Enumeration of antigen-specific IgE responses at the single-cell level by an ELISA plaque assay

Journal of Immunological Methods, 135 (1990) 129-138

129

Elsevier JIM05776

Enumeration of antigen-specific IgE responses at the single-cell level by an ELISA plaque assay * Swey-Shen Chen Department of Pathology, Case Western Reserve University, Cleveland, OH 44106, U.S.A.

(Received 12 April 1990, revised received 24 August 1990, accepted 30 August 1990)

In vitro elicitation and enumeration of antigen-specific IgE-secreting cells in antigen-primed murine lymophocyte cultures can be reproducibly and accurately quantitated. Rat anti-mouse IgE monoclonal antibodies (Mab anti-e) were developed. Antigen-specific IgE-secreting cells can be detected on antigen or MAb anti-e coated nitrocellulose discs pasted to 24-well culture plates. This IgE ELISA-plaque assay (EPe) is more reliable than antigen capture or total IgE ELISAs in monitoring de novo induction of antigen-specific IgE memory responses in cultures of antigen-primed lymphocytes elicited with antigens. Key words: ELISA; IgE; Monoclonal antibody; In vitro elicitation

Introduction

Immunoglobulin E (IgE) mediates key events in tissue injury and regional immunological defense. High levels of IgE are produced in helminthic parasite infection and IgE-mediated allergy disCorrespondence to: S.-S. Chen, Department of Veterinary Sciences, IANR, Department of Pathology and Microbiology, Medical Center, and School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583-0907, U.S.A. * This publication was supported by NIH Grants AI-22569 and P30-AR39750, and an Arthritis Investigatorship. Abbreviations: ABC, avidin biotinylated enzyme complex; AP-GARaG, alkaline phosphatase coupled to goat anti-rat IgG; AP-SAGG, alkaline phosphate swine anti-goat IgG; BCIP/rNBT, 5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium; b.g., background; b-GAME, biotinylated goat anti-mouse IgE; biotin-X-NHS, long chain N-hydroxysuccinimide ester; CsA, cyclosporin A; DNP, dinitrophenol; DNP-KLH, dinitrophenylated-keyhole limpet hemocyanin; EPe, IgE ELISA plague assay; ELISA, enzyme-linked immunosorbant assay; GAME, goat anti-mouse IgE; GAMG1, goat anti-mouse IgG1; GAM/~,goat anti-mouse IgM; GARaG, goat anti-rat IgG; MAb anti-e, rat monoclonal antibody against mouse IgE; NC, nitrocellulose; NP, nitrophenol; PCA, passive cutaneous anaphylaxis; PNPP, p-nitrophenyl phosphate; RAG, ragweed; SA-AP, streptavidin-alkaline phosphatase; SAGG, swine anti-goat IgG.

orders. Numerous in vitro cell culture systems and assays in rodents and humans have been developed for studying mechanisms of spontaneous or nonantigen-specific IgE production; however, due to the difficulties of detecting antigen-specific IgE, it has not been possible to study cellular and molecular mechanisms of antigen-stimulated IgE antibody production in vitro. ELISA-plaque (EP) assay has gained increasing importance in assessing immunoglobulin and cytokine production on a single cell level (Sedgwick and Holt, 1983; Holt et al., 1984; Czerkinsky et al., 1983, 1988; Moeller and Borrebaeck, 1985; Skidmore et al., 1989). To this aim, we prepared rat anti-mouse IgE monoclonal antibodies (MAb anti-e) and are able to elicit antigen-specific IgE production in vitro, and these antigen-specific IgE secreting cells can be detected by an MAb anti-e based ELISA-plaque assay (EPe). Materials and methods Animals

2-month-old female L o u / c N rats were a generous gift of Dr. Richard Smith, Case Western Re-

0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

130 serve University. The rats were raised and maintained by Dr. Smith throughout the study. CAF1 and BALB/c mice were purchased from Jackson Laboratories (Bar Harbor, ME).

Immunization The rats were immunized twice with 10 /xg of mouse monoclonal anti-DNP IgE (Liu et al., 1980; Chen et al., 1984) in complete Freund's adjuvant, 10/~g of IgE in incomplete Freund's adjuvant and 10/~g soluble IgE i.v. Rats were rested and boosted with 100 ~tg of IgE in 4 mg AI(OH)3 i.p. and later with 50 #g of soluble IgE i.v. Spleen cells of immunized Lou rats were fused with P3X63Ag 8.653 plasmacytoma immunoglobulin nonsecretor cells obtained from the Salk Institute. The plates were coated with 50 /~1 of different classes of monoclonal or myeloma immunoglobulins at 1 /xg/ml. Supernatants from confluent hybridoma cultures were diluted one to five, tested for activities against each immunoglobulin isotype, and developed with alkaline phosphatase coupled to goat anti-rat IgG (Ap-GARaG). Female CAF 1 mice were primed with 1 /~g KLH in 2 mg AI(OH)3 i.p. 7 days later, mice were boosted with 1 /~g KLH in 2 mg AI(OH)3, mice were rested for 7 days, and boosted again in 1/~g KLH in 2 mg AI(OH)3. One groups of mice received three daily injections of cyclosporin A (CsA) at 150 mg/kg i.m., prior to antigen priming. This protocol was shown to optimally induce antigen-specific IgE responses (Chen, 1988; Chen et al., 1989). Reagents Monoclonal anti-DNP IgM, IgG1, IgG2b, IgE and anti-ragweed (RAG) IgE were affinity purified as described (Liu et al., 1980; Chen et al., 1984). Murine anti-NP IgE hybridoma 91.58 (Bose et al., 1984) was a generous gift of Dr. E. Rector of the University of Winnipeg, and was purified on an NP-BSA-Sepharose 4B column. Rabbit anti-mouse DNP-specific IgE (RAME) and goat anti-mouse DNP-specific IgE (GAME) hyperimmune sera were prepared as described (Liu et al., 1980; Chen et al., 1984), and rendered IgE isotype specific by sequential affinity adsorption on Sepharose 4B columns coupled with normal mouse sera, IgG and IgA, and finally adsorbed and eluted

from monoclonal anti-RAG IgE-Sepharose 4B column. Pure myeloma immunoglobulins MOPC 315(X, a), MOPC21(x, Yl), MOPC195(x, ~2b), and RPC20 (X2) were obtained from Organon Teknika (Malveran, PA). Rat IgG was obtained from Axell (Westbury, NY). A panel of rabbit anti-rat immunoglobulin isotype-specific reagents made by Dr. H. Bazin was obtained from Dr. R. Smith, including anti-IgG2a (SLG2a-9), antiIgG2b (SLG2b-9), anti-IgG2c (SLG2c-5), antiIgM (SLM1), anti-IgA (SLA1), and anti-IgE (SLE1). EM95 supernatant, a rat anti-mouse IgE monoclonal antibody product, was generously provided by Drs. M. Baniyash and Z. Eshhar at Weizrnann Institute (Baniyash and Eshhar, 1984). Affinity purified goat anti-rat IgG (GARaG), goat anti-mouse IgG1 (GAMG1) and goat anti-mouse IgM (GAM/~) were obtained from Southern Biot e c h n o l o g y A s s o c i a t e s (SBA, Birmingham, AL). 5-bromo-4-chloro-3-indolyl phosphate/ nitroblue tetrazolium (BCIP/NBT) substrate was obtained from Kirkegaard & Perry Laboratories (Gaithersburg, MD). p-nitrophenyl phosphate (DNPP) was obtained from Sigma Co. (St. Louis, MO). Streptavidin-alkaline phosphatase (SA-AP) was obtained from Zymed (San Francisco, CA). Alkaline phosphatase-swine anti-goat IgG (APSAGG), and lactoperoxidase was obtained from Boehringer-Mannheim (Indianapolis, IN). Biotin long chain N-hydroxy succinimide ester (biotin-XNHS) was obtained from Calbiochem (La Jolla, CA). Dulbecco minimal essential medium (DMEM) was obtained from M.A. Bioproducts (Walkersville, MD). Fetal calf serum was obtained from HyClone Laboratories (Logan, UT).

Biotinylation of rat monoclonal antibodies Mab anti-e from clone B 12 or BF 815 was affinity purified by rabbit anti-rat IgG coupled Sepharose 4B and extensively dialysed in 1 × PBS at pH 7.8 without azide. 4/~1 of biotin-X-NHS at 10 mg/ml in 100% dimethylsulfoxide (DMSO) was added to 100/~1 of MAb anti-e at 3 mg/ml in PBS. The reaction mixture was left at 4°C for 12 h, and dialyzed against PBS. Radioimmunoassay 50 ~1 of MAb anti-e at 10-50 /zg/ml were added to 96-wells (Falcon Microtest III) overnight

131 at 4°C. Plates were b l o c k e d b y 1% B S A a n d 0.1% T w e e n 20, a n d 50 /~1 o f a n t i - D N P m o u s e I g E or I g G were a d d e d to i n d i v i d u a l wells at different c o n c e n t r a t i o n s a n d i n c u b a t e d at 37°C for 30 min. T h e wells were w a s h e d a n d i n c u b a t e d with 5 0 / x l of r a d i o l a b e l e d antigen, D N P - B S A at 100,000 c p m (20 n g / m l ) at 37°C for 30 min. T h e plates were w a s h e d a n d dried, i n d i v i d u a l wells were cut a n d c o u n t e d in a M i c r o m e d i c g a m m a - c o u n t e r .

w i t h I g E s t a n d a r d s at a p p r o p r i a t e dilutions. Biot i n y l a t e d M A b anti-e was a d d e d at 1 ~ g / m l . Avidin-biotinylated alkaline phosphatase enzyme c o m p l e x ( A B C ) ( A B C - A P kits, A K - 5 0 0 0 , Vector Labs, Burlingame, C A ) was then a d d e d for 30 n'fin at 37°C, w a s h e d a n d followed b y a d d i t i o n of 50 #1 of P N P P at 2 m g / m l . Results were assessed 30 m i n later a t A414 nrn.

Total IgE sandwich assay

ELISA

96-well plates were c o a t e d with 50 btl M A b anti-e at 10 # g / m l o v e r n i g h t at 4°C, washed, b l o c k e d a n d followed b y the a d d i t i o n of s t a n d a r d I g E or cell s u p e r n a t a n t s at a p p r o p r i a t e dilutions. 50 /tl of b i o t i n y l a t e d g o a t a n t i - m o u s e I g E (bG A M E ) at 1 / ~ g / m l or a different b i o t i n y l a t e d M A b anti-e at 1 / t g / m l were a d d e d , a n d the p l a t e was w a s h e d a n d d e v e l o p e d with A B C as above.

A 4-layer E L I S A for evaluating the anti-e activities of h y b r i d o m a s u p e r n a t a n t s : 50/~1 of m u r i n e m o n o c l o n a l a n t i b o d i e s or m y e l o m a i m m u n o globulins of different classes a n d subclasses were a d d e d to 96-well plates at 1 # g / m l overnight at 4°C. The plates were w a s h e d a n d blocked. 50/L1 of h y b r i d o m a s u p e m a t a n t s were then a d d e d at 1 : 5 for 45 min at 37°C a n d washed. Plates were w a s h e d a n d 50 ~1 of G A R a G at 1 / t g / m l were a d d e d for 45 m i n at 37°C. F i n a l l y , 50 / d of A P - S A G G at 1 : 5 0 0 were a d d e d for 45 m i n at 37°C. Plates were w a s h e d a n d d e v e l o p e d with 50 /~1 of P N P P at 2 m g / m l at p H 9.8. T h e r e a c t i o n was s t o p p e d 30 m i n later b y a d d i n g 50/~1 of 2 N N a O H . Plates were r e a d at A414 in a T i t e r t e k M u l t i s k a n 96-well plate reader.

Antigen capture assay 96-well p l a t e s were c o a t e d with 50/~1 of one of the M A b anti-e: BF815, A12, B12, G S 6 at 10 / ~ g / m l o v e r n i g h t at 4°C, washed, b l o c k e d a n d followed b y the a d d i t i o n of s t a n d a r d a n t i - D N P I g E or cell s u p e r n a t a n t s at a p p r o p r i a t e dilutions for 4 h at r.t. R a d i o l a b e l l e d or b i o t i n y l a t e d antigens, D N P - B S A or K L H , were then a d d e d at 4 ° C overnight, washed, a n d the c p m a n d O D values were assessed b y a g a m m a - c o u n t e r or b y an E L I S A reader. T h e results were expressed as c p m or O D values, or c a l c u l a t e d a c c o r d i n g to a s t a n d a r d curve

Antigen-specific IgE assay 50/~1 of D N P - B S A at 10 ~ g / m l were a d d e d to 96-well plates overnight at 4°C. T h e plates were w a s h e d a n d blocked. T h e wells were then a d d e d

TABLE I DETERMINATION OF ISOTYPES SPECIFICITIES OF HYBRIDOMA SUPERNATANTS The plates were coated with 50/tl of different classes of monoclonal or myeloma immunoglobulins at 1/zg/ml. Supernatants were diluted 1/5, tested for activities against each immunoglobulin product, and developed with AP-GARaG. The OD reading at 414 nm was recorded. Sup (1/5) of hybridoma cell lines OD

AntiDNP IgE (r, e)

AntiRAG IgE (x, e)

Anti-NP IgE (~, e)

Heat-treated anti-DNP (x, e)

2ME-treated anti-DNP (K, e)

AntiDNP IgG1 (K,)'1)

AntiDNP IgG2b (r, YEb)

AntiDNP IgA (~, a)

AntiDNP IgM (h, la)

RPC20 (X2)

FF3 AF5 DC3 JB3 BF8 SC10

> 1.5 > 1.5 > 1.5 >1.5 >1.5 >1.5

> 1.5 > 1.5 > 1.5 >1.5 >1.5 >1.5

> 1.5 > 1.5 > 1.5 >1.5 >1.5 >1.5

< 0.1 < 0.1 < 0.1 <0.1 <0.1 <0.1

< 0.1 < 0.1 < 0.1 <0.1 <0.1 <0.1

< 0.1 < 0.1 < 0.1 <0.1 <0.1 <0.1

< 0.1 < 0.1 < 0.1 <0.1 <0.1 <0.1

< 0.1 < 0.1 < 0.1 <0.1 <0.1 <0.1

< 0.1 < 0.1 < 0.1 <0.1 <0.1 <0.1

< 0.1 < 0.1 < 0.1 <0.1 <0.1 <0.1

132

based on the capture of DNP-BSA by anti-DNP IgE.

IgE producing cell cultures Single cell suspensions were prepared from female CAF 1 mice immunized with K L H in AI(OH)3 with or without CsA. 5 x 106 cells were cultured in 0.5 ml of D-MEM with 10% FCS in each inner Marbrook vessel, and the outer Marbrook vessel was filled with 10 ml of D - M E M with 10% FCS. K L H antigens at 0.1-10 /~g/ml were added at the initiation of cell cultures, and on day 7 cells were harvested from the cultures and assayed for K L H specific IgE producing cells by the ELISA-plaque assay described below. In some cultures, cells were washed twice in D-MEM with 10% FCS after 2 days of antigen stimulation, and cell pellets were resuspended and recultured in new Marbrook vessels. Washed cells were further incubated for five more days, and cells were harvested and assayed for K L H specific IgE producer cells. For nonantigen-specific IgE production, spleen cells are stimulated with LPS (20 /tg/ml) and IL-4 at different concentrations, or stimulated with IL-4 alone.

addition of B C I P / N B T substrate for 30 min at r.t. The reaction was stopped by rinsing the plates, and the paper discs were left dry. Round blue plaques were easily visualized without magnification.

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ELISA-plaque assay (EP) Nitrocellulose (NC) membrane (0.45 /~M pore size, 47 mm diameter) was obtained from MFS (Dublin, CA). The membrane was pasted with photomount from 3M (St. Paul, MN) in 60 X 15 mm tissue culture plates (Falcon 3002) directly, or cut in 15 mm in diameter and pasted to the 24-well Linbro plates. N C discs were prewetted in PBS for 1 min and incubated with 0.3 ml of antigen at 100 # g / m l for 30 min at r.t. The wells were rinsed and blocked with Blotto from Carnation Co. (Los Angeles, CA) for 30 min at r.t., rinsed with PBS twice, and incubated with cells in 0.5 ml D - M E M / 1 0 % FCS for 4 - 6 h at 37 ° C in a 10% CO 2 incubator. Cyclohexamide was added at 1 0 / ~ g / m l to inhibit de novo protein synthesis in antigen coated control wells (Czerkinsky et al., 1988). Cells were then removed from the discs by rinsing 5 x in PBS, and the plates were incubated with 0.1 ml of biotinylated MAb anti-e overnight at 4°C. The wells were washed, and incubated with streptavidin-AP for 1 h at r.t. followed by

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Fig. 1. Isotype specificity of M a b antiie: lack of inhibition with myeloma immunoglobulins. The plates were coated with DNPBSA at 10 lag/m1, and a D N P IgE was added at 100 n g / m l . Different concentrations of M a b anti-e at 0.25 lag, 1 lag, and 2 lag/ml were preincubated with equal volumes of M O P C 21 (A), M O P C 195 (B), or M O P C 104E ( C ) at 0.1 mg and 1 m g / m l . The plates were developed with A P - G A R a G .

133

Results

Generation of rat anti-mouse IgE monoclonal antibodies Hybridoma supernatants were tested against a panel of monoclonal or myeloma immunoglobulins of different isotypes. As shown in Table I, six representative supernatants from FF3, AF5, DC3, JB3, BF8, and SC10 displayed high anti-e activities against aDNP-IgE(K, e), aRAG-IgE(K, e), and aNP-IgE()t, e); but not aDNP-IgGI(K, Ta), ~xDNP-IgG2b(x, 72b), ctDNP-IgA M O P C 315(X, a), or RPC20(X2). These anti-es did not react with heat-denatured and 2ME-reduced IgE. B12 and BF815 are subclones derived from AF5 and BF8 respectively, and are of the rat IgG2a subclass. Fig. 1 demonstrated the specificity of

MAb anti-e. 50/~1 of MAb anti-e (B-12 and BF815) from 0.25 #g to 2 /~g/ml were incubated with equal volumes of MOPC 21(~, 71), MOPC 195(r, Tzb), and MOPC 104E(?,, /~) at 0.1 mg and 1 mg/ml at 37°C for 30 min. The incubation mixture was then added to a plate pre-coated with 10 #g DNP-BSA and 100 ng of IgE. MAb anti-e bound to the IgE plate without hindrance despite a preincubation with 40-4000-fold molar excess of myeloma proteins of different classes (Figs. 1A1C), or with IgE depleted normal mouse sera (data not shown). IgE standard ELISAs (I) Antigen-specific IgE assay. Plates were coated with DNP-BSA, followed by a titration of txDNP IgE, and developed with biotinylated B12

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Fig. 2. IgE standard curves. A: antigen-specific ELISA: the plates were coated with DNP-BSA at 10/~g/rrd, followed by addition of IgE and biotinylated B12. B: single MAb, total IgE sandwich assay: the plates were coated with B12 at 10 #g/rnl, followed by addition of IgE and b-GAME C: double MAb, total IgE sandwich assay: the plates were coated with BF815, followed by addition of IgE and b-EM95. D: antigen capture assay: the plates were coated with B12 at 10 or 50/~g/ml, followed by addition of anti-DNP IgE at 100 ng/rrd or Ig(31 at 10 #g/ml, and 50,000 cprn of radiolabeled DNP-KLH.

134 (b-B12) at 2 /~g/ml and ABC. As shown in Fig. 2A, this assay detected IgE at 3-6 n g / m l (0.15 to 0.3 ng/assay). (II) Total IgE sandwich assays. Plates were coated with MAb anti-e followed by a titration of etDNP IgE, and developed with biotinylated goat anti-mouse IgE (b-GAME), or by a previously determined non-competing biotinylated MAb anti-e. Fig. 2B showed a standard curve with B12 and b-GAME, which detected IgE at 0.4 n g / m l (20 pg/assay) with 8-18 h incubation steps. Fig. 2C showed a standard curve with two MAb anti-e: BF815 and b-EM95 with 2 h incubation steps. (111) Antigen capture assay. Fig. 2 D showed that monoclonal anti-DNP IgE, but not DNPspecific IgG1 was bound to MAb anti-e (B12) coated well, and the bound DNP-specific IgE in turn captured radioactive antigen, DNP-BSA.

IgE ELISA-plaque assay (EPe) CsA was shown to optimally stimulate Agspecific IgE responses (Chen, 1988; Chen et al., 1989). CAF 1 mice were pretreated with CsA and primed with 1 /~g K L H in 2 mg alum. K L H primed cells were cultured in Marbrook vessels, and boosted with antigens. Cell were harvested on day 7 and overlayed onto NC discs coated with K L H antigen in 24 wells. As shown in Fig. 3, antigen-specific IgE responses can be enumerated at the single cell level by the EP assay. Antigenspecific IgE, secreted by the plasma cell, formed a concentric diffusion gradient, and was efficiently captured by the antigen adsorbed to the NC discs. Biotinylated BF815 reacted with IgE bound to N C solid phase, and the dye deposit resulted from a reaction of B C I P / N B T substrate and conjugate of SA-AP bound to biotinylated MAb anti-e. Nonantigen-specific IgE plasma cells, stimulated by IL-4 were detected by N C discs coated with BF815 (Fig. 3(1)); and antigen stimulation lead to detection of more antigen-specific IgE plasma cells on N C discs coated with K L H antigen (Fig. 3(2) vs. 3(4)). The IgE isotype specificity of EPe was ascertained. As shown in Table II, G A M E blocked 82% of KLH-specific IgE responses and MAb anti-e: BF815 inhibited 70% of IgE responses but neither anti-isotype affected KLH-specific IgG1 responses; likewise GAMG1 inhibited 58% of IgG1

Fig. 3. Detection of EPe. CAF1 mice were primed with KLH and CsA. 5 × 106 spleen cells were set in each Marbrook vessel. On day 7, lymphocytes were pooled from three vessels and plated on a single NC disc. 1: total IgE: BF 815 coating, cells stimulated with IL-4 at 250 U/ml, b-GAME. 2: KLH-specific EPe: KLH coating, cells in medium alone, b-BF815. 3: blank control: BF815 coating, no cells, b-GAME. 4: KLH-specific EPe: KLH coating, cells stimulated with KLH at 1 /~g/ml, b-BF 815. responses, but not IgE responses, and GAM/~ affected neither IgE nor IgG1 responses. Antigen-specific IgE was synthesized de novo by IgEsecreting plasma cells; and preformed cytophilic IgE on lymphocytes did not interfere with the plasma cell assay, since cyclohexamide added at 10 ~ g / m l during the assay completely abrogated the formation of EPe (data not shown). The antigen dose-response curve was shown in Fig 4. Optimal KLH-specific IgE responses were observed in cultures stimulated with 1/~g/ml K L H for 48 h; and the magnitude of total IgE responses assessed on MAb anti-e coated N C discs was

135 T A B L E II E F F E C T OF ANTI-ISOTYPES OF Ag-SPECIFIC ANTIB O D Y P R O D U C T I N G CELLS ASSAYED BY EP 5 x 106 i m m u n e spleen cells were stimulated with K L H at 1 / t g / m l . Cells were harvested on day 7 after antigen stimulation. For IgE assay, one half of the cells from each culture was added to antigen-coated N C discs and incubated in the present of goat anti-mouse isotypes at 2-20 /~g/ml, or BF815 at 20 /xg/ml for 4 h at 37°C in a 10% CO 2 incubator. For IgG1 assay, 1/100 of the cells from each culture were added to antigen-coated N C discs, and incubated with different anti-isotypes as above. Biotinylated BF815 or G A M G 1 at 1 /~g/ml was added to plates overnight at 4°C for developing antigenspecific EPe or EPy1. The rest of the procedures were performed as described in the materials and methods section. Incubation of anti-isotype specific antibodies

GAME BF815 GAMG1 GAM#

N u m b e r of EP EPe

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Dosage of KLH (p,g/ml) Fig. 4. Dose-response curve of EPe in antigen-stimulated in vitro cultures. C A F 1 mice received three bi-weekly priming of 1 # g K L H in 2 m g AI(OH) 3. 5 x 106 spleen ceils were set in each Marbrook vessel stimulated with antigen at different dosages from 0.1 /.tg to 10 ~ g / m l for 2 days. Cells were washed and recultured. Antigen-specific and total EPe were enumerated on day 7 after the initiation of cultures.

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comparable to that of antigen-specific IgE responses observed in KLH coated discs. Fig. 5 showed the kinetics of appearance of IgE-secreting cells. Antigen-specific EPe rose significantly on day 5, peaked on day 7, and steeply declined on day 10 after antigen stimulation (Fig. 5A); these 300 270 240 210 180 150. 120, 9060300

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Fig. 5. Kinetics of EPe in antigen-stimulated in vitro cultures. 5 X 106 K L H primed C A F 1 spleen cells were set in Marbrook vessels with continuous antigen stimulation at 1 # g / m l . Background (b.g.) antigen-specific EPe as well as total EPe were enumerated on day 5, 7, and 10.

antigen-specific IgE producers apparently were also detected by a total EPe assay on NC discs coated with BF 815 (Fig. 5B). The augmented total IgE plaque responses observed on day 5 may be due to bystander activation of IgE B cells of undertermined specificies during the process of antigen stimulation. In our hand, the EPe assay is far superior to regular IgE ELISA in quantitating Ag-specific IgE in Ag-stimulated cell cultures. As shown in Table III, antigen-specific EPe increased 3.3-5.8-fold in cultures stimulated with antigens for 2 days, whereas the level of total IgE in antigen-stimulated cultures was not significantly different from control cultures (Table III: groups 2, 3, vs. 1). Likewise, antigen-specific EPe increased 5-7.2fold in cultures stimulated with antigen from day

136 TABLE III COMPARISON OF IgE RESPONSES DETECTED BY DIFFERENT METHODS OF IMMUNOASSAYS CAF1 mice were primed with three bi-weekly i.p. injections of 1 /xg KLH in 2 mg AI(OH)3. 5 × 106 spleen cells were initiated in Marhrook vessels, and stimulated with KLH at 100 ng or 1/~g/ml for 2 days or 7 days. Ceils were harvested on day 7 after initiation of cultures. Antigen-specific IgE plasma cells were enumerated by EPe assay; antigen-specific IgE in the culture supernatants was determined by antigen capture ELISA with wells coated with BF815. Total IgE sandwich assay was performed with BF815 and b-GAME. These two conventional ELISAs were described in details in the materials and methods section Group

Antigen stimulation (duration)

1

None (background)

2 3 4 5

Ag-specific Epe

Ag-specific IgE (O.D.)

Total IgE (ng/ml)

32

0.117

142

KLH, 100 ng/ml (d0-d2) KLH, 1/~g/ml (d0-d2)

104 185

0.086 0.220

104 195

KLH, 100 ng/ml (d0-d7) KLH, 1 g g / m l (d0-d7)

229 159

0.189 0.077

355 215

0 to day 7, whereas total IgE increase only 2.12.5-fold to that of control (Table III: groups 4, 5 vs. 1). Although MAb anti-e based antigen capture assay detected monoclonal anti-DNP IgE in the standard assay, this assay failed to consistently detect antigen-specific IgE despite that there was 3.3- or 5-fold increase of IgE-secreting cells by EPe assay (Table III: groups 2 and 5). We suspected that antigen-IgE complexes may either fail to bind biotinylated antigens, or may be cleared more rapidly; however, washing antigens from cultures day 2 after antigen stimulation did not improve detection of IgE by the antigen capture assay (Table III: groups 2,3, vs. 4, 5); moreover, the performance of this assay was not improved by depleting cell supernatants of IgG via adsorption to protein G-Sepharose 4B, or by increasing the dosage of coating MAb anti-e (data not shown).

Discussion

The studies of mechanisms of antigen-specific IgE production in experimental animals were greatly facilitated by a semiquantitative technique of passive cutaneous anaphylaxis (PCA) (Ovary, 1964). However, it has been difficult to directly detect antigen-specific IgE in vivo or in vitro by quantitative ELISA methods. The precursor frequency of IgE producing B cells is low (Teale et al., 1981), and the secreted antigen-specific IgE

molecules inherit a short biological half-life (Haba et al., 1985), and are greatly outnumbered by antigen-specific IgA and IgG molecules present concomitantly in biological fluids. The EP technique was originally devised as a substitution for the haemolytic plaque-forming cell assay (Czerkinsky et al., 1973; Sedgwick and Holt, 1973). We developed rat MAb anti-e, and applied these reagents for detecting antigen-specific IgE secreting plasma cells. The success of detecting EPe on NC discs can be attributed to the following two reasons. First, greater efficiency of antigen or MAb anti-e coating was achieved through adsorption to NC discs. MoeUer and Borrebaeck (1985) and our lab (data not shown) indicated that antigen adsorption capacity on NC discs can increase 100fold to that on polystyrene wells. High density of antigen coating on a large available surface area may be particularly important for detecting IgE responses, since IgE molecules are present in exceedingly lower molar concentration as compared to the major classes of IgM, IgA and IgG antibodies. The problem of limiting antigenic epitopes available for IgE on conventional plastic surface was circumvented by direct antigen coating to NC discs. Second, and perhaps more importantly, any immunoglobulin secreting cell of a given class has equal chance of occupying an antigen-coated surface area on NC disc. IgE molecules secreted from the plasma cell formed a concentric diffusion

137 gradient around the center of the cell, and were efficiently captured by N C disc-bound antigen or M A b anti-e in the vicinities. Even though I g G and IgA antibody secreting cells were usually present several hundred- or even thousand-fold higher in numbers (data not shown), the secreted antigenspecific I g G and IgA were readily bound by the coated antigen and were unable to compete and displace IgE at a distance. The sharpest image of EPe was formed after cells were incubated on discs for 4 - 6 h, and the blurred images of EPe after overnight incubation may either be due to the motion of the cells or to displacement of IgE by major classes of immunoglobulins diffused into the reaction centers the IgE plasma cells following prolonged incubation. Antigen-specific EPe directed against BSA, OVA and Sendai virus can also be routinely carried out in the laboratory (data not shown). Holt et al. (1984) had been first able to measure murine IgE secreting spleen cells by EP technique, recently, Heusser et al. (1989) was also able to demonstrate a memory in vitro murine IgE response to benzyl penicillin by this technique with antigen directly coated on petri plate (Czerkinsky et al., 1983; Sedgwick and Holt, 1983). Antigenspecific EP of other isotypes can be easily performed on antigen-coated N C discs, and revealed by biotinylated isotype-specific antibodies. We have tested several different MAb anti-e: A12, B12, GS6, BF815, BF8.123, EM95 or a mixture of these MAbs in the antigen capture assay. Although several MAb anti-e antibodies were capable of binding standard IgE reagents, which in turn captured radioactive or biotinylated D N P BSA, these capture assays did not consistently detect antigen-specific IgE present in culture supernatants or sera. Perhaps, unlike Hirano et al. (1989), our MAb anti-e may react with a determinant on IgE, and interfere with their subsequent antigen binding capacities. Alternatively, the detection of antigen-specific IgE by conventional ELISA may be interfered by basal IgE of undetermined specificities normally present in culture supernatants and sera; these nonantigen-specific IgE may pre-empt IgE binding sites on M A b anti-e, and hinder the detection of antigen-specific IgE. We propose that the EPe technique is the

method of choice to precisely quantitate antigenspecific IgE-secreting cells. This assay circumvents the problems of epitope pre-emption by major classes of antibodies inherent in antigen-specific conventional ELISAs, as well as gives consistent measurement of antigen-specific IgE regardless of the basal IgE level in biological fluids.

Acknowledgements The authors gratefully acknowledge the critical comments of the manuscript by Dr. Zoltan Ovary at New York University, and Dr. Michael Caufield at the Cleveland Clinic. The authors also gratefully acknowledge the consultation of Dr. Michael Caufield on procedures of ELISA-plaque, the technical assistance of Ms. Y.-Y. Qian, Mr. G.S. Stanescu and Mr. A.E. Magalski, and the preparation of this manuscript by Ms. Tonya Marshall.

References Baniyash, M. and Eshhar, Z. (1984) Inhibition of IgE binding to mast cells and basophils by monoclonal antibodies to murine IgE. Eur. J. Immunol. 14, 799. Bose, R., Bundesen, P.G., Horford-Stevens, V., Stefura, W.P., Rector, E. and Sehon, A.H. (1984) Immunochemicalproperties of some monoclonal IgE antibodies to 4-hydroxy-3nitrophenylacetyl (NP). Immunology 53, 81. Chen, S.-S., Liu, F.-T. and Katz, D.H. (1984) Cellular and molecular mechanisms of murine IgE class-restricted tolerance induced by neonatal administration of soluble or cell bound IgE. J. Exp. Med. 160, 953. Chen, S.-S. (1988) Cyclosporin A acts as a potent costimulator in up-regulation of IgE antibody synthesis and expression of Fc receptor for IgE-A unifying hypothesis of the cellular mechanisms of CsA. Transpl. Proc. 22 (suppl. 2), 92. Chen, S.-S., Stanescu, G., Magalski, A.E. and Qian, Y.-Y. (1989) Cyclosporin A is an adjuvant in murine IgE antibody responses. J. Immunol. 142, 4225. Czerkinsky, C.C., Nilsson, L.-A., Nygren, H., Ouchterlony, O. and Tarkowski, A. (1983) A solid-phase enzyme-linked immunospot (EL1SPOT) assay for enumeration of specific antibody-secreting cells. J. Immunol. Methods 65, 109. Czerkinsy, C., Andersson, G., Ekre, H.-P., Nilsson, L.-A., Klareskog, L. and Ouchterlony, O. (1988) Reverse ELISPOT assay for clonal analysis of cytokine production. I. Enumeration of gamma-interferon-secreting cells. J. Immunol. Methods 110, 29. Haba, S., Ovary, Z. and Nisonoff, A. (1985) Clearance of IgE

138 from serum of normal and hybridoma-bearing mice. J. Immunol. 134, 3291. Heusser, C.H., Wagner, K., Brinkmann, V., Severinson, E. and Blaser R. (1989) Establishment of a memory in vitro murine IgE response to benzyl penicillin and its resistance to suppression by anti-IL-4 antibody. Int. Arch. Allergy Appl. Immunol. 90, $1, 45. Hirano, T., Yamakawa, N., Miyajima, H., Maeda, K., Takai, S., Udea, A., Taniguchi, O., Hashimoto, H., Hirose, S., Okumura, K. and Ovary, Z. (1989) An improved method for the detection of IgE antibody of defined specificity by ELISA using rat monoclonal anti-IgE antibody. J. Immunol. Methods 119, 145. Holt, P.G., Sedgwick, J.D., O'Leary, C., Krska, K. and Leivers, S. (1984) Long-lived IgE-and IgG-secreting cells in rodents manifesting persistent antibody responses. Cell. Immunol. 89, 281. Liu, F.-T., Bohn, T.W., Ferry, E.L., Yamamoto, H., Molinaro, C.A., Sherman, L.A., Klinman, N.R. and Katz, D.H. (1980)

Monoclonal DNP-specific murine IgE antibody: preparation, isolation and characterization. J. Immunol. 124, 2728. Moeller, S.A. and Borrebaeck, C.A.K. (1985) A filter immunoplaque assay for the detection of antibody-secreting cells in vitro. J. lmmunol. Methods 79, 195. Ovary, Z. (1964) Passive cutaneous anaphylaxis. In: J.F. Ackroyd (Ed.), Immunological Methods, C.I.O.M.S. Symposium. Blackwell, Oxford, p. 259. Sedgwick, J.D. and Holt, P.G. (1983) A solid-phase immunoenzymatic technique for the enumeration of specific antibody-secreting ceils. J. Immunol. Methods 57, 301. Skidmore, B.J., Stamnes, S.A., Townsend, K., Glasebrook, A.L., Sheehan, K.C.F., Schreiber, R.D. and Chiller, J.M. (1989) Enumeration of cytokine-secreting cells at the single-cell level. Eur. J. Immunol. 19, 1591. Teale, J.M., Liu, F.-T. and Katz, D.H. (1981) A clonal analysis of the IgE response and its implications with regard to isotype commitment. J. Exp. Med. 153, 783.