Detection of plaque-forming cells by a latex bead technique

Journal o f Immunological Methods, 49 (1982) 283--292 Elsevier Biomedical Press

283

DETECTION OF PLAQUE-FORMING CELLS BY A LATEX BEAD TECHNIQUE

OMAR B AGAS R A and IVAN DAMJANOV

Department o f Pathology and Laboratory Medicine, Hahnemann Medical College, Philadelphia, PA 19102, U.S.A. (Received 31 July 1981, accepted 2 October 1981)

A new latex bead technique for measuring the plaque-forming cell (PFC) responses to bacterial antigens is described. This technique has been designed for the study of antigens that cannot be readily coated onto SRBC but may also be used for antigens that adsorb onto SRBC as well. Application of the latex bead technique for the study of PFC response of hamsters to Treponema reiter antigen is described in detail. Using SIII, an antigen that readily adsorbs to SRBC, we have compared the latex bead technique and the conventional SRBC-PFC technique and found that the latex bead technique is more sensitive than the conventional technique. The technique can be used for direct and indirect PFC assays. Technical details for the optimal performance of the latex bead PFC assay are outlined.

Key words: plaque-forming cells -- latex beads -- Treponema pallidum

INTRODUCTION

Several techniques referred to as plaque-forming cell (PFC) assays are currently in use for the detection of antibody-forming cells. Most PFC assays are done in agar or agarose (Jerne and Nordin, 1963) b u t may also be performed between glass slides (Cunningham and Szenberg, 1968), in carboxymethyl cellulose (Nossal et al., 1970) or on poly-L-lysine conjugated sheep e r y t h r o c y t e (SRBC) monolayers (Kennedy and Axelrad, 1971; Baker et al., 1973). All the present techniques utilize red blood cells either as an antigen or a vehicle for soluble antigen(s), which are coated onto the red blood cells. However, there are many antigens which cannot be coated onto the red b l o o d cells surface due to their unique chemical nature (Schwartz and Braun, 1965; Golub et al., 1968; McAlack et al., 1970; Jerne et al., 1974), or because they could n o t be coupled in sufficient density (Pasanen and Makela, 1969; Noar et al., 1971). In the present report we describe a new PFC assay for detection o f antibody-forming cells sensitized with antigens that cannot be readily adsorbed onto the SRBC. However, this new tech0022-1759/82/0000--0000]$02.75 © 1982 Elsevier Biomedical Press

284 nique can be used for antigens that adsorb readily to SRBC as well. We show t h a t our technique is more sensitive than the standard PFC assay based on coating of SRBC with the same antigen. MATERIALS AND METHODS

Animals Male inbred LSH/Ss Lak hamsters were obtained from Charles River Breeding Laboratory, Wilmington, MA. Hamsters weighing 90--110 g were housed 6--8 per cage.

Antigens Treponema reiter (T. phagedenis) was grown in serum-containing broth as described previously (Baseman et al., 1979). Treponemes were sonicated extensively by a sonicator (Fisher Scientific Co.) and a homogeneous bacterial cell suspension was obtained by passing the homogenate through a series of filters (3.0 gm -* 1.0 pm -~ 0.8 pm -~ 0.40 pm -~ 0.22 pm MiUipore filters). Protein concentration of final filtrate was assayed by the Lowry et al. (1951) m e t h o d . PVP (polyvinyl pyrollidone) a T-independent antigen was obtained from Sigma. Purified pneumococcal polysaccharide type III (SIII) was kindly provided by Dr. Phillip Baker, NIH. The immunological properties of SIII from this source have been described previously (Baker et al., 1973).

Immunization Bacterial extracts were diluted in sterile saline and the desired dosage (see legends to figures and tables) was injected intraperitoneally (i.p.) in 0.3 ml volume. SIII was injected i.p. in the a m o u n t of 0.5 pg/hamster in 0.25 vol. The PFC assays were performed 5 days after immunization unless otherwise specified.

Coating o f SRBC and latex beads with antigens SRBC were coated with SIII according to the m e t h o d described by Baker et al. (1973). Briefly, 1 ml of SIII 1 mg/ml was added to 1 ml of 20% washed SRBC and vortexed for 5 min. Then 1 ml of 0.1% chromium chloride (CrC13) in sterile saline was added, mixed and incubated for 10 min at room temperature. The cells were washed 3 times with PBS followed by centrifugation at 600 X g for 5 min. These cells were diluted in Hank's balanced salt solution (HBSS) to 1% before use. PVP was bound to tannic acid-treated SRBC by the previously described m e t h o d (Rotter and Trainen, 1974). SIII, PVP or trepomenal antigens were incubated with 8 X 101° beads/ml (Dow Diagnostics, diameter 1.01--1.74 pm) in glycine buffer pH 8.6 (Sigma Chemical Co., St. Louis, MO) for 15 min with intermittent vortexing at

285 r o o m temperature. The beads were counted in a h e m o c y t o m e t e r and their concentration was adjusted as desired. The beads were washed 3 times in PBS followed b y centrifugation at 1500 X g for 10 min and diluted in HBSS to a final concentration as desired (see legends of tables or figures). Preparation o f cell suspensions PFC assays were performed 5 days after immunization. Single cell suspensions were prepared b y gently teasing spleen or lymph node fragments apart in chilled culture medium (RPMI 1640 + 10% FCS + 50 pg/ml Geramicin) and subsequently filtering the cell clumps through 60-mesh stainless steel screens. Cell debris were removed by incubating the cell suspensions at r o o m temperature and allowing them to settle for 10 min. Cells were washed 2 times by centrifugation at 400 X g for 10 min and resuspended in serum-free medium. Antigen-coated SRBC were coupled to 60 nm X 15 nm Coming tissue culture dishes with poly-L-lysine (MW 40,000, Sigma) according to the method described b y Kennedy and Axeirad (1971). Cell suspensions from immunized animals in medium mixed with 100 pl of complement were subsequently plated over the SRBC monolayers and incubated at 37°C for 45 min. In order to measure the PFC response against SIII or treponemal antigen b y the latex bead method, latex beads were mixed with SRBC at the SRBC to latex bead ratio (E/L ratio) of 1 : 50. Glass tubes (13 mm X 75 mm) containing 50 #1 of 10% SRBC (2 X 109/ml) and 20 pl of guinea pig complement (C) and 100 #1 of latex beads (5 X 101°/ml) conjugated with antigen were mixed on a Vortex. One hundred pl of cells (1 X 107 cells/ml) plus 700 pl of 1% Seaplaque agarose (Marine Colloids, Rockville, ME) was added at 47°C, mixed and immediately poured into premarked, frosted clean glass slides. After solidification ( 2 ~ 3 min), slides were incubated in a humidified a t m o sphere at 37°C for 4 h. Plaques were enumerated with the aid of a dissecting microscope. In order to measure the indirect plaques (Fig. 6), 10 pl of rabbit-anti-hamster IgG (Cappel Laboratories, Cochranville, PA) serum were added just before the addition of cells. The indirect PFC were enumerated b y the following formula: total number of PFC - - n u m b e r of direct PFC = number of indirect plaques. RESULTS PFC response to SIII The results of the PFC assays are presented in Table 1. Our data show that the latex bead m e t h o d is more sensitive than the conventional SRBC technique. Using the latex bead technique, numerous plaques were obtained at all 4 levels of antigen concentrations. The technique gives satisfactory results even with 0.125/~g/ml of SIII antigen used for coating whereas with the standard technique considerably larger amounts of antigen were needed to obtain the same number 0f plaques. The plaques obtained with the latex bead technique were of the same size as the plaques obtained b y the conventional technique. The plaques could be counted b y naked eye or under a dissecting microscope with either bright or dark field illumination. The plaques were also easily recognizable b y light

286 TABLE 1 C o m p a r i s o n o f P F C assays using t h e c o n v e n t i o n a l S R B C c o a t i n g t e c h n i q u e a n d t h e l a t e x bead technique. Concentration of antigen ( m g / m l ) used for c o a t i n g

1.0 0.5 0.25 0.125 0 PVPcontrol

No. o f P F C (± S.E.M.)/106 splenic cells SIII c o a t e d o n to S R B C b y CrC13 a

SIII a d s o r b e d o n t o latex b e a d s

70 ± 18 67 +- 2.3 31 ± 2.1 3+_ 1.0 2+_ 2 5+- 3

89 +- 6.9 86 -+ 4.2 88 +- 4.8 76-+ 2.8 8+_2 11-+4

a Technique of Kennedy and Axelrad (1971). E / L r a t i o was 1 : 50 in glycine b u f f e r p H 8.6.

microscopy (Fig. 1). At higher magnification, one could identify the antibody producing cell surrounded by latex beads (Fig. 2) in the center of the hemolytic plaque.

Fig. 1. Bright field light m i c r o s c o p y view o f l a t e x bead h e m o l y t i c p l a q u e (× 90).

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Fig. 2. High power view of a latex bead hemolytic plaque. Note the centrally located antibody secreting cell (arrow) surrounded by latex beads (× 380).

TABLE 2 Comparison of PFC responses to treponemal antigens using conventional SRBC coating technique and the latex bead technique. Dilution of antigen used for immunization a

No. of PFC (_+ S.E.M.)/106 splenic cells Antigen coated onto SRBC by

1:100 1:10 1:102 1:103 1 : 104 1:10 s 0

CrCI3

tannic acid

6+1 8_+3 13+3 22_+2 16 _+3 4_+1 3_+1

5_+2 4_+2 7_+3 9_+I 11 _+4 2_+1 4_+1

Antigen adsorbed onto latex beads

3_+ 1 29+ 6 110_+16 272_+28 109 _+19 48_+ 7 6_+ 2

extract containing 12.6 mg/ml protein diluted in PBS buffer and injected i.p. into hamsters. E/L ratio was I : 50.

a T. r e i t e r

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PFC responses to T. reiter antigens Table 2 contains the data obtained with spleen cells of animals immunized with T. reiter antigen in 10-fold serial dilutions. No PFC response could be detected by the conventional techniques of coating of antigen to SKBC because we could n o t coat the SRBC with the treponemal antigen. This is in keeping with the results of Sequeira and Eldridge (1973) who have successfully attached the T. pallidum antigen to formalinized, tanned turkey RBC b u t could n o t coat the native RBC. Using the latex bead technique, a bellshaped PFC response was obtained. Ionic and buffer conditions for adsorption o f antigens onto latex particles T. reiter extract, containing approximately 1 mg of protein/ml, was used to test the effects of pH and the composition of various buffers on the efficiency o f adsorption of the antigen to latex particles. As may be seen from Table 3, an adequate PFC response may be obtained using PBS, glycine and borate buffer. The pH of PBS was varied from 7.2 to 9.0 w i t h o u t significant effect on the PFC assay. Ratio of SRBC to latex particles (ElL ratio) for optimal PFC response In order to determine E/L ratio for the optimal PFC response, latex particles in increasing numbers were added to a constant number of SRBC. As

TABLE 3 C o m p a r a t i v e e f f i c i e n c y o f a d s o r p t i o n o f T. reiter a n t i g e n s t o latex beads in various buffers. D i l u t i o n of a n t i g e n used for i m m u n i z a t i o n

Source of PFC

1 : 10 °

Spleen LN a Spleen LN Spleen LN Spleen LN Spleen LN Spleen LN Spleen LN

1 : 10 1 : 102 I : 103 1 : 104 I : l0 s 0

No. o f P F C / 1 0 6 cells in various b u f f e r s PBS a p H 7.2

PBS pH 8.0

PBS p H 9.0

Glycine p H 8.6

Borate pH 8.0

6 5 11 20 160 122 201 311 66 162 48 98 5 2

8 9 22 41 143 108 192 318 58 185 26 79 6 3

6 11 19 37 129 117 226 381 56 155 18 90 8 5

7 3 27 43 147 116 229 441 79 203 55 117 5 3

3 5 12 17 110 97 129 291 36 133 16 65 8 4

a LN, inguinal a n d m e s e n t e r i c l y m p h n o d e ; PBS, p h o s p h a t e - b u f f e r e d saline. E / L ratio was 1 : 50.

289 TABLE 4 T h e e f f e c t o f various S R B C t o l a t e x b e a d s r a t i o s ( E / L ratios) o n P F C response, i : 103 d i l u t i o n of T. reiter e x t r a c t was used for i m m u n i z a t i o n a n d t h e c o n t r o l a n i m a l s were i n j e c t e d w i t h saline. A n t i g e n was a d s o r b e d o n l a t e x particles in glycine b u f f e r at p H 8.6. Source of PFC

Immunized -- spleen Immunized -- lymph node N o n - i m m u n i z e d spleen

No. o f P F C / 1 0 6 cells. E / L r a t i o 1:1

1 : 10

1:20

1 : 50

1 : 100

1 : 200

0 1 1

16 22 3

43 51 4

117 206 8

122 191 8

93 116 9

may be seen from Table 4 the maximal number of PFC was detected at E/L ratios of 1 : 50--1 : 100. Autoagglutination of latex beads was noticed at higher E/L ratios (1 : 100--1 : 200). Autoagglutinated latex beads (Fig. 3) obscured the plaques and made the interpretation of findings equivocal. We have, therefore, decided to designate the E/L ratio of 1 : 50 as optimal for this test.

Fig. 3. H e m o l y t i c p l a q u e , s t a i n e d w i t h h e m a t o x y l i n a n d eosin, s h o w i n g a u t o a g g l u t i n a t i o n o f l a t e x h e a d s a t high c o n c e n t r a t i o n o f a n t i g e n ( 1 0 m g / m l ) (X 320).

290 TABLE 5 Direct a n d i n d i r e c t P F C r e s p o n s e s to T. reiter a n t i g e n . 1 : 1 0 0 0 d i l u t i o n o f a n t i g e n e x t r a c t was used for i m m u n i z a t i o n o n day 0. E / L r a t i o was 1 : 50 in glycine b u f f e r p H 8.6. R a b b i t a n t i - h a m s t e r IgG ( 1 0 p l / t u b e ) was used in o r d e r t o d e t e c t t h e i n d i r e c t ( P F C ) response. Day o f assay

Source of PFC

No. o f P F C (_+ S.E.M.)/106 cells Direct

Indirect

4

Spleen LN a

65 -+ 10 90 -+ 11

15 N.D.

-+ 3

5

Spleen LN

365 + 19 610+-42

905 45

_+ 88 -+17

6

Spleen LN

55 -+ 3.5 701-+17

126 109

_+ 11 -+23

7

Spleen LN

25 -+ 2.8 170-+28.2

71.5 + 3 165 _+21.2

8

Spleen LN

6 -+ 2 9 -+ 1

13 29

-+ 1 -+ 5

a LN, inguinal a n d m e s e n t e r i c l y m p h nodes.

Comparison o f direct and indirect PFC response using the latex bead technique In order to establish whether the latex bead technique can be used for indirect PFC, hamsters were immunized with T. reiter extract (1 : 1000 dilution) and both the direct and indirect PFC response were measured 4--8 days after immunization. From Table 5 it may be seen t h a t an adequate PFC response was obtained in both the direct and indirect PFC assay. The maximal PFC response for spleen cells was noticed on day 5, and on day 6 for the lymph node cells.

TABLE 6 P F C r e s p o n s e to SIII c o a t e d to l a t e x b e a d s in various c o n c e n t r a t i o n s . Concentration of antigen(mg/ml) 0.2 1 5 10 0

No. o f P F C (-+ S . E . M . ) / 1 0 6 splenic cells

A p p e a r a n c e of p l a q u e s

4 9 -+ 22 78 -+ 12

Clear, circular

1 6 -+ 4 18 -+ 9 4-+2

Granular, poorly demarcated, i n d i s t i n c t , o f t e n elliptical

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PFC response at various concentrations of antigen used for adsorption to latex beads SIII in various concentrations was adsorbed onto latex particles keeping all the other conditions optimal. As it may be seen in Table 6, low concentrations of antigen give better results than higher concentrations. Due to autoagglutination of latex particles at higher concentrations, the plaques become indistinct, have a granular texture and are often elliptical or elongated and not circular as under optimal conditions. It thus appears that low concentrations of antigen give optimal results. DISCUSSION

The original hemolytic plaque-forming assay developed by Jerne and his associates (Jerne and Nordin, 1963; Jerne et al., 1963) has greatly contributed to our understanding of the basic immunologic phenomena involving the antibody producing cells and their precursors. This PFC was originally established for detection of antibodies present on the surface of SRBC and was later modified for the detection of antigens that could be coupled to SRBC or other erythrocytes (Jerne et al., 1974). However, even with this modification, the technique can be used only for the study of a small number of antigens, primarily those that could be coupled to SRBC by CrCI3, tannic acid or by diazotation (Jerne et al., 1974). Antigens that could not be coupled to SRBC could not be studied by these techniques. Therefore, it has so far been impossible to adequately perform the PFC assay for various bacterial, parasitic and tumor antigens that cannot be attached to SRBC. Other techniques, such as bacteriolytic PFC assay (Schwartz and Braun, 1965; McAlack et al., 1970) and tumor cytolytic plaque-forming assay (Fuji et al., 1971) have been devised for the study of these antigens, In general, these assays have been found to be irreproducible (Jerne et al., 1974). In the present paper, we describe a new PFC technique useful for studying the responses to antigens that may not be readily coupled to SRBC by standard means. In our studies we have used treponemal antigens, i.e., bacterial wall antigens that could not be adsorbed to SRBC (Tomizawa and Kasamatsu, 1966; Shore, 1974). We have shown that this antigen readily adsorbs onto latex beads and that a latex bead PFC may be used to detect immune cells secreting antibodies specific for this antigen. Our additional findings (not reported here) indicate that other bacterial and tumor antigens could be used in the same manner. Although we do not know the exact mechanism of plaque formation, we believe that the hemolysis of SRBC was initiated by formation of antigenantibody complexes on the surface of latex beads and local activation of the complement cascade. Although activated complement proteins attached to antigen-antibody complexes bound to latex beads and were thus 'immobilized' they apparently lysed the nearby SRBC as 'innocent bystanders.' Dif-

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fusion of activated hemolytic complement complexes from the center of the plaques obviously occurred but was of limited extent. In this paper we also show t h a t the latex bead PFC assay may be used for antigens that adsorb to SRBC as well. From our data it appears that the latex bead PFC assay is more sensitive than the conventional PFC with antigen coated directly to SRBC. Although we do n o t know the reasons for this higher sensitivity, we think that the porous latex beads bind more antigen per surface area than the glycocalyx covered SRBC. It is also possible t h a t antigens bound to the latex beads have more antigenic determinants available for reaction with antibodies than the antigens coating the SRBC. ACKNOWLEDGEMENTS

The authors wish to t h a n k Ms. Barbara Walker for secretarial help, Mr. Shahab Hashemi for the photography, and Ms. Cynthia Myers and Ms. Bernice Uppright for technical assistance. This work was supported by PHS Grants NIH CA23097 and GM29040 and an institutional grant from Hahnemann Medical College. REFERENCES Baker, P.J., N.D. Reed, P.W. Stashak, D.K. Amsbaugh and B. Prescott, 1973, J. Exp. Med. 137, 1431. Baseman, J.B., J.G. Nichols and S. Mogerdey, 1979, Infect. Immunol. 23, 392. Cunningham, A.J. and A. Szenberg, 1968, Immunology 14, 599. Fuji, H., M. Zaleski and F. Milgrom, 1971, Transplant. Proc. 3, 852. Golub, E.S., R.I. Mishell, W.O. Weigle and R.W. Dutton, 1968, J. Immunol. 100, 133. Jerne, N.K. and A.A. Nordin, 1963, Science 140, 405. Jerne, N.K., A.A. Nordin and C. Henry, 1963, in: Cell Bound Antibodies, eds. B. Amos and H. Koprowski (Wistar Inst. Press, Philadelphia, PA) p. 109. Jerne, N.K., C. Henry, A.A. Nordin, H. Fuji, A.M.C. Koros and I. Lefkovits, 1974, Transplant. Rev. 18, 130. Kennedy, J.C. and M.A. Axelrad, 1971, Immunology 20, 253. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, J. Biol. Chem. 193, 265. McAlack, R.F, J. Cerny, J.L. Allen and H. Friedman, 1970, Science 168, 141. Noar, D., C. Henry, H.H. Fudenberg, 1971, J. Immunol. 107,302. Nossal, G.J.V., A.E. Bussard, H. Lewis and J.C. Mazic, 1970, J. Exp. Med. 131,894. Pasanen, V.J. and O. Makela, 1969, Immunology 16, 399. Rotter, V. and N. Trainen, 1974, Cell. Immunol. 13, 76. Schwartz, S.A. and W. Braun, 1965, Science 149, 200. Sequeira, P.J.L. and A.E. Eldridge, 1973, Br. J. Vener. Dis. 49,242. Shore, R.N., 1974, Arch. Dermatol. 109,854. Tomizawa, T. and S. Kasamatsu, 1966, Jap. J. Med. Sci. Biol. 19, 305.