzyme-linked immunosorbent assays (ELISAs) have been developed to detect Cryptococcus neoformans capsular polysaccharides from the four serotypes A, B, C and D. The ELISAs avoid the problem of unreliable polysaccharide binding to polystyrene plates by using MAbs to capture and immobilize the polysaccharide antigen. The presence of polysaccharide is detected using MAbs of a different isotype from that of the capture MAb. T h e capturing MAbs are themselves immobilized on the plates using commercial goat anti-mouse polyclonal sera. The MAbs bind to the glucuronoxylomannan component of cryptococcal polysaccharide. The ELISAs can be used to measure the concentration of polysaccharide in biological fluids and are potentially useful tools for basic research and clinical studies. Key words: Cryptococcus neoformans; Monoclonalantibody, mouse; Immunoglobulin,mouse; ELISA; Polysaccharide,cryptococcal
Introduction The fungus Cryptococcus neoformans causes life-threatening meningoencephalitis in up to 10% of patients with the acquired immunodeficiency syndrome (Zuger et al., 1986). This fungus is
Correspondence to: M.D. Scharff, Director, Cancer Center, Albert Einstein College of Medicine, 1300 Morris Park Ave., Chanin Room 330, Bronx, NY 10461, USA. * This work was supported by N1H grants CA09173 and CA39838. A.C. is supported by a Pfizer Postdoctoral Fellowship. J.M. is supported by NIH training grant 2T32C809173-16. M.D.S. is supported in part by the Harry Eagle Chair in Cancer Research from the Women's Division of the Albert Einstein College of Medicine. Abbreviations: BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; GXM, glucaronoxylomannan; MAb, monoclonal antibody; PBS, phosphate-buffered saline.
unusual in that it has a large polysaccharide capsule (Diamond, 1985). The capsular polysaccharide contains glucuronoxylomannnan ( G X M ) and galactoxylomannan fractions (Cherniak et al., 1982). GXM, the major component, consists of a mannose backbone substituted with xylose, glucuronic, and mannose residues (Battacharjee et al., 1985; Turner and Cherniak, 1990). Structural differences in the G X M are the basis for the differentiation of cryptococcal strains into four major serotypes known as A, B, C, and D (Cherniak et al., 1980; lkeda et al., 1982; Kozel, 1989). In the United States the majority of cryptococcal infections are caused by serotypes A and D (Diamond, 1985). Cryptococcal polysaccharide causes a variety of immunological effects including immune paralysis (Kozel et al., 1977), complement activation (Marcher et al., 1978), inhibition of leukocyte
migration (Diamond et al., 1985) and enhancement of HIV infection in vitro (PettoelloMantovani et al., 1992). lmmunoprecipitation (Neill et al., 1951), latex agglutination (Bloomfield et ai., 1963), complement fixation (Bennett et al., 1964), hemagglutination inhibition (Kozel and Cazin, 1972), counterimmunoelectrophoresis (Macconi et al., 1977), and ELISA (Scott et al., 1980, 1981; Eckert and Kozel, 1987; Todaro-Luck et al., 1989; Gade et al., 1991) have been used to detect polysaccharide, in human infections the polysaccharide antigen accumulates in tissues, serum, and cerebrospinal fluid (Bloomfield et al., 1963; Bennett et al., 1964). Detection of polysaccharide antigen is used for the diagnosis of cryptococcal infections (Bloomfield et al., 1963), and changes in :.ntigen concentration during treatment are indicators of prognosis (Diamond and Bennett, 1974). Latex bead agglutination assays are routinely used to detect polysaccharide antigen in clinical practice (Kaufman and Blumer, 1968). Polysaccharides, including those from C. neoformans, often bind poorly to the polystyrene support used for ELISAs and passive adsorption can be unreliable for their attachment to microtiter plates (Callahan et al., 1979; MelvilleSmith and Sheffield, 1980; Scott et al., 1981; Leinonen and Frash, 1982; Reiss et al., 1984; Cherniak et al., 1988; Casadevall et al., 1992). Enhanced cryptococcal polysaccharide binding to polystyrene can be achieved by priming with polylysine (Eckert and Kozel, 1987) or with protein adipic acid dihydrazide derivatives (Cherniak et al., 1988). Enhanced binding can also be achieved by capturing the polysaccharide with specific antibodies bound to polystyrene (Scott et al., 1981; Todaro-Luck et al., 1989; Gade et al., 1991). We describe several sensitive double sandwich ELISAs using mouse monoclonal antibodies (MAbs) of different isotype for the capture and detection of cryptocoecal polysaccharides.
Materials and methods
Strains and polysaccharides Capsular polysaccharide was isolated from the supernatant of 10-14-day-old cultures of C. neo-
formans grown in Sabouraud Dextrose broth (Difco Laboratories, Detroit, MI) as described (Kozel and Cazin, 1972; Dromer et al.0 1987), C. neofonnans strains of serotype A, B, C, and D were obtained from the American Type Culture Collection (ATCC; Rockville, MD), and these are ATCC numbers 24064, 24065, 24066, and 24067, respectively. Polysaccharide concentrations were determined by the phenol-sulfuric acid method (Dubois et al., 1956). Antibodies MAbs 21D2 (IgMk), 7B13 (IgMA), 4D4 (IgMk), 2HI (IgGlk), and 18G9 (IgAk) have been described (Casadevall and Scharff, 1991; Casadevall et al., 1992). These MAbs were generated in the Hybridoma Facility of the Cancer Center at the Albert Einstein College of Medicine. MAbs 21D2, 4D4, 2HI, and 18G9 bind to the capsular polysaccharides from the four serotypes and react with the GXM fraction (Casadevall et al., 1992). However, MAb 21D2 binds to a different epitope than MAbs 2HI, 4D4, and 18G9 (Casadevail et al., 1992). MAbs 2HI, 4D4, and 18G9 have similar if not identical fine specificity (Casadevall et al., 1992; J. Mukherjee, unpublished data). MAb 7B 13 binds only to the capsular polysaccharide of the D (24067) strain and recognizes a different epitope than the other MAbs (Casadevall and Scharff, 1991; Casadevall et al., 1992). Thus the MAbs utilized in this work recognize three different epitopes defined by MAbs 21D2, 2HI (4D4 and 18G9) and 7B13. Antibody concentrations were determined by ELISA relative to standards of the same isotype of known concentration. MAb solutions were made by dilution of ascites fluid or concentrated hybridoma supernatants in 1% BSA in PBS. Hybridoma supernatants were concentrated by filtration through an Amicon filter with a molecular exclusion limit of 100 kD. ELISAs. Assays were done in 96 well microtiter polystyrene plates (Corning Glass Works No. 25801-96, Corning, NY). Two types of ELISA were used. One.type of ELISA used plates passively coated with cryptococcal polysaccharide as described (Dromer et al., 1987; Casadevall and Scharff, 1991). The second type of ELISA used mouse MAbs to capture and detect cryptococcal
min (BSA) and 0.5% horse serum in PBS. Third, a murine anti-cryptococcal M A b of the isotypc recognized by the goat anti-mouse sera was a d d e d in 1% BSA in PBS. Fourth, the cryptococcal polysaccharide .solution was added. Fifth, a second murine anti-cryptococcal M A b o f ~, different isotype than that used for capture was added. Sixth, alkaline p h o s p h a t a s e conjugated goat antimouse specific for the second M A b was added. Lastly, the p h o s p h a t a s e substra~u, p-nitrophcnyl p h o s p h a t e (1 m g / m l in 0.001 M MgCI z, 0.05 M N a 2 C O 3, p H 9.8) was a d d e d and the absorbance at 405 n m was measured in a E L 320 Microplate reader (BioTek Instruments, Winooski, VT). Each
polysaccharide, and is similar in configuration to double antibody sandwich ELISAs used by others (Scott et al., 1981; Todaro-Luck et al., 1989). T h e capturing (bottom) murine M A b s were themselves immobilized by coating the plate with goat anti-mouse isotype specific antibody. All commercial goat anti-mouse antibodies were obtained from FisherBiotech (Orangeburg, NY). T h e protocol for the capture E L I S A s was as follows. First, the polystyrene plates were coated with 50 /zl o f 0 . 2 / z g / m l goat anti-mouse isotype specific polyclonal antibody in 0.02 M p h o s p h a t e - b u f f e r e d saline p H 7.2 (PBS). Second, the plates were blocked with a solution o f I % bovine serum albu-
SYMBOL 1.501 1 20 [-- 24064 (A)
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,,~ 2H1 (IgG1) POLYSACCHARIDE y 21D2 (IgM)
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,~ 2H1 (IgG1) POLYSACCHARIDE ~I" 4D4 (tgM) y GAM-IglVl I I I I I I I I ALKP-GAM-IgM 4D4 (IgM) POLYSACCHARIDE T 2H1 (19GI)
¥ G,,~-~G~ / I I I I I I I
Fig. I. Passive adsorption (triangles) and MAb capture (square and circles) ELISAs for the detection of cryptococcal polysaccharide from the four serotype strains 24064 (A), 24065 (B), 24066 (C), and 24067 (D). The diagram on the right shows the configuration of the ELISAs corresponding to the symbols.The passive adsorption ELISAs used polysaccharide attached to the polysterene support by incubation of polysaccharide solutions in PBS. Thc capture ELISAs used MAbs to immobilize the polysaceharide. The antibody concentrations were: 0.2 p.g/ml fi,r the polyclonal goat anti-mouse (GAM) isotype specific scra used to immobilize the capture MAb; 5 #g/ml for the capture MAb; l0 p.g/ml for the detection MAb; and 0.2 p.g/ml for the alkaline phosphatase conjugated goat anti-mouse (AP-GAM). The optical density values are the average of 6-8 independent wells. The four strains were studied on different days and hence the optical density signals arc not directly comparable for the four serotypes.
step was followed by incubation for 1-1.5 h at 37°C. Overnight incubations at 4°C can also be used. The plates were washed in a Titertek Microplate washer 120 (Flow Laboratories) between each step with a solution of 0.05% Tween.20 (polyoxyethylenesorbitan monolaurate) in PBS. Each plate was washed three times between each step with the exception of the last step before the addition of the p-nitrophenyl substrate when the plates where washed five times. We added 0.010 M NaN 3 to washing, blocking and antibody solutions as a bacteriostatic agent.
simply diluting concentrated hybridoma supernatants or ascites fluid in 1% BSA in PBS. The presence of polysaccharidc is detected by the use of a second mouse MAb (top MAb) which is of a different isotype than that of the capture MAb. We studied various combinations of lgM, lgG~, and IgA MAbs for capture and detection. All combinations successfully detected the presence of polysaccharide. Fig. 1 shows binding curves obtained with passive adsorption and with the MAb capture ELISAs for various combinations of MAbs to the polysaccharides from strains 24064 (A), 24065 (B), 24066 (C), and 24067 (D). Two IgM (4D4 and
Results
21D2) and one IgG t (2HI) were used. The configurations of the ELISAs are shown in Fig. 1. The passive adsorption ELISAs (open and closed triangles) revealed marked differences in the concentration of polysaccharide detected with the detection threshold being D < < A < < B < C. For strain 24067 (D) the sensitivity of the passive adsorption EL1SA (triangles) was comparable or superior to that observed with MAb capture ELISAs (square and circles). For strains 24064 (A), 24065 (t3), and 24066 (C), the MAb capture ELISAs were much more sensitive than the pas-
The capture ELISA has a double antibody 'sandwich' configuration and uses mouse MAbs of different isotypes to immobilize and detect cryptococcal polysaccharide. The capture MAbs are themselves intmobilized using commercial goat anti-mouse polyclonal antisera specific for the isotype of the capture MAb. The use of commercial goat anti-mouse sera eliminates the need to purify the mouse MAbs for use in this assay. In fact the MAb solutions were made by
SYMBOL
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,~ 7B13 (IgM) POLYSACCHARIDE y 2H1 (IgG1)
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,ix 2H1 (IgG1) POLYSACCHARIDE T 7B13 (IgM) y GAM-IgM .,.//////
Fig. 2. Capture ELISAs using MAbs 7B13 (IgM) and 2HI (IgG 0 for the detection of strain 24067 (D) polysaccharide. The configurationof the ELISAs are shown on the right side of the figure. The concentrations of antibodies used were: 0.2 Izg/ml for the polyclonal goat anti-mouse (GAM) isotype specific sera; 5 i.tg/ml for the capture (bottom) mouse MAb; 5 /zg/ml for the detection (top) mouse MAb; and 0.2 /tg/ml for the alkaline phosphatase conjugated goat anti-mouse (AP-GAM). The optical density values are the averageof 5-6 independent wells.
siv¢ adsorption ELISAs. For strain 24067 (D) the optical density signal decreased for the higher polysaccharide concentrations suggesting a prozone-like effect that could potentially yield a false negative result. For strains 24064 (A) and 24066 (C) the ELISA using MAb 21D2 (IgM) for capture and MAb 2HI (IgG I) for detection were more sensitive that the other combinations (squares, Fig. 1). However, for strains 24065 (B) and 24067 (D) the ELISA using MAb 4D4 (lgM) for capture and MAb 2H1 (IgG I) for detection were more sensitive than the other combinations (circles, Fig. 1). An ELISA using the combination of MAb 18G9 (IgA) and MAb 2H1 (IgG I) was also studied but this ELISA was generally less sensitive than that using MAbs 4D4 and 2HI (data not shown). Comparison of the capture ELISAs alternating MAbs 4D4 (lgM) and 2HI (lgG t) as the capture and detection antibodies (open and closed circles) reveals that in all cases the use of MAb 4D4 for capture was more sensitive, presumably because the polyvalence of the lgM class increases the amount of polysaccharide immobilized on the plate. The MAb capture ELISAs were more senstrive for the polysaccharide of strain 24064 (A) than for the other strains. This is consistent with the fact that the MAbs used have higher apparent affinity for the 24064 (A) polysaccharide than for those of the other strains (J. Mukherjee, unpublished data; Casadevall et al., 1992).
The combination of MAbs 7B13 (lgM,~) and 2HI (lgGiK) was also studied. MAbs 7B13 and 2HI bind to different epitopes and double sandwich ELISAs using MAbs of different epitope specificity have the theoretical advantage that they avoid using the same ¢pitope for capture and detection which could potentially decrease sensitivity (Buttler et al., 1984). Fig. 2 shows binding curves with the MAbs 7B13 and 2HI as capture and detection antibodies respectively, and vice versa. This ELISA has comparable sensitivity to that using MAbs which bind to the same epitope (such as 4D4 and 2H 1; see Fig. 1) but is likely to be of limited usefulness given the narrow serotype specificity of MAb 7B13. To determine whether human or mouse sera interfered with the MAb captlire ELISA we compared the binding curves of polysaccharide dissolved in sera relative to polysaccharide in 1% BSA in PBS (Fig. 3). The resulting binding curves are very similar if not identical, indicating the absence of interfering substances in these sera. The capsular polysaccharides of C. neoformans are known to accumulate in the supcrnatant of liquid cultures (Kozel and Cazin, 1971). The ELISA using MAb 4D4 for capture and MAb 2HI for detection (open circles, Fig. 1) was used to measure the concentration of strain 24067 (D) polysaccharide in culture broth as a function of time and yeast cell density (Fig. 4). The polysaccharide was measured directly in the Sabouraud's
=~, 2.80 c~ 2.40] ~'~ 2.00
rl BSA Humanserum • Mousesera . . / /
~ 1.60 Z 1.20
ELIS._~.AA ALKP-GAM-IgG1 2H1(IgG1) POLYSACCHARIDE y 4D4(IgM) y GAM-IgM
~
J
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~ . .~.""
i M . : . : . . . = _ A _ . . . . : . . . . , ~ 'i~ . ~l
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l
i
l
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ililnll
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Fig. 3. Measurement of strain 24064(A) polysaccharidedissolvedin human serum, BALB/c mouse sera, and 1% BSA in PBS using the capture ELISAwith MAb 4D4 for capture and MAb 2HI for detection. The configurationof the ELISA sandwich is shown at right. The optical densityvaluesarc the averageof 6-8 independent wells.
but rather rises dramatically several days after the onset of the stationary growth phase.
Discussion
I
200.
.lO
150,
~1~.
10 3
1
234
5
67
8
9
DAY(S) OF CULTURE
Fig. 4. Accumulatiol~of soluble polysaccharide in the supernatant of a 24067 (D) strain liquid culture with time. The yeast were counted in a hemocytometer. The culture was grown iu Sabouraud's broth at 35°C with shaking at 120 ray/rain. The polysaccharide concentration was measured using a capture ELISA with MAb 4D4 for capture and MAb 2H1 for detection (For configuration see diagram in Fig. 1; open circle.) The right vertical axis denotes the concentration of yeast/ml and the closed circles indicate the yeast cell counts for the day of culture. The left vertical axis denotes the concentration of cryptococcal polysaccharide in tLg/ml and the bars indicate the concentration of polysaccharide for the day of culture. broth medium without purification. The data show that the polysaceharide concentration in the supernatant does not increase linearly with time,
We describe a set of double antibody 'sandwich' ELISAs for the detection of cryptococcal polysaccharide using mouse MAbs. In developing the ELISAs we used unpurified ascites or hybridoma supernatants and commercial goat mouse specific antisera. In principle, the use of commercial goat antisera to immobilize the capture mouse MAb can be dispensed with by purifying the mouse MAbs and adsorbing them directly to polystyrene plates. The ELISAs work remarkably well despite the complex nature of the antibody sandwich which involves the interaction of four antibodies and antigen. The alkaline phosphatase reagents produced strong and easily measured optical density signals (Fig. 1). MAb-based ELISAs have several advantages over other immunodetection methods including latex bead agglutination. MAbs are temporally invariant reagents which are theoretically available in unlimited supply. As a result, MAb-based ELISAs are not subject to the type of variation reported for polyclonal sara-based latex bead assays (Wu and Koo, 1983). In contrast to bead agglutination, ELISAs are suitable for automation and provide quantitative information. MAbbased ELISAs may also be less vulnerable to false positive results caused by rheumatoid factor than polyclonal sera-based latex bead assays (Stockman and Roberts, 1983). However, MAbbased assays have the inherent disadvantage that a single epitope is reeognized. This limitation may be important since C. neoformans strains exhibit considerable antigenic variation (Ikeda et ai., 1982; Spiropulu et ai., 1990). Another potential problem is the use of MAbs binding to the same epitope or to spatially close epitopes for the capture and detection of antigen, since the MAbs could compete a n d / o r cause steric hindrance which could result in lower sensitivity (Buttler et al., 1983). This does not appear to be the case in our assay even though MAbs 2H1, 18G9, and 4D4 have the same fine specificity (Casadevall et al., 1992). The good results obtained using MAbs
with the same fine specificity for capture and detection probably reflect the fact that our antigen is a high molecular weight polysaccharide (200-800 kDa) with a repeating structure that must have many identical epitopes per molecule (Battacharjee et ai., 1984). In fact, the sensitivity of the ELISA using MAb 4D4 for capture and MAb 2H1 for detection of serotype A polysaccharide is comparable to that reported for a polyclonal sera-based ELISA (Scott et al., 1980). MAb capture ELISAs were clearly superior to passive adsorption ELISAs for the detection of polysaccharides from the 24064 (A), 24065 (B), and 24066 (C) strains. Passive adsorption ELISAs showed marked differences in the polysaccharide detection threshold for the four C. neoformans strains used. For the passive adsorption ELISAs using MAbs 2H1 and 4D4 the detection threshold for polysaccharide detection was D < < A < < B < C. Since both 4D4 and 2HI have greater apparent affinity for A than for the other serotypes (Casadevall et al., 1992), these differences in detection threshold presumably reflect differences in the ability of these polysaccharides to bind polystyrene plates or differences in the epitope availability of polystyrene absorbed polysaccharide. Cryptococcal polysaccharides have been reported to bind poorly to polystyrene (Scott et al., 1981; Reiss et al., 1984; Cherniak et ai., 1988). it is noteworthy that the G X M s of serotypes D and C are the least and most heavily substituted, respectively, with xylose and glucuronic acid residues (Battacharjee et al., 1984). Thus the degree of backbone substitution correlates inversely with MAb detection in passive absorption ELISAs, suggesting that the more highly substituted polysaccharides (B and C) either do not bind well to polystyrene or undergo structural changes on binding such that their epitopes are altered or not accessible. In this regard, a MAb has been described which binds cryptococcal polysaceharide by immunodiffusion but not in ELISA (Spiropulu et al., 1989). The significant decrease in the optical density observed for the 24067 (D) passive adsorption ELISA at the high polysaccharide concentrations (Fig. 1, bottom panel) resembles a prozone phenomenon and suggests that the conformation of the polysaccharide bound to the polystyrene is important for
MAb binding. The MAb capture ELISAs were less susceptible to prozone-like effects. In summary, we describe a versatile ~ t of mouse MAb-based ELISAs for the detection and measurement of cryptococcal polysaccharidc. These ELISAs can be useful in studies of cryptococcal pathogenesis, of capsular assembly, and of the many immunological phenomena attributed to the polysaccharide. The MAb capture ELISAs provide an attractive method for the measurement of polysaccharide in biological fluids because no purification of the polysaccharide is needed, in addition to polysaccharide detection, MAb capture can be used to immobilize polysaccharides to polysterene support for .-;creening of hybridoma supernatants, for studies of antibody specificity, and for human serology studies. The MAb capture ELISAs readily detect polysaccharide in sera and have potential clinical applications.
Acknowledgements The authors thank Manxia Fan for expert technical assistance in some aspects of this work and Dr. Lionel Zuckier for critical reading of the manuscript.
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Callahan, 111. L.T., Woodhour, A.F.. Meekcr, J.B. and Hilleman, M.R. (1979) Enzyme-linked immunosorbent assay for measurement of antibodies against pneumococcal polysacchar(de antigens: comparison with rodioimmunoassay. J. Clin. Microbiol. 10, 459. Casadevall, A. and Scharff. M.D. (1991)The mouse antibody response to infection with Co'ptoeoccus neoformans: V H and Vt. usage in polysaccharide binding antibodies. J. Exp. Med. 174, 151. Casadevall, A., Mukherjee, J., Devi, S.J.N., Scheerson, R., Robbins, J.B. and ScharfL M.D. (1992) Antibodies elicited by a Ctyptococcus neoformans glucuronoxylomannantetanus toxoid conjugate vaccine have the same specificity as those elicited in infection. J. Infect Dis. (in press). Cherniak. R.. Reiss, E.. Slodki, M.E.. Plattner, R.D. and Blumer, S.O. (1980) Structure and antigenic activity of the capsular polysaccharide of Cryptococcus neoformans serotype A. Mol. lmmunol. 17, 1{125. Chcrniak, R., Reiss. E. and Turner, S.H. (19821A galactoxylomannan antigen of CrYl]tOcoccttsneoformans serotype A. Carbohydr. Res. 102 239. Cherniak, R., Cheeseman, M.M., Reyes, G.H.. Reiss, E. and Todaro, F. (1988) Enhanced binding of capsular polysacchar(des of Cryptococcus neoformans to polystyrene microlitration plates h)r enzyme-linked immunosorbent assay. Diagn. Clin. Immunol. 5, 344. Diamond, R.D. and Bennett, J.E. (19741 Prognostic factors in cryplococcal ~!eningitis. Ann. Intern. Med. 80, 176. Diamond, R.D. (1985) Co,ptococcus neoformans. In: G.L. Mandell, R.G. Gordon, and J.E. Bennett (Eds.), Principles and Practice of Infectious Diseases. John Wiley, New York, p. 1460. Dromcr, F., Salamero, J., Contrcpnis, A., Carbon, C. and Yen(, P. (19871 Productkm, Characterization, and antmody specificity of a mouse monoclonal antibody reactive with Co'ptococcus neoformatts capsular polysaccharide. Infect. Immun. 55, 742. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, R.A. and Smith, F. (19561 Color(metric method for the determination of sugars and related substances. Anal. Chem. 28, 3511. Eckert, T.F. and Kozel, T.R. (1987) Production and characterization of monoclonal antibodies specific for Cryptococcus neoformans capsular polysaccharide. Infect. Immun. 55, 1857. Gade, W., Hinnefeld, S.W. and Yi, A. (19911 The detection ~md quantitation of cryptococcal antigen: a comparison of a new EIA test and latex agglutination. Am. Clin. Lab. 10, 28. Kozel, T.R. and Cazin. J.R. (1971) Noncncapsulated variant cf Cryptococcus neoformans. I. Virulence studies and characterization of solubTe polysaccharide. Infect. Immun. 3, 287. Kozel, T.R., Gulley, W.F. and Cazin, Jr., J. (1977) Immune response to Cryptococcus ncoformans soluble polysaccharide: immunological unresponsiveness. Infect. lmmun. 18, 701. Kozel, T.R. (1989) Antigenic structure of Cryptococcus neo-
formans capsular polysaccharide. In: E. Kurstak tEd.), Immunology of Fungal Diseases. Marcel Dekker, New York - Bosel, p. 63. Ikeda, R., Shinoda, T., Fukazawa, Y. and Kaufman, L. (1982) Antigenic characterization of Cryptococcus neofornlans serotypes and its application to serotyping of clinical isolates. J. Clin. Microbiol. 16, 22. Leinonen, M. and Frasch, C.E. 0982) Class-specific antibody response to Group B Neisseria motingitides capsular polysaccharide: use of polylysine precoating in an enzymelinked immunosorbent assay. Infect. lramun. 38, 1203. Maccani. J.E. (1977) Detection of cryptococeal polysaccharide using counterimmunoelectrophoresis. Am. J. Clin. Pathol. 68, 38. Macher, A.M., Bennett. J.E., Gad(k, J.E, and Frank, M.M. (19781 Complement depletion in cryptococcal sepsis. J. lmmunol. 120, 1686. Melville-Smith, M. and Sheffield, F. (19811) Enzyme-linked immunosorbent assays for the estimation of immune responses to pneumococcal pnlysaccharides. J. Biol. Stand. 8, 317. Murphy, J.W. and Moorhead,J.W. (19821 Regulation of cellmediated immunity in cryptococcosis I. induction of specific afferent T-suppressor cells by cryptocoecal antigen. J. Immunol. 128, 276. Neill, J.M., Sugg, J.Y. and McCaulcy, D.W. (19511 Serologicady reactive material in the spinal fluid, blood, and urine from a human case of cryptococcosis itorulosis). Proc. Soc. Exp. Biol. Med. 77, 775. Pettoello-ManRwani, M., Casadevall, A., Kollman, T., Rub(nstein, A. and Goldstein, A. (19921 Enhancement of HIV-I infection by the capsular polysaccharide of Cryptococcus neoformans. Lancet 339, 21. Scott, E.N., Muchmore, H.G. and Felton, F.G. (19801 Comparison of enzyme immunoassay and latex agglutination for detection of Cryptococcits neoformans antigen. Am. J. Clin. Pathol. 6, 790. Scott, E.N., Muchmore, H.G. and Felton, F.G. O9811 Enzyme linked immunoabsorbent assays in murine cryptococeosis. Sabouraudia 19, 257. Stockman, L. and Roberts, G.D. (1983) Specificity of the latex test for cryptococcal antigen: a rapid, simple method for eliminating interference factors. J. Clin. Microbiol. 17, 945. Spiropulu, C., Eppard, R.A., Otteson, E. and Kozel, T.R. (1989) Antigenic variation within serotypes of Cryptococcus neoformans detected by monoclonal antibodies specific for the caosular polysaccharide. Infect. Immun. 57, 32411. Todaro-Luck, F., Reiss, E., Cherniak, R. and Kaufman, L. (19891 Characterization of Cryptococcus neoformans capsular glucuronoxylomannan polysacchuride with monoclonal antibodies. Infect. immun. 57, 3882. Turner, S.H. and Cherniak, R. (19901 Multiplicity in the structure of the glucuronoxylomannan of Cryptococcus neoformans. In: J.P. Latge and D. Boucias (Eds.), Fungal Cell Wall and Immune Response. Springer-Verlag, Berlin Heidelberg, p. 124. Reiss, E., Cherniak, R., Eby, R. and Kaufman, L. (19841 -
Enzyme immunodetection of lgM to galactoxylomannan of Cryptococcus neo[ormans. Diagn. Immunol. 2, 1119. Wu. T.C. and Koo. S.Y. (1983) Comparison of three commercial cryptococcal latex kils for detection of ¢r,yptococeal antigen. J. Clin. Microbiol. 18, 1127.
Zuger, A., lx~uic, E., Ilolzman, R.S., Simbcrkoff, M.S. and Rahal, J,J. 1I~1S6)('r~ptococcal disease in patients with the acquired immunodeficiency syndrome: diagnostic features and outcome of trcatmenl. Ann. Intern. Mcd. 1114,234.
27
© 1992 Elsevier Science Publishers B.V. All rights reserved 0022-1759/92/$05.00
JIM 06413
Monoclonal antibody based ELISAs for cryptococcal polysaccharide * A r t u r o C a s a d e v a l l a, J e a n M u k h e r j e e b a n d M a t t h e w D. S e h a r f f ~ a Dit'ision of Infectioas Diseasesof the Depatlment of Medicine, and h Department of Cell Biology of the Albert Einstein School of Medicine. 1300 Morris Park Ave., Bronx. IVY 10461, USA
(Received 20 February 1992,revised received 9 April 1992,accepted 13 April 1992)
Mouse monoclonal antibody (MAb)-based enzyme-linked immunosorbent assays (ELISAs) have been developed to detect Cryptococcus neoformans capsular polysaccharides from the four serotypes A, B, C and D. The ELISAs avoid the problem of unreliable polysaccharide binding to polystyrene plates by using MAbs to capture and immobilize the polysaccharide antigen. The presence of polysaccharide is detected using MAbs of a different isotype from that of the capture MAb. T h e capturing MAbs are themselves immobilized on the plates using commercial goat anti-mouse polyclonal sera. The MAbs bind to the glucuronoxylomannan component of cryptococcal polysaccharide. The ELISAs can be used to measure the concentration of polysaccharide in biological fluids and are potentially useful tools for basic research and clinical studies. Key words: Cryptococcus neoformans; Monoclonalantibody, mouse; Immunoglobulin,mouse; ELISA; Polysaccharide,cryptococcal
Introduction The fungus Cryptococcus neoformans causes life-threatening meningoencephalitis in up to 10% of patients with the acquired immunodeficiency syndrome (Zuger et al., 1986). This fungus is
Correspondence to: M.D. Scharff, Director, Cancer Center, Albert Einstein College of Medicine, 1300 Morris Park Ave., Chanin Room 330, Bronx, NY 10461, USA. * This work was supported by N1H grants CA09173 and CA39838. A.C. is supported by a Pfizer Postdoctoral Fellowship. J.M. is supported by NIH training grant 2T32C809173-16. M.D.S. is supported in part by the Harry Eagle Chair in Cancer Research from the Women's Division of the Albert Einstein College of Medicine. Abbreviations: BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; GXM, glucaronoxylomannan; MAb, monoclonal antibody; PBS, phosphate-buffered saline.
unusual in that it has a large polysaccharide capsule (Diamond, 1985). The capsular polysaccharide contains glucuronoxylomannnan ( G X M ) and galactoxylomannan fractions (Cherniak et al., 1982). GXM, the major component, consists of a mannose backbone substituted with xylose, glucuronic, and mannose residues (Battacharjee et al., 1985; Turner and Cherniak, 1990). Structural differences in the G X M are the basis for the differentiation of cryptococcal strains into four major serotypes known as A, B, C, and D (Cherniak et al., 1980; lkeda et al., 1982; Kozel, 1989). In the United States the majority of cryptococcal infections are caused by serotypes A and D (Diamond, 1985). Cryptococcal polysaccharide causes a variety of immunological effects including immune paralysis (Kozel et al., 1977), complement activation (Marcher et al., 1978), inhibition of leukocyte
migration (Diamond et al., 1985) and enhancement of HIV infection in vitro (PettoelloMantovani et al., 1992). lmmunoprecipitation (Neill et al., 1951), latex agglutination (Bloomfield et ai., 1963), complement fixation (Bennett et al., 1964), hemagglutination inhibition (Kozel and Cazin, 1972), counterimmunoelectrophoresis (Macconi et al., 1977), and ELISA (Scott et al., 1980, 1981; Eckert and Kozel, 1987; Todaro-Luck et al., 1989; Gade et al., 1991) have been used to detect polysaccharide, in human infections the polysaccharide antigen accumulates in tissues, serum, and cerebrospinal fluid (Bloomfield et al., 1963; Bennett et al., 1964). Detection of polysaccharide antigen is used for the diagnosis of cryptococcal infections (Bloomfield et al., 1963), and changes in :.ntigen concentration during treatment are indicators of prognosis (Diamond and Bennett, 1974). Latex bead agglutination assays are routinely used to detect polysaccharide antigen in clinical practice (Kaufman and Blumer, 1968). Polysaccharides, including those from C. neoformans, often bind poorly to the polystyrene support used for ELISAs and passive adsorption can be unreliable for their attachment to microtiter plates (Callahan et al., 1979; MelvilleSmith and Sheffield, 1980; Scott et al., 1981; Leinonen and Frash, 1982; Reiss et al., 1984; Cherniak et al., 1988; Casadevall et al., 1992). Enhanced cryptococcal polysaccharide binding to polystyrene can be achieved by priming with polylysine (Eckert and Kozel, 1987) or with protein adipic acid dihydrazide derivatives (Cherniak et al., 1988). Enhanced binding can also be achieved by capturing the polysaccharide with specific antibodies bound to polystyrene (Scott et al., 1981; Todaro-Luck et al., 1989; Gade et al., 1991). We describe several sensitive double sandwich ELISAs using mouse monoclonal antibodies (MAbs) of different isotype for the capture and detection of cryptocoecal polysaccharides.
Materials and methods
Strains and polysaccharides Capsular polysaccharide was isolated from the supernatant of 10-14-day-old cultures of C. neo-
formans grown in Sabouraud Dextrose broth (Difco Laboratories, Detroit, MI) as described (Kozel and Cazin, 1972; Dromer et al.0 1987), C. neofonnans strains of serotype A, B, C, and D were obtained from the American Type Culture Collection (ATCC; Rockville, MD), and these are ATCC numbers 24064, 24065, 24066, and 24067, respectively. Polysaccharide concentrations were determined by the phenol-sulfuric acid method (Dubois et al., 1956). Antibodies MAbs 21D2 (IgMk), 7B13 (IgMA), 4D4 (IgMk), 2HI (IgGlk), and 18G9 (IgAk) have been described (Casadevall and Scharff, 1991; Casadevall et al., 1992). These MAbs were generated in the Hybridoma Facility of the Cancer Center at the Albert Einstein College of Medicine. MAbs 21D2, 4D4, 2HI, and 18G9 bind to the capsular polysaccharides from the four serotypes and react with the GXM fraction (Casadevall et al., 1992). However, MAb 21D2 binds to a different epitope than MAbs 2HI, 4D4, and 18G9 (Casadevail et al., 1992). MAbs 2HI, 4D4, and 18G9 have similar if not identical fine specificity (Casadevall et al., 1992; J. Mukherjee, unpublished data). MAb 7B 13 binds only to the capsular polysaccharide of the D (24067) strain and recognizes a different epitope than the other MAbs (Casadevall and Scharff, 1991; Casadevall et al., 1992). Thus the MAbs utilized in this work recognize three different epitopes defined by MAbs 21D2, 2HI (4D4 and 18G9) and 7B13. Antibody concentrations were determined by ELISA relative to standards of the same isotype of known concentration. MAb solutions were made by dilution of ascites fluid or concentrated hybridoma supernatants in 1% BSA in PBS. Hybridoma supernatants were concentrated by filtration through an Amicon filter with a molecular exclusion limit of 100 kD. ELISAs. Assays were done in 96 well microtiter polystyrene plates (Corning Glass Works No. 25801-96, Corning, NY). Two types of ELISA were used. One.type of ELISA used plates passively coated with cryptococcal polysaccharide as described (Dromer et al., 1987; Casadevall and Scharff, 1991). The second type of ELISA used mouse MAbs to capture and detect cryptococcal
min (BSA) and 0.5% horse serum in PBS. Third, a murine anti-cryptococcal M A b of the isotypc recognized by the goat anti-mouse sera was a d d e d in 1% BSA in PBS. Fourth, the cryptococcal polysaccharide .solution was added. Fifth, a second murine anti-cryptococcal M A b o f ~, different isotype than that used for capture was added. Sixth, alkaline p h o s p h a t a s e conjugated goat antimouse specific for the second M A b was added. Lastly, the p h o s p h a t a s e substra~u, p-nitrophcnyl p h o s p h a t e (1 m g / m l in 0.001 M MgCI z, 0.05 M N a 2 C O 3, p H 9.8) was a d d e d and the absorbance at 405 n m was measured in a E L 320 Microplate reader (BioTek Instruments, Winooski, VT). Each
polysaccharide, and is similar in configuration to double antibody sandwich ELISAs used by others (Scott et al., 1981; Todaro-Luck et al., 1989). T h e capturing (bottom) murine M A b s were themselves immobilized by coating the plate with goat anti-mouse isotype specific antibody. All commercial goat anti-mouse antibodies were obtained from FisherBiotech (Orangeburg, NY). T h e protocol for the capture E L I S A s was as follows. First, the polystyrene plates were coated with 50 /zl o f 0 . 2 / z g / m l goat anti-mouse isotype specific polyclonal antibody in 0.02 M p h o s p h a t e - b u f f e r e d saline p H 7.2 (PBS). Second, the plates were blocked with a solution o f I % bovine serum albu-
SYMBOL 1.501 1 20 [-- 24064 (A)
[ .~
.o
....I-
.,o_-o ~
F.o-
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,~
........
,,. ~'
l
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/ P/
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01 .
~ . , ,
~
,-,,..,~.:, - ......
/ ° ' °f
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x - - . ----'-'~
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........... ...o- -o--
.o--'.-~< - Z~
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....I O.3O 0
~
,,~ 2H1 (IgG1) POLYSACCHARIDE y 21D2 (IgM)
y C~M-~M
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~lsot
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/J~ 4D4 (IgM)
POLYSACCHARIDE / / / / / / / /
p..-"°- - o - --o
2.501 2ooi- 2,~or~(c)
,~ 2H1 (kJG1)
,~ ALKP-GAM-IgM •
-o
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,~ ALKPGAM'IgG1 A
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,~ 2H1 (IgG1) POLYSACCHARIDE ~I" 4D4 (tgM) y GAM-IglVl I I I I I I I I ALKP-GAM-IgM 4D4 (IgM) POLYSACCHARIDE T 2H1 (19GI)
¥ G,,~-~G~ / I I I I I I I
Fig. I. Passive adsorption (triangles) and MAb capture (square and circles) ELISAs for the detection of cryptococcal polysaccharide from the four serotype strains 24064 (A), 24065 (B), 24066 (C), and 24067 (D). The diagram on the right shows the configuration of the ELISAs corresponding to the symbols.The passive adsorption ELISAs used polysaccharide attached to the polysterene support by incubation of polysaccharide solutions in PBS. Thc capture ELISAs used MAbs to immobilize the polysaceharide. The antibody concentrations were: 0.2 p.g/ml fi,r the polyclonal goat anti-mouse (GAM) isotype specific scra used to immobilize the capture MAb; 5 #g/ml for the capture MAb; l0 p.g/ml for the detection MAb; and 0.2 p.g/ml for the alkaline phosphatase conjugated goat anti-mouse (AP-GAM). The optical density values are the average of 6-8 independent wells. The four strains were studied on different days and hence the optical density signals arc not directly comparable for the four serotypes.
step was followed by incubation for 1-1.5 h at 37°C. Overnight incubations at 4°C can also be used. The plates were washed in a Titertek Microplate washer 120 (Flow Laboratories) between each step with a solution of 0.05% Tween.20 (polyoxyethylenesorbitan monolaurate) in PBS. Each plate was washed three times between each step with the exception of the last step before the addition of the p-nitrophenyl substrate when the plates where washed five times. We added 0.010 M NaN 3 to washing, blocking and antibody solutions as a bacteriostatic agent.
simply diluting concentrated hybridoma supernatants or ascites fluid in 1% BSA in PBS. The presence of polysaccharidc is detected by the use of a second mouse MAb (top MAb) which is of a different isotype than that of the capture MAb. We studied various combinations of lgM, lgG~, and IgA MAbs for capture and detection. All combinations successfully detected the presence of polysaccharide. Fig. 1 shows binding curves obtained with passive adsorption and with the MAb capture ELISAs for various combinations of MAbs to the polysaccharides from strains 24064 (A), 24065 (B), 24066 (C), and 24067 (D). Two IgM (4D4 and
Results
21D2) and one IgG t (2HI) were used. The configurations of the ELISAs are shown in Fig. 1. The passive adsorption ELISAs (open and closed triangles) revealed marked differences in the concentration of polysaccharide detected with the detection threshold being D < < A < < B < C. For strain 24067 (D) the sensitivity of the passive adsorption EL1SA (triangles) was comparable or superior to that observed with MAb capture ELISAs (square and circles). For strains 24064 (A), 24065 (t3), and 24066 (C), the MAb capture ELISAs were much more sensitive than the pas-
The capture ELISA has a double antibody 'sandwich' configuration and uses mouse MAbs of different isotypes to immobilize and detect cryptococcal polysaccharide. The capture MAbs are themselves intmobilized using commercial goat anti-mouse polyclonal antisera specific for the isotype of the capture MAb. The use of commercial goat anti-mouse sera eliminates the need to purify the mouse MAbs for use in this assay. In fact the MAb solutions were made by
SYMBOL
121
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~=E 0.8
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lo,.Hi~-~4~.~rl°'~l'lllnll ,i,ilull l JJ,llul • 0.001 0.01 0.1 1 10 [24067 (D) Polysaccharide] (p.g/ml)
ALKP-GAM-IgM
,~ 7B13 (IgM) POLYSACCHARIDE y 2H1 (IgG1)
.--_>" 1.0
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ELISA
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,ix 2H1 (IgG1) POLYSACCHARIDE T 7B13 (IgM) y GAM-IgM .,.//////
Fig. 2. Capture ELISAs using MAbs 7B13 (IgM) and 2HI (IgG 0 for the detection of strain 24067 (D) polysaccharide. The configurationof the ELISAs are shown on the right side of the figure. The concentrations of antibodies used were: 0.2 Izg/ml for the polyclonal goat anti-mouse (GAM) isotype specific sera; 5 i.tg/ml for the capture (bottom) mouse MAb; 5 /zg/ml for the detection (top) mouse MAb; and 0.2 /tg/ml for the alkaline phosphatase conjugated goat anti-mouse (AP-GAM). The optical density values are the averageof 5-6 independent wells.
siv¢ adsorption ELISAs. For strain 24067 (D) the optical density signal decreased for the higher polysaccharide concentrations suggesting a prozone-like effect that could potentially yield a false negative result. For strains 24064 (A) and 24066 (C) the ELISA using MAb 21D2 (IgM) for capture and MAb 2HI (IgG I) for detection were more sensitive that the other combinations (squares, Fig. 1). However, for strains 24065 (B) and 24067 (D) the ELISA using MAb 4D4 (lgM) for capture and MAb 2H1 (IgG I) for detection were more sensitive than the other combinations (circles, Fig. 1). An ELISA using the combination of MAb 18G9 (IgA) and MAb 2H1 (IgG I) was also studied but this ELISA was generally less sensitive than that using MAbs 4D4 and 2HI (data not shown). Comparison of the capture ELISAs alternating MAbs 4D4 (lgM) and 2HI (lgG t) as the capture and detection antibodies (open and closed circles) reveals that in all cases the use of MAb 4D4 for capture was more sensitive, presumably because the polyvalence of the lgM class increases the amount of polysaccharide immobilized on the plate. The MAb capture ELISAs were more senstrive for the polysaccharide of strain 24064 (A) than for the other strains. This is consistent with the fact that the MAbs used have higher apparent affinity for the 24064 (A) polysaccharide than for those of the other strains (J. Mukherjee, unpublished data; Casadevall et al., 1992).
The combination of MAbs 7B13 (lgM,~) and 2HI (lgGiK) was also studied. MAbs 7B13 and 2HI bind to different epitopes and double sandwich ELISAs using MAbs of different epitope specificity have the theoretical advantage that they avoid using the same ¢pitope for capture and detection which could potentially decrease sensitivity (Buttler et al., 1984). Fig. 2 shows binding curves with the MAbs 7B13 and 2HI as capture and detection antibodies respectively, and vice versa. This ELISA has comparable sensitivity to that using MAbs which bind to the same epitope (such as 4D4 and 2H 1; see Fig. 1) but is likely to be of limited usefulness given the narrow serotype specificity of MAb 7B13. To determine whether human or mouse sera interfered with the MAb captlire ELISA we compared the binding curves of polysaccharide dissolved in sera relative to polysaccharide in 1% BSA in PBS (Fig. 3). The resulting binding curves are very similar if not identical, indicating the absence of interfering substances in these sera. The capsular polysaccharides of C. neoformans are known to accumulate in the supcrnatant of liquid cultures (Kozel and Cazin, 1971). The ELISA using MAb 4D4 for capture and MAb 2HI for detection (open circles, Fig. 1) was used to measure the concentration of strain 24067 (D) polysaccharide in culture broth as a function of time and yeast cell density (Fig. 4). The polysaccharide was measured directly in the Sabouraud's
=~, 2.80 c~ 2.40] ~'~ 2.00
rl BSA Humanserum • Mousesera . . / /
~ 1.60 Z 1.20
ELIS._~.AA ALKP-GAM-IgG1 2H1(IgG1) POLYSACCHARIDE y 4D4(IgM) y GAM-IgM
~
J
0.80
O F- 0.40 o
~ . .~.""
i M . : . : . . . = _ A _ . . . . : . . . . , ~ 'i~ . ~l
0.0001
0.001
l
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l
i
l
l
-
0.01 0.1 1 [Polysaccharide](gg/ml)
I
ililnll
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Fig. 3. Measurement of strain 24064(A) polysaccharidedissolvedin human serum, BALB/c mouse sera, and 1% BSA in PBS using the capture ELISAwith MAb 4D4 for capture and MAb 2HI for detection. The configurationof the ELISA sandwich is shown at right. The optical densityvaluesarc the averageof 6-8 independent wells.
but rather rises dramatically several days after the onset of the stationary growth phase.
Discussion
I
200.
.lO
150,
~1~.
10 3
1
234
5
67
8
9
DAY(S) OF CULTURE
Fig. 4. Accumulatiol~of soluble polysaccharide in the supernatant of a 24067 (D) strain liquid culture with time. The yeast were counted in a hemocytometer. The culture was grown iu Sabouraud's broth at 35°C with shaking at 120 ray/rain. The polysaccharide concentration was measured using a capture ELISA with MAb 4D4 for capture and MAb 2H1 for detection (For configuration see diagram in Fig. 1; open circle.) The right vertical axis denotes the concentration of yeast/ml and the closed circles indicate the yeast cell counts for the day of culture. The left vertical axis denotes the concentration of cryptococcal polysaccharide in tLg/ml and the bars indicate the concentration of polysaccharide for the day of culture. broth medium without purification. The data show that the polysaceharide concentration in the supernatant does not increase linearly with time,
We describe a set of double antibody 'sandwich' ELISAs for the detection of cryptococcal polysaccharide using mouse MAbs. In developing the ELISAs we used unpurified ascites or hybridoma supernatants and commercial goat mouse specific antisera. In principle, the use of commercial goat antisera to immobilize the capture mouse MAb can be dispensed with by purifying the mouse MAbs and adsorbing them directly to polystyrene plates. The ELISAs work remarkably well despite the complex nature of the antibody sandwich which involves the interaction of four antibodies and antigen. The alkaline phosphatase reagents produced strong and easily measured optical density signals (Fig. 1). MAb-based ELISAs have several advantages over other immunodetection methods including latex bead agglutination. MAbs are temporally invariant reagents which are theoretically available in unlimited supply. As a result, MAb-based ELISAs are not subject to the type of variation reported for polyclonal sara-based latex bead assays (Wu and Koo, 1983). In contrast to bead agglutination, ELISAs are suitable for automation and provide quantitative information. MAbbased ELISAs may also be less vulnerable to false positive results caused by rheumatoid factor than polyclonal sera-based latex bead assays (Stockman and Roberts, 1983). However, MAbbased assays have the inherent disadvantage that a single epitope is reeognized. This limitation may be important since C. neoformans strains exhibit considerable antigenic variation (Ikeda et ai., 1982; Spiropulu et ai., 1990). Another potential problem is the use of MAbs binding to the same epitope or to spatially close epitopes for the capture and detection of antigen, since the MAbs could compete a n d / o r cause steric hindrance which could result in lower sensitivity (Buttler et al., 1983). This does not appear to be the case in our assay even though MAbs 2H1, 18G9, and 4D4 have the same fine specificity (Casadevall et al., 1992). The good results obtained using MAbs
with the same fine specificity for capture and detection probably reflect the fact that our antigen is a high molecular weight polysaccharide (200-800 kDa) with a repeating structure that must have many identical epitopes per molecule (Battacharjee et ai., 1984). In fact, the sensitivity of the ELISA using MAb 4D4 for capture and MAb 2H1 for detection of serotype A polysaccharide is comparable to that reported for a polyclonal sera-based ELISA (Scott et al., 1980). MAb capture ELISAs were clearly superior to passive adsorption ELISAs for the detection of polysaccharides from the 24064 (A), 24065 (B), and 24066 (C) strains. Passive adsorption ELISAs showed marked differences in the polysaccharide detection threshold for the four C. neoformans strains used. For the passive adsorption ELISAs using MAbs 2H1 and 4D4 the detection threshold for polysaccharide detection was D < < A < < B < C. Since both 4D4 and 2HI have greater apparent affinity for A than for the other serotypes (Casadevall et al., 1992), these differences in detection threshold presumably reflect differences in the ability of these polysaccharides to bind polystyrene plates or differences in the epitope availability of polystyrene absorbed polysaccharide. Cryptococcal polysaccharides have been reported to bind poorly to polystyrene (Scott et al., 1981; Reiss et al., 1984; Cherniak et ai., 1988). it is noteworthy that the G X M s of serotypes D and C are the least and most heavily substituted, respectively, with xylose and glucuronic acid residues (Battacharjee et al., 1984). Thus the degree of backbone substitution correlates inversely with MAb detection in passive absorption ELISAs, suggesting that the more highly substituted polysaccharides (B and C) either do not bind well to polystyrene or undergo structural changes on binding such that their epitopes are altered or not accessible. In this regard, a MAb has been described which binds cryptococcal polysaceharide by immunodiffusion but not in ELISA (Spiropulu et al., 1989). The significant decrease in the optical density observed for the 24067 (D) passive adsorption ELISA at the high polysaccharide concentrations (Fig. 1, bottom panel) resembles a prozone phenomenon and suggests that the conformation of the polysaccharide bound to the polystyrene is important for
MAb binding. The MAb capture ELISAs were less susceptible to prozone-like effects. In summary, we describe a versatile ~ t of mouse MAb-based ELISAs for the detection and measurement of cryptococcal polysaccharidc. These ELISAs can be useful in studies of cryptococcal pathogenesis, of capsular assembly, and of the many immunological phenomena attributed to the polysaccharide. The MAb capture ELISAs provide an attractive method for the measurement of polysaccharide in biological fluids because no purification of the polysaccharide is needed, in addition to polysaccharide detection, MAb capture can be used to immobilize polysaccharides to polysterene support for .-;creening of hybridoma supernatants, for studies of antibody specificity, and for human serology studies. The MAb capture ELISAs readily detect polysaccharide in sera and have potential clinical applications.
Acknowledgements The authors thank Manxia Fan for expert technical assistance in some aspects of this work and Dr. Lionel Zuckier for critical reading of the manuscript.
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