Generation of high-affinity monoclonal antibodies of IgG class against native β-d -glucans from basidiomycete mushrooms

Process Biochemistry 51 (2016) 333–342

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Generation of high-affinity monoclonal antibodies of IgG class against native ␤-d-glucans from basidiomycete mushrooms Magda C. Semedo a,b , Amin Karmali a,b,∗ , Sónia Martins a,b , Luís Fonseca c a Chemical Engineering and Biotechnology Research Center and Departmental Area of Chemical Engineering of Instituto Superior de Engenharia de Lisboa, R. Conselheiro Emídio Navarro, 1, 1959-007 Lisbon, Portugal b Centre for the Research and Technology of Agro-Environment and Biological Sciences, Universidade de Trás-os-Montes e Alto Douro, Quinta de Prados, Apartado 1013, 5001-801 Vila Real, Portugal c Department of Bioengineering, Centre for Biological and Chemical Engineering of Instituto Superior Técnico, Av. Rovisco Pais, 1, 1049-001 Lisbon, Portugal

a r t i c l e

i n f o

Article history: Received 8 October 2015 Received in revised form 26 November 2015 Accepted 27 November 2015 Available online 2 December 2015 Keywords: Hybridoma technology Mabs of IgG class Extracellular ␤-d-glucans from Pleurotus ostreatus Basidiomycete mushrooms Specificity and cross-reactivity Epitope analysis of Mabs

a b s t r a c t ␤-d-glucans from basidiomycete strains are powerful immunomodulatory agents in several clinical conditions. Therefore, their assay, purification and characterization are of great interest to understand their structure-function relationship. Hybridoma cell fusion was used to raise monoclonal antibodies (Mabs) against extracellular ␤-d-glucans (EBGs) from Pleurotus ostreatus. Two of the hybridoma clones (1E6 1E8 B5 and 3E8 3B4) secreting Mabs against EBGs were selected. This hybridoma cell line secreted Mabs of the IgG class which were then purified by hydroxyapatite chromatography to apparent homogeneity on native and SDS-PAGE. Mabs secreted by 1E6 1E8 B5 clone were found to recognize a common epitope on several ␤-d-glucans from different basidiomycete strains. This Mab exhibited high affinity constant (KA ) for ␤-d-glucans from several mushroom strains in the range of 3.20 × 109 ± 3.32 × 103 –1.51 × 1013 ± 3.58 × 107 L/mol. Moreover, they reacted to some heat-treated ␤d-glucans in a different mode when compared with the native forms; these data suggest that this Mab binds to a conformational epitope on the ␤-d-glucan molecule. The epitope-binding studies of Mabs obtained from 1E6 1E8 B5 and 3E8 3B4 revealed that the Mabs bind to the same epitope on some ␤-dglucans and to different epitopes in other antigen molecules. Therefore, these Mabs can be used to assay for ␤-d-glucan from basidiomycete mushrooms. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Medicinal mushrooms have been known for several millennia, and they play a major role in several clinical conditions such as cancer, human immunodeficiency virus (HIV) syndrome, diabetes, neurological diseases, and immunological disorders [1,2]. Most of such medicinal mushrooms belong to the basidiomycetes class

Abbreviations: BRM, biological response modifiers; EBG, extracellular ␤-d-glucan; ELISA, enzyme-linked immunosorbent assay; HAT, hypoxanthine–aminopterin–thymidine; IBG, intracellular ␤-d-glucans; IEF, isoelectric focusing; Mabs, monoclonal antibodies; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PDA, potato dextrose agar; PEG, polyethylene glycol; PR-Mabs, polyol-responsive MAbs; SDS, sodium dodecyl sulfate; MES, 2-(N-morpholino) ethanesulfonic acid. ∗ Corresponding author at: Chemical Engineering and Biotechnology Research Center and Departmental Area of Chemical Engineering of Instituto Superior de Engenharia de Lisboa, R. Conselheiro Emídio Navarro, 1, 1959-007 Lisbon, Portugal. Fax: +351 21831 7267. E-mail addresses: [email protected], [email protected] (A. Karmali). http://dx.doi.org/10.1016/j.procbio.2015.11.029 1359-5113/© 2015 Elsevier Ltd. All rights reserved.

and contain useful nutrients namely enzymes, lipids, ␤-d-glucans, lectins, and secondary metabolites [3,4]. As isolation of purified forms of ␤-glucans of basidiomycete mushroom is difficult, there is limited information on their structure and functions in the relevant literature [4,5]. In fact, the isolated product is a heterogeneous mixture of ␤-glucans of different sizes, either bound to proteins or in free forms [4,5]. The specific assay of ␤-d-glucans obtained from mushroom sources is of great interest to monitor their levels in fungal strains as well as to find novel sources of ␤-d-glucans with unique biological activities. Some studies have conducted assay of ␤-d-glucans via colorimetric and fluorimetric methods using phenol–sulfuric acid and some dyes as well as aniline blue, respectively [6–9]. However, there is only one report on the immunochemical assay of mushroom ␤-dglucans using polyclonal antibodies against lentinan [10]. To the authors’ knowledge, there are no reports about the production of monoclonal antibodies (Mabs) against basidiomycete mushroom ␤-glucans as well as their use in the assay of such polysaccharides.

M. C. Semedo et al. / Process Biochemistry 51 (2016) 333–342

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3

0.0

15

2

14

1

13

0

12

-1

11

-2

ln(A550nm )

-0.5

-1.0

-1.5

-2.0

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-3.0

ln(Concentration of Viable Cells)

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ln(Antibody Activity)

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ln(Con centration of viable ce lls ) ln(A550nm ) ln(Antibo dy Activity)

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-4 0

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Time (Days) Fig. 1. Hybridoma growth curve and Mab production. Tissue culture microplates were seeded with hybridoma cell line 1E6 1E8 B5 in RPMI medium containing gentamicin at 1 × 105 cells/ml. The analysis of cellular viability was carried out by trypan blue and MTT methods. The antibody activity of the culture supernatants was determined by indirect ELISA.

Mabs can be obtained by hybridoma technology using a suitable antigen, and can be used for the detection and quantification of ␤-glucan from novel mushroom strains using immunochemical assays. Nevertheless, these ␤-glucans from mushroom strains can be purified by immunoaffinity chromatography using polyolresponsive Mabs (PR-Mabs) as the immunoaffinity ligand [11]. These PR-Mabs allow the release of the antigen under mild conditions by disrupting the Ag–Ab complex in the presence of a polyol and/or salt [12]. Moreover, these antibodies can be used as powerful tools to detect ␤-glucans with triple helix as their tertiary structure, which exhibit anti-tumor activity in human carcinoma cell lines. In fact, anti-tumor activity of ␤-glucans has been associated with their triple helical structure by many authors [4,5,7,9]. The present work involves the use of extracellular ␤-d-glucans (EBGs) from Pleurotus ostreatus as the antigen for the production of Mabs by hybridoma technology. These Mabs will be purified by hydroxyapatite chromatography, and the purified form will be characterized in terms of class, subclass, relative molecular mass (Mr), specificity, selectivity, affinity, conformational probes, and epitope-binding studies using a battery of different antigens isolated from basidiomycete mushroom strains. 2. Materials and methods 2.1. Chemicals Potato dextrose agar (PDA) medium was supplied by HiMedia Laboratories (Mumbai, India). Milk whey was supplied by a local manufacturer. ␤-1,3-d-glucan from Euglena gracilis; ␤-d-glucan from barley; laminarin from Laminaria digitata; chitosan from shrimp; starch, acacia gum, cellulose, dextran sulfate, chondroitin sulfate, heparin and Congo red dye; Complete Freund’s adjuvant (CFA); polyethylene glycol (PEG) 3000; rabbit anti-mouse IgG-alkaline phosphatase conjugate; p-nitrophenyl phosphate; anti-mouse polyvalent immunoglobulin antibody; hypoxathine–aminopterin–thymidine (HAT) and hypoxathine–thymidine (HT) media; bovine serum albumin (BSA); isotyping kit ISO-2 for enzyme-linked immunosorbent assay (ELISA); and ampholine (pH range: 3.5–10) were purchased

from Sigma–Aldrich (St Louis, MO, USA). A sample of myeloma cell line (Sp2/0Ag14) was obtained from the American Type Culture Collection (ATCC, USA). Membranes (P10) for ultrafiltration units were supplied by Merck Millipore (Darmstadt, Germany). BioWhittakerTM RPMI 1640 (with 25 mM HEPES, 4(2-hydroxyethyl)-1-piperazineethanesulfonic acid; l-glutamine; 100 units/mL penicillin; and 50 ␮g/mL streptomycin) was obtained from Lonza Ltd. (Visp, Switzerland), and fetal bovine serum (FBS) was obtained from GIBCO® —Life Technologies Corp. (USA). The mouse IgG ELISA kit was purchased from Life Diagnostics cat number 5015-1 (West Chester, USA). Polystyrene 96-well MicroWellTM PolySorp® flat bottom plates were purchased from Thermo ScientificTM NuncTM (Nunc 475094; MA, USA). Hydroxyapatite (CHT Type II, particle size 40 ␮m) and nitrocellulose transfer membranes were supplied by BioRad Laboratories, Inc. (Hercules, USA). All disposable plasticware were obtained from Orange Scientific (Braine-l’Alleud, Belgium), and all other reagents were of analytical grade.

2.1.1. Balb/c mice Female mice of inbred strain Balb/c were obtained from Instituto Gulbenkian de Ciência (Oeiras, Portugal).

2.1.2. Basidiomycete mushroom strains P. ostreatus and Ganoderma carnosum were obtained as described previously [7]. Young fruit body powders of G. lucidum, Hericium erinaceus, Coriolus versicolor, Lentinula edodes, P. ostreatus, Inonotus obliquus, Auricularia auricula, Grifola frondosa, Polyporus umbellatus, Cordyceps sinensis, Poria cocos, and Agaricus blazei young fruit body powders were obtained from the Mycology Research Laboratory, Ltd. (UK).

2.2. Methods 2.2.1. Production and extraction of EBG from P. ostreatus The strains of P. ostreatus and G. carnosum were maintained at 4 ◦ C on PDA.

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0.9 0.8

IBG from P. ostreatus FKOH 4 ºC

IBG from P. ostreatus FKOH 37 ºC

EBG from P. ostreatus FKOH 4 ºC

EBG from P. ostreatus FKOH 37 ºC

A

0.7

A415

0.6 0.5 0.4 0.3 0.2 0.1 0.0 1

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48 72 Immobilization Time (h)

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A. auricula FKOH 4 ºC

A. auricula FKOH 37 ºC

H. erinaceus FKOH 4 ºC

H. erinaceus FKOH 37 ºC

B

A415

1.5

1.0

0.5

0.0 1

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48 72 Immobilization Time (h)

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2.0 1.8

C. sinensis FKOH 4 ºC

C. sinensis FKOH 37 ºC

G. frondosa FKOH 4 ºC

G. frondosa FKOH 37 ºC

C

1.6 1.4

A415

1.2 1.0 0.8 0.6 0.4 0.2 0.0 1

24

48 72 Immobilization Time (h)

96

120

Fig. 2. Effect of time and temperature (4 and 37 ◦ C) on immobilization of antigens. FKOH fractions of ␤-glucans (20 ␮g) were adsorbed as antigens on Polysorp® microtiter plates. A—EBG and IBG from Pleurotus ostreatus ; B—␤-d-glucans from Auricularia auricula and Hericium erinaceum; and C—␤ -d-glucans from Cordyseps sinensis and Grifola frondosa.

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Table 1 Purification of Mabs from 1E6 1E8 B5 against EBGs from P. ostreatus by hydroxyapatite type II chromatography. Purification steps

Total protein (mg)

Total Mab activity (A units)

Specific content of IgG (mg/mg protein)

Recovery (%)

Purification factor

1. Culture supernatant 2. Column eluate

0.32 0.06

19.35 10.35

8.77 × 10−2 6.15 × 10−1

100 53.49

1 7.28

P. ostreatus was grown for 14 days by submerged fermentation as described previously [7,13]. Mycelial biomass was separated from the culture broth as mentioned previously [7,13].

2.2.5. Production of Mabs against EBG from P. ostreatus The EBG precipitated from the P. ostreatus sample was used as the antigen, and Mabs were produced by hybridoma technology. Female (4-week-old) BALB/c mice were immunized by intraperitoneal injections on day 0 with 50 ␮g of EBG. Subsequently, three immunizations were carried out at 3-week intervals with the same amount of antigen. Three days after the last immunization, mice were bled, and the antibody titer was determined by ELISA. Afterward, the spleen cells (2.5 × 108 ) were fused with Sp2/0 Ag 14 (3.37 × 107 ) in the presence of PEG 3000. The selection of hybrids was carried out in HAT medium, and the positive hybrids were cloned by the limiting dilution method and Mabs were produced from two clones (1E6 1E8 B5 and 3E8 3B4) as described previously [14].

2.2.2. Extraction of intracellular ˇ-d-glucans G. carnosum was grown in media containing 1 g/L KH2 PO4 , 4 g either rice bran or rice husk, and 2 g/L MgSO4 at pH 5.8, 200 rpm, and 25 ◦ C for 10 days by submerged fermentation. Mycelial biomass from the fermentation broth of P. ostreatus and G. carnosum was separated by suction in a Buchner funnel as reported previously [13]. Several fractions of ␤-d-glucans were extracted using the procedure reported previously [7]. Briefly, the aqueous extraction from the mycelial biomass pellet obtained from P. ostreatus and G. carnosum and from young fruit body powders of G. lucidum, H. erinaceus, C. versicolor, L. edodes, P. ostreatus, I. obliquus, G. frondosa, A. auricula, P. umbellatus, C. sinensis, P. cocos, and A. blazei was first carried out with cold water at room temperature. After centrifugation, the supernatant (FW1) was separated from the residue that was extracted with boiling water, and then obtained by centrifugation (FW2). The remaining residue was subsequently extracted with KOH, HCl, and NaOH, and the extraction fractions were designated as FKOH, FHCl, and FNaOH, respectively. These extraction steps were also carried out for EBG from P. ostreatus, which was obtained by precipitation with 95% (v/v) ethanol.

2.2.6. Indirect ELISA The culture supernatants were screened for antibody activity by ELISA. PolySorp® microplate wells were incubated overnight at 4 ◦ C with EBG from P. ostreatus as the antigen (50 ␮g/well) and the microtiter plates were blocked with 1% (w/v) BSA. Rabbit anti-mouse IgG-alkaline phosphatase conjugate was used as the secondary antibody and p-nitrophenyl phosphate as the substrate [14]. One antibody unit is defined as the amount of Mab required to cause a change in absorbance of 1.0 per 30 min at 415 nm due to the effect of rabbit anti-mouse IgG-alkaline phosphatase conjugate activity on p-nitrophenyl phosphate under the standard conditions of ELISA. All screenings tests were carried out in triplicates. 2.2.7. Hybridoma growth curve and Mab production Hybridoma growth curve and Mab production was carried out by seeding 96-well tissue culture plate with hybridoma cell line 1E6 1E8 B5. The analysis of cell viability was carried out by trypan blue and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) methods. The antibody activity of cell culture supernatants was determined by indirect ELISA as described earlier.

2.2.3. Assay of total ˇ-d-glucans The phenol–sulfuric acid method was used for assay of the total ␤-d-glucans obtained from several mushroom samples using polygalacturonic acid as the standard [6].

2.2.4. Congo red assay for specific determination of ˇ-d-glucans with triple-helical structure Congo red colorimetric assay was carried out to determine the concentration of ␤-d-glucans with triple-helical structure in mushroom samples, as described previously using ␤-d-glucan from barley as the standard [7].

2.2.8. Adsorption of ˇ-glucans in ELISA microtiter plates The effect of temperature (4 and 37 ◦ C) and time (1, 24, 48, 72, 96, and 120 h) of antigen adsorption on flat-bottom PolySorp® 96well plates was investigated by indiect ELISA method. The following samples were used as antigens: FKOH samples from EBGs and IBGs

Table 2 Affinity constant (KA ) for native and denatured ␤-d-glucan with cooling and without cooling. Affinity constant (KA ) (L/mol) 100 ◦ C

Antigens

Native

EBG from Pleurotus ostreatus FHCl from Pleurotus ostreatus FHCl from Hericium erinaceus FKOH from Coriolus versicolor FHCl from Grifola frondosa FKOH from Agaricus blazei FKOH from Poria cocos FHCl from Auricularia auricula FKOH from EBG from Pleurotus ostreatus FNaOH from EBG from Pleurotus ostreatus FHCl from Lentinula edodes FKOH from Polyporus umbellatus

1.51 × 10 ± 3.58 × 10 1.98 × 1010 ± 1.07 × 104 3.30 × 1010 ± 1.52 × 105 1.73 × 1011 ± 6.61 × 105 8.49 × 1010 ± 5.42 × 105 1.11 × 1010 ± 4.24 × 104 2.15 × 1010 ± 6.25 × 104 1.66 × 1011 ± 7.25 × 105 9.57 × 1010 ± 2.62 × 105 1.15 × 1010 ± 3.28 × 105 2.59 × 1011 ± 4.24 × 105 3.20 × 109 ± 3.32 × 103

a

ND stands for not determined.

13

7

100 ◦ C + cooling

3.08 × 10 ± 1.19 × 10 5.71 × 109 ± 1.07 × 104 5.04 × 1012 ± 1.96 × 107 1.88 × 1011 ± 6.11 × 105 1.02 × 1011 ± 3.12 × 105 1.57 × 1011 ± 4.81 × 105 NDa ND ND ND ND ND 11

6

1.15 × 1010 ± 3.28 × 104 2.21 × 1011 ± 2.78 × 105 4.12 × 1010 ± 1.23 × 105 1.65 × 1011 ± 1.04 × 106 1.34 × 1011 ± 3.53 × 105 1.68 × 1011 ± 5.25 × 105 ND ND ND ND ND ND

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0.8 (NH 4)2SO4 0M (NH40M )2SO4

A

(NH 0.1M 0.14)2SO4 M (NH 4)2SO4

0.7

(NH 4)2SO4 0.254M 0.25 M (NH )2SO4 (NH4)2SO4 0.5M 0.5 M (NH 4)2SO4

0.6

(NH 4)2SO4 0.754M 0.75 M (NH )2SO4

1 M (NH41M )2SO4 (NH4)2SO4

A415

0.5 0.4 0.3 0.2 0.1 0.0 0

10

20 30 Propylene glycol (%, v/v)

40

50

0.8

0% PG

B

10% PG

0.7

20% PG 30% PG

0.6

40% PG 50% PG

A415

0.5 0.4 0.3 0.2 0.1 0 0

0.25

0.5 [(NH4)2SO4] (M)

0.75

1

Fig. 3. ELISA-elution assay to identify and characterize PR-Mabs. ELISA-elution assay was carried out using varying concentrations of propylene glycol and a suitable salt. EBG from P. ostreatus (25 ␮g/well) was immobilized on PolySorp® microtiter plate and incubated with Mab 1E6 1E8 B5. A—The antigen–antibody complex was then treated with (NH4 )2 SO4 (0–1.0 M) in the presence of 0,10, 20, 30, 40, and 50% (v/v) propylene glycol for 30 min at room temperature. B—The antigen–antibody complex was then treated with propylene glycol (0–50%) in the presence of 0, 0.25, 0.5, 0.75, and 1.0 M (NH4 )2 SO4 for 30 min at room temperature.

obtained from P. ostreatus, H. erinaceus, A. auricula, G. frondosa, and C. sinensis. The indirect ELISA method was performed as described earlier. 2.2.9. Immunoglobulin class and subclass and immunoglobulin assay The immunoglobulin class and subclass were determined both by ELISA and Ouchterlony double-diffusion analysis and immunoglobulin assay was performed as described previously [14,15]. 2.2.10. Purification of Mabs from hybridoma clone 1E6 1E8 B5 by hydroxyapatite chromatography The culture supernatant containing Mabs (5 mL diluted with an equilibration buffer in 1:2 ratio) was applied to a column (1 × 5 cm) packed with ceramic hydroxyapatite (Type II, particle size 40 ␮m) that was previously equilibrated with 10 mM sodium phosphate buffer at pH 6.7 with 50 mM 2-(N-morpholino) ethanesulfonic acid (MES). The Mabs were eluted from the column with a linear gradi-

ent of sodium phosphate buffer (10–500 mM) at pH 6.7 at a flow rate of 15 mL/h, and the fractions (1.5 mL) were collected and analyzed for antibody activity and protein concentration.

2.2.11. MTT and protein assays The viability of hybridoma cell lines in the culture medium was carried out by a colorimetric assay based on the MTT method, and the protein concentration was quantified by Coomassie blue dyebinding method with BSA as the protein standard, respectively [13,16].

2.2.12. Electrophoretic analysis of purified Mabs from hybridoma clone 1E6 1E8 B5 and isoelectric focusing Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), native PAGE of Mabs from hybridoma clone 1E6 1E8 B5 and analytical isolecetric focusing (IEF) were carried out as mentioned previously [14].

M. C. Semedo et al. / Process Biochemistry 51 (2016) 333–342

1.8

500

8

1.6 400

6

300

4 200

2 100

1.4

[Phosphate Buffer] mM

Antibody Activity (U/fraction)

Antibody Activity (U/fraction) [Phosphate buffer] A280nm

1.2 1.0 0.8

A280nm

338

0.6 0.4 0.2

0

0 0

20

40

60

0.0

80

Fraction N. (1.5 mL) Fig. 4. Purification of Mabs against EBG from Pleurotus ostreatus by hydroxyapatite chromatography. The culture supernatant was applied to a column packed with ceramic hydroxyapatite (Type II) and Mabs were eluted from the column with a linear gradient of sodium phosphate buffer (10–500 mM) at pH 6.7.

2.2.13. ELISA-elution assay The screening for polyol responsiveness by the ELISAelution assay was carried out in triplicates to select PR-Mabs for immunoaffinity chromatography as described previously [14]. The ELISA-elution assay was carried out by treating the antigen–antibody complex with a combination of salt and polyol before the addition of enzyme-conjugated secondary antibody as described previously [14].

2.2.14. Analysis of Mab specificity Mab specificity was determined by Western blotting using EBGs from P. ostreatus as the antigen. PAGE of EBG was carried out with a linear gradient of 2.5–10% (v/v) of acrylamide with Tris–glycine buffer system at 120 V at room temperature. The gel was cut into two halves whereby one half was stained with 0.2% (w/v) toluidine blue in 0.1 M acetic acid for 20 min. The gel was destained for 90 min in 3% (v/v) acetic acid with constant shaking. The background was eliminated by washing the gel with water. The other half was used for electrophoretic transfer of the samples to a nitrocellulose membrane (8.3 × 6 cm). The membranes were analyzed by ELISA with Mabs from hybridoma clone 1E6 1E8 B5, as reported previously [14].

2.2.15. Analysis of Mab cross-reactivity with other polysaccharides The interaction of Mabs with several ␤-d-glucans was investigated by indirect ELISA as described earlier. The following samples were used as the source of antigen: extraction fractions (i.e., FW1, FW2, FKOH, FHCl, and FNaOH) of IBGs obtained from P. ostreatus, G. carnosum, G. lucidum, H. erinaceus, C. versicolor, L. edodes, P. ostreatus, I. obliquus, G. frondosa, A. auricula, P. umbellatus, C. sinensis, P. Cocos, and A. blazei. However, the commercial sources of polysaccharides were also used as the source of antigen such as ␤-d-glucan from barley; ␤-1,3-glucan from E. gracilis; starch, cellulose, and chitosan from shrimp; acacia gum, laminarin from L. digitata, dextran sulfate, chondroitin sulfate, and heparin.

2.2.16. Quantification of affinity constant (KA ) for Mabs using different antigens The affinity constant (KA ) of Mabs was determined using the simple antibody dilution analysis method by ELISA [17]. Briefly, the ELISA plates precoated for 120 h at 4 ◦ C with a constant concentration of EBGs from P. ostreatus (50 ␮g/well), FHCl from P. ostreatus, FHCl from H. erinaceus, FHCl from G. frondosa, FHCl from A. auricula, FHCl from L. edodes, FKOH from C. versicolor, FKOH from A. blazei, FKOH from P. cocos, FKOH from P. umbellatus, FKOH from EBG from P. Ostreatus, and FNaOH from EBG from P. ostreatus were incubated with serial concentrations of 1E6 1E8 B5 Mabs. The sigmoid curves were plotted to represent the relationship of A415 value versus the decreasing concentration of Mabs. The concentration of immunoglobulin was determined at each antibody dilution as mentioned earlier. The half-maximum antigen binding was obtained for all the selected curves, and the KA values were determined using GraphPad Prism software v.6.05. 2.2.17. Interaction of Mabs with native and heat-treated antigens The interaction of Mabs with native and heat-treated ␤-glucans was also studied by indirect ELISA. The EBGs from P. ostreatus and the extraction fractions (FW1, FW2, FKOH, FHCl, and FNaOH) from EBGs obtained from P. ostreatus, G. carnosum, G. lucidum, H. erinaceus, C. versicolor, L. edodes, I. obliquus, A. auricula, G. frondosa, P. umbellatus, C. sinensis, P. cocos, and A. blazei were incubated in a thermocycler at 100 ◦ C for 1 h. In order to study Mab reactivity, the antigen was immobilized either immediately in the microtiter plates after heating or it was cooled in an ice bath for 10 min before immobilization. In the present assay, the EC50 is defined as the amount of Mab required to give a 50% response on the doseresponse sigmoid curve plotted with increasing concentration of Mabs [18]. The EC50 values were determined using GraphPad Prism software v.6.05. 2.2.18. Epitope studies of Mabs by additive binding assay The indirect ELISA additivity test was carried out to perform epitope analysis using Mabs obtained from hybridoma clones 1E6 1E8 B5 and 3E8 3B4 and a panel of antigen molecules as described previously [19] with some modifications. PolySorp® 96-

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Fig. 5. Characterization of purified preparation of Mab 1E6 1E8 B5 by hydroxyapatite chromatography. A—SDS-PAGE of purified Mabs and cultured supernatants using a 12.5% separating gel. Lanes: 1 and 3—eluted fractions 15 and 16 (1 ␮g); 2—culture supernatant (4 ␮g). Lane 4—Molecular weight markers and B—Native PAGE of purified Mab and cultured supernatant using a 7.5% separating gel. Lanes: 1 and 2—elution fractions (1 ␮g); 2—culture supernatant (3 ␮g). The molecular weights of protein markers are represented on the left.

well microtiter plates were coated with EBGs obtained from P. ostreatus (50 ␮g/well), FNaOH from L. edodes, FKOH from C. versicolor, FKOH from C. sinensis, FKOH from P. umbellatus, FKOH from H. erinaceus, FHCl from A. auricula, FHCl from P. cocos, and FHCl from G. frondosa incubated for 120 h at 4 ◦ C. The microtiter plates were blocked with 1% (w/v) BSA in PBS and incubated at room temperature. After 1 h, a pair of Mabs was added in combination to additive group wells and individually to nonadditive group wells, and the microtiter plates were developed as described previously. 3. Results and discussion 3.1. Production and Purification of Mabs Our aim was the production of Mabs against the intact and native forms of ␤-glucans from mushrooms. The conjugation of

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␤-glucans with protein (i.e., BSA) would change the native conformation of ␤-glucans, which is a disadvantage from the antigen structural point of view. Therefore, we decided to use the native and intact ␤-glucans as the antigen in the immunization process. Regarding the production of Mabs, 33% of the total culture wells exhibited hybrid growth, and 6% of the total wells contained antiEBG-secreting hybrids. One positive hybrid cell line was selected for cloning purposes by limiting dilution method. Fifteen clones secreting Mabs of IgG class against EBG were obtained. Two of these clones were selected (i.e., 3E8 and 1E6) and cloned again. The cloning of 3E8 resulted in clone 3E8 3B4, whereas that of clone 1E6 1E8 B5 yielded 1E6. This hybridoma clone 1E6 1E8 B5 secreting Mabs against EBG was selected for production, purification, and characterization purposes. The hybridoma growth curve of clone 1E6 1E8 B5 revealed typical growth phases such as lag, exponential, and stationary phases (Fig. 1). Conversely, the cellular viability measured by MTT method was linearly related to the antibody concentration as determined by indirect ELISA at A415 (Fig. 1). Preliminary experiments carried out with indirect ELISA exhibited low vales at A415 (data not shown). After several trials, we decided to use Polysorp® microtiter plates that exhibited high adsorption capacity for ␤-glucans compared with the standard ELISA microtiter plates. Furthermore, we also investigated the effect of time and temperature on the immobilization of ␤-glucans obtained from several basidiomycete strains as antigens on these microtiter plates. The data revealed that the signal obtained in indirect ELISA was highest at higher temperature and longer periods of antigen immobilization for EBGs from P. ostreatus, whereas IBGs exhibited higher signal at 37 ◦ C and shorter periods of immobilization (Fig. 2A). However, the data obtained for A. auricular and H. erinaceus revealed the highest signal at higher temperature and longer periods of ␤-d-glucan immobilization, as shown in Fig. 2B. It can be observed in Fig. 2C that the highest value was obtained at lower temperature (i.e., 4 ◦ C) and longer periods of ␤-d-glucan immobilization for C. sinensis but at higher temperature (i.e., 37 ◦ C) and shorter periods of immobilization for G. frondosa. During the course of this investigation, Mabs suitable for immunoaffinity chromatography were selected. Such Mabs are known as PR-Mabs that bind antigen tightly, but the Ag–Ab complex is disrupted under mild and non-denaturing elution conditions [11,12]. Therefore, the hybridoma cell line (1E6 1E8 B5) secreting Mabs were screened by ELISA-elution assay, wherein the Ag–Ab complexes are disrupted by propylene glycol in the presence of (NH4 )2 SO4 and thus yield Mabs (Fig. 3). In the present work, the hybridoma clone 1E6 1E8 B5 was found to secrete PR-Mabs against ␤-d-glucans, as the dissociation of the Ag–Ab complex required 10% propylene glycol and 0.25 M (NH4 )2 SO4 as observed in the marked decrease in A415 (Fig. 3A). However, Fig. 3B presents the reverse situation whereby the concentration of (NH4 )2 SO4 varied at a fixed concentration of propylene glycol. The data presented in Fig. 3B is in agreement with those in Fig. 3A, as the dissociation of the Ag–Ab complex also required 0.25 M (NH4 )2 SO4 and 10% propylene glycol. These data strongly support the fact that the Mabs obtained from 1E6 1E8 B5 are PR-Mabs, and that these can be used as ligands in immunoaffinity chromatography [11,12]. The hybridoma cell line (1E6 1E8 B5) secreting Mabs of IgG2 class and subclass were purified in a single step by hydroxyapatite (Type II) chromatography (Fig. 4) with a final recovery of antibody activity of about 55% and a purification factor of 7 (Table 1). To the author´ıs knowledge, this is the first report about the production and characterization of Mabs of IgG class against ␤-glucans from basidiomycete mushroom strains.

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A

120 100

Reactivity (%)

80 60 40 20 0

B

EBG from P. ostreatus

Starch

Cellulose

Chitosan

Laminarin β-1,3-glucan β-D-glucan

Dextran Sulfate

Acacia Gum Chondroitin sulfate

Heparin

450 400

Reactivity (%)

350

Control

FW1

FW2

FKOH

FHCl

FNaOH

300 250 200 150 100 50 0

Control

IBG from P. IBG from L. edodes ostreatus G.carnosum

H. erinaceus

C. C. sinensis G. frondosa A. blazei versicolor

I. obliquus

P. umbellatus

P. cocos

A.auricula

Fig. 6. Selectivity of Mabs for polysaccharides. Indirect ELISA method was carried out and EBG (25 ␮g) from P. ostreatus was used as the control sample which represents 100% Mab reactivity. A—Several commercial polysaccharides (25 ␮g) as the antigens and B—Several fractions of ␤-glucans (25 ␮g) from mushroom strains as the antigens.

3.2. Characterization of Mabs The purified preparation of Mab from 1E6 1E8 B5 was analyzed by SDS-PAGE and native PAGE; a single protein band and two protein bands with Mr of 21.32, 50.63, and 155 kDa, respectively, were obtained (Fig. 5). These data are in agreement with the literature as Mabs of IgG class have light and heavy chains with Mr of 25 and 50 kDa, respectively, and the entire native molecule has an Mr of 150 kDa. The Mr value obtained for the purified Mab on native PAGE was also confirmed by gel filtration chromatography on Sephacryl S-300HR, which revealed a Mr value of 160 kDa (data not shown). Ouchterlony double-diffusion analysis of purified Mabs was carried out in the presence of rabbit anti-mouse immunoglobulin heavy chains; the results suggest that the Mab 1E6 1E8 B5 is of IgG2 class and subclass as a precipitin line was observed against mouse immunoglobulin heavy chain (data not shown). The monoclonality of the antibody was analyzed by IEF, which revealed single bands with pI values in the range of 6.7–7.0 (data not shown). Similar band patterns were also obtained with other Mabs by IEF as described in the literature [20]. This band pattern may be explained on the basis of microheterogeneity in antibody populations that results in charge alteration due to loss of amide group from asparagine/glutamine residues and different degree of glycosylation (i.e., N-acetylneuraminic acid) [20,21]. The specificity of Mabs was determined by Western blotting using a crude sample of EBGs from P. ostreatus, and a sin-

gle positive reaction band was observed on the nitrocellulose paper, which corresponded to the band of alcian blue staining (data not shown). The binding of Mab 1E6 1E8 B5 to EBG from P. ostreatus as well as to other ␤-glucans from different basidiomycete strains was investigated by indirect ELISA. The affinity constant (KA ) for these ␤-glucans was found to be in the range of 3.20 × 109 ± 3.32 × 103 –1.51 × 1013 ± 3.58 × 107 L/mol (Table 2). These data suggest that there was no significant difference between the epitope recognized by this Mab on EBG from P. ostreatus and that in ␤-glucans from different basidiomycete strains (Table 2). Moreover, these data strongly suggest that this Mab can be used in the development of biosensors for ␤-glucan assay, as this Mab exhibits high affinity for these polysaccharides. However, the heat treatment of ␤-glucans decreased the affinity constant (KA ) for ␤glucans obtained from P. ostreatus, whereas it was found to increase for H. erinaceus, G. frondosa, and A. blazei (Table 2). The effect of cooling after the heat treatment at 100 ◦ C on KA showed that the latter increased for ␤-glucans from P. ostreatus, G. frondosa, and A. blazei, but decreased for H. erinaceus. Conversely, the KA value did not change significantly for C. versicolor (Table 2). The epitope analysis of Mabs from 1E6 1E8 B5 and 3E8 3B4 was carried out in terms of the additivity index (AI) parameter; that the results showed that the Mabs bind to the same epitope in ␤-glucans from P. ostreatus, L. edodes, C. versicolor, P. cocos, P. umbellatus, H. erinaceus, G. frondosa, and C. sinensis, as AI < 50% (Table 3). However, the data presented in Table 3 revealed that these Mabs were

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Table 3 Epitope analysis from indirect ELISA additivity test of Mabs 1E6 1E8 B5 and 3F8 3B4 using Additivity Index (AI) parameter with different ␤-D-glucans from basidiomycete mushroom strains as the antigens. ␤-d-glucan antigens

AI (%)

EBG from P. ostreatus FNaOH from L. edodes FKOH from C. versicolor FKOH from C. sinensis FKOH from P. umbellatus FHCl from A. auricula FHCl from P. cocos FKOH from H. erinaceus FHCl from G. frondosa

−19.26 −37.49 1.36 −35.66 1.97 65.77 −53.60 −7.72 −0.51

sulfate, and heparin revealed cross-reactivity to a certain extent, that is, up to 20% (Fig. 6A). However, ␤-glucans from other basidiomycete mushroom strains exhibited high cross-reactivity in the range of 5–400% compared with the original ␤-glucan used as the immunization agent (Fig. 6B). It is not possible to compare these results with the data in the literature, as there are no published reports regarding the production, purification, and characterization of Mabs against basidiomycete mushroom ␤-glucans. 3.4. Mabs as powerful probes for subtle changes in the structural conformations of native ˇ-glucans

Fig. 7. Reactivity of Mabs to native and heat-treated forms of ␤-glucans and Congo red assay for ␤-glucan with triple-helical structure. Native ␤-glucan fractions (25 ␮g) from mushroom strains were heated at 100 ◦ C and the antigens were immobilized to microtiter plates as described in Section 2. Binding of Mabs to native and heat-treated forms of ␤-glucans was tested with indirect ELISA. The changes in the ␤-glucan conformation were detected using the Congo red assay for the native and heat-treated forms of ␤-glucans. A—Reactivity of Mabs to EBG from P. ostreatus as well as the Congo red assay for ␤-glucans; B—Reactivity of Mabs to FHCl from H. erinaceus as well as the Congo red assay for ␤-glucans; and C—Reactivity of Mabs to FHCl from G. frondosa as well as the Congo red assay for ␤-glucans.

bound to different epitopes on ␤-glucans from A. auricula as the AI value > 50% [19]. 3.3. Mab Selectivity for polysaccharides The selectivity studies were carried out for Mab 1E6 1E8 B5 which revealed that ordinary polysaccharides such as starch, cellulose, chitosan, and laminarin exhibited no cross-reactivity compared with the ␤-glucans from P. ostreatus, whereas ␤-1,3-dglucan from E. gracilis and acacia gum, dextran sulfate, chondroitin

The Mab-combining sites on proteins may be classified into continuous or discontinuous epitopes, depending on the linearity of the epitope-forming amino acids. Most protein epitopes are discontinuous and consist of 2–5 short stretches of amino acid residues that are distant in the polypeptide chain and are brought together at protein surface due to protein folding [22]. Heating is responsible for the changes in the secondary and tertiary structures of ␤-glucans [23,24]. Because most ␤-glucans exhibit triple-helical structure, we decided to investigate the Mab reactivity toward heat-treated ␤-glucans compared with the native forms (Fig. 7). The results were expressed as the fraction between EC50 determined on the sigmoid dose-response curves obtained with native and heat-treated ␤-glucans (i.e., percentage reactivity of Mab on the yaxis). The Mab 1E6 1E8 B5 revealed a lower affinity for heat-treated ␤-glucans from P. ostreatus compared with the native ␤-glucans (Fig. 7A). However, this Mab exhibited a higher affinity for the heat-treated ␤-glucans from G. frondosa and H. erinaceus than for the native ␤-glucans (Fig. 7B and C). The effect of cooling after the heat treatment on the EC50 values revealed that the Mab did not detect cooled ␤-glucans from P. ostreatus, whereas it exhibited a higher and lower affinity for ␤-glucans from G. frondosa and H. erinaceus, respectively (Fig. 7). The Mab reactivity data were correlated with Congo red assay that only detects and quantifies ␤-glucans with a triple-helical structure [7,9]. Congo red assay did not detect heat-treated ␤-glucans from P. ostreatus, which suggests that the heat treatment disrupted its triple-helical structure (Fig. 7A). Conversely, Congo red assay detected higher amounts of heat-treated ␤-glucans from G. frondosa and H. erinaceus compared with the native forms (Fig. 7B and C). The effect of cooling after the heat treatment on the ␤-glucan levels determined by Congo red assay revealed that the cooled ␤-glucans from P. ostreatus were not detected; however, the assay yielded lower values for ␤-glucans from G. frondosa and H. erinaceus compared with the heat-treated forms (Fig. 7). Therefore, these data strongly suggest that the Mabs detected changes in the conformation in ␤-glucans from basidiomycete strains due to heat and cooling treatments (Fig. 7). There are several reports in the literature which present a strong evidence for the changes in the conformation of the heat-treated ␤-glucans obtained from several mushroom strains due to their structural

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transition from triple helix to random coil [23,24]. It is not possible to compare these results with the data in the literature as there are no published reports regarding the use of Mabs for determining the changes in the conformations of ␤-glucans of basidiomycete mushrooms. Similar results have been reported by other investigators on Mabs raised against porcine growth hormone, which detected conformational changes in the antigen molecule [25]. However, some investigators have found that some Mabs raised against native proteins exhibited preferential binding to the denatured form of proteins [26]. 4. Conclusions This is the first report on the production and characterization of Mabs of IgG 2 class and subclass against ␤-glucans from basidiomycete mushroom strains. These Mabs exhibited a very high affinity constant (KA ) for the ␤-glucans; these can be used to design an immunosensor to monitor and quantify these polysaccharides from novel mushroom strains. Moreover, since it detects changes in the conformation of ␤-d-glucan molecules, this biosensor would be very useful to find novel polysaccharides with different biological activities. The PR-Mabs can be used as affinity ligands in immunoaffinity chromatography for the purification of ␤-d-glucan. It is worth noting that purification of ␤-d-glucans to apparent homogeneity is essential for studying their structure-function relationship in some clinical disorders. Acknowledgments We would like to thank Fundac¸ão para a Ciência e a Tecnologia/MCES (Portugal) for their financial support (PTDC/AGRAAM/74526/2006; PEst-OE/EQB/UI0702/2012-2014). References [1] H. Chen, S. Li, Polysaccharides from medicinal mushrooms and their antitumor activities, in: K.G. Ramawat, J.-M. Mérillon (Eds.), Polysaccharides, Springer International Publishing, Switzerland, 2015 (Chapter 61). [2] L. Ren, C. Perera, Y. Hemar, Antitumor activity of mushroom polysaccharides: a review, Food Funct. 3 (2012) 1118–1130. [3] E. Guillamón, A. García-Lafuente, M. Lozano, M. D´ıArrigo, M.A. Rostagno, A. Villares, J.A. Martínez, Edible mushrooms: role in the prevention of cardiovascular diseases, Fitoterapia 81 (2010) 715–723. [4] F.Y. Kagimura, M.A.A. da Cunha, A.M. Barbosa, R.F.H. Dekker, C.R.M. Malfattid, Biological activities of derivatized d-glucans: a review, Int. J. Biol. Macromol. 72 (2015) 588–598. [5] S.P. Wasser, Current findings, future trends, and unsolved problems in studies of medicinal mushrooms, Appl. Microbiol. Biotechnol. 89 (2011) 1323–1332. [6] T. Masuko, A. Minami, N. Iwasaki, T. Majima, S.-I. Nishimura, Y.C. Lee, Carbohydrate analysis by a phenol–sulfuric acid method in microplate format, Anal. Biochem. 339 (2005) 69–72.

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