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Production and characterization of mouse monoclonal antibodies recognizing multiple subclasses of human IgG.

Abstract

Different IgG subclass profiles are produced in response to different antigenic stimuli in a variety of diseases. IgG subclass levels may reflect disease severity. Quantification of IgG subclasses depends on the availability of specific Monoclonal antibodies (MAbs). In the present study seven hybridoma clones producing MAbs reactive with multiple subclasses of human IgG were established. Splenocytes from Balb/c mice immunized with Fc fractions of human IgG1 or IgG2 myeloma proteins were fused with mouse myeloma cells. Fused cells were selected and cloned by limiting dilution assay. Antibody secreting cells were screened by Enzyme-linked immunosorbent assay (ELISA) and the specificity of secreted MAbs was further analyzed, using a panel of purified human myeloma paraproteins of different IgG subclasses by ELISA and immunoblotting. Cross-reactivity to immunoglobulins (Igs) of other species was studied by indirect ELISA using serum samples collected from 9 animals. The MAbs were found to react with triple IgG subclasses, including IgG1,2,4 (n=4) and IgG1,2,3 (n=3). Immunoblotting studies revealed recognition of linear (n=4) or conformational (n=3) epitopes by these MAbs. The most abundant cross-reactivity (71.4%) was observed with monkey Ig while no cross-reactivity was detected with hen and cat sera. The MAbs mostly displayed a restricted pattern of cross-reactivity and one of them did not bind to any of the animal sera tested. The affinity constant of 3 MAbs was measured by ELISA. Based on the data obtained from this study, mouse MAbs reactive with multiple human IgG subclasses are directed to a variety of immunogenic epitopes, mostly shared with IgG of other species. These MAbs are valuable tools for purification of non-reactive IgG subclasses through negative affinity chromatography. These MAbs could also provide an opportunity for epitope mapping of the Fc region of IgG, as well as serological phylogenetic studies.

Avicenna / Med Biotech 2010; 2(1): 37-45

Keywords: ELISA, Hybridoma, IgG, Isotype, Monoclonal antibody, Subclass

Introduction

IgG, the most abundant Ig present in human serum and extravascular fluids, is subdivided in to four subclasses (IgG 1-4)based on antigenic differences between their heavy chain constant regions (1). Each subclass has different biological characteristics (2) and a particular profile of effector functions (3).

Immunoglobulin subclasses are differentially regulated in response to different antigenic stimuli (4), (5). Thus different patterns of IgG subclasses (6-8) have been reported to be produced in a variety of diseases. Moreover, there is a relationship between IgG subclass levels and severity of diseases (9), (10). Therefore IgG subclasses have a great diagnostic and prognostic value (11). Analysis of the IgG subclass response to different antigens of a microorganism may reveal the profile of T helper associated cytokine response (12) and hence is helpful for designing a potential vaccine (13). Quantification of IgG subclass levels depends on the availability of specific MAbs. Assay sensitivity could be improved by application of high affinity MAbs recognizing appropriate epitopes (14).

Since human IgG subclasses are highly homologous with almost 95% aminoacid sequence identity (15), some human IgG subclass restricted mouse MAbs may recognize epitopes shared by multiple IgG subclasses. Such MAbs are valuable tools for epitope mapping of human IgG isotype and may recognize isotypic or isoallotypic markers within IgG subclasses.

The aim of the present study is production and characterization of MAbs with specificity for multiple IgG subclasses.

Materials and Methods

Preparation of purified human IgG subclasses

A panel of 27 different purified human IgG myeloma proteins of known IgG subclasses and light chain types was employed in this study. These myeloma proteins, obtained from patients with multiple myeloma, were either purified by Diethyl amino ethyl (DEAE) cellulose (Whatmann, UK) chromatography or by affinity chromatography using Staphylococcal protein A (SPA) or Streptococcal protein G (SPG) Sepharose 4B (Pharmacia, Sweden). The heavy chain and light chain isotypes and subclasses of myelomas were identified using specific mouse monoclonal antibodies including: AF6 (IgM), 8a4 (IgG), 2D7 (IgA), JA 11 (IgD), C4 ([lambda]), 6e1 ([kappa]), JL512 (IgG1), GOM2 (IgG2), ZG4 (IgG3) and RJ4 (IgG4), kindly provided by Professor R. Jefferis and Dr. M. Goodal (Department of Immunology, University of Birmingham, UK).

Polyclonal IgG was isolated from normal serum with SPG-Sepharose and polyclonal IgG3 was isolated, as break-through fraction, from polyclonal IgG by SPA-Sepharose column. Fc and Fab and F (ab') 2 fragments were produced from several purified human myeloma proteins of each of the IgG subclasses, by pepsin and papain digestion (16). Digested fragments were isolated by affinity chromatography with SPA-Sepharose column.

Animal sera

Sera from human and nine animals were prepared from their clotted blood. The animal species used in this study were chicken, rabbit, guinea pig, cat, dog, sheep, goat, horse and monkey. The human serum was used as a control.

Production and selection of hybridomas

Balb/c mice (8-12 weeks of age) were immunized with four intraperitoneal injections of Fc fragments of human IgG 1 or IgG2 myeloma proteins emulsified in Freund's complete adjuvant (Sigma, USA) (first injection) or incomplete adjuvant (Sigma) (other injections) (50 [micro]g every 2 weeks). Three days after the last injection, spleen cells were fused with SP2/0 myeloma cells (NCBI 129, National Cell Bank of Iran, Pasteur Inst. of Iran, Tehran), using polyethyleneglycol (PEG 1500) (Sigma). Hybridomas were grown in DMEM culture medium (Sigma) containing 20% Fetal Calf Serum (FCS) (Seromed, Germany), penicillin 100 IU/ml) and streptomycin (100 pg/m1) and supplemented with hypoxantine (1x[10.sup.-4] M). aminopterin (4x[10.sup.-7] M) and thymidine (1.6x[10.sup.-5] M) (HAT) (Sigma).

Ten to fourteen days after fusion, secreting hybrids were identified by analysis of culture supernatants by the ELISA technique described below. Selected antibody producing cultures were cloned by limiting dilution process according to the conventional methods (17). Clones secreting antibody of desired reactivity were expanded in 25 and 75 [cm.sup.2] flasks (Nunc, Denmark), harvested and cryopreserved in 40% FCS, 10% Dimethylsulfoxide (DMSO) (Sigma) and 50% RPMI-1640 culture medium (Sigma).

Detection of specificity of MAbs by indirect enzyme-linked immunosorbent assay (ELISA)

Specificity of MAbs was determined by ELISA technique as described elsewhere (18). Briefly, microtiter polystyrene plates (Maxisorp, Nunc, Denmark) were coated with 1-10 [micro]g/ml of purified myeloma IgG subclasses or polyclonal IgG in PBS (0.15 M, pH=7.2). Then 0.05 ml of culture supernatant was added. Appropriate dilution of HRP-conjugated sheep anti mouse Ig (prepared in our lab) was then added and the reaction revealed with 0-phenylenediamine dihydrochloride (OPD) (Sigma) substrate. Finally, the reaction was stopped with 20% H2SO4 and the optical density (OD) measured by a multiscan ELISA reader (Organon Teknika, Box-tel, Belgium) at 492 nm.

Isotype determination of MAbs by capture ELISA

Goat antimouse IgG 1, IgG2a, IgG2b, IgG3, IgA and IgM at 1/1000 dilution, were adsorbed on to the wells of a microtitre ELISA plate (Nunc). Isotypes of MAbs in culture supernatants were determined according to the ELISA technique mentioned above.

Affinity constant ([K.sub.aff]) determination by ELISA

We determined the [K.sub.aff] by ELISA technique as described elsewhere (19). Briefly, ELISA plates (Nunc) precoated with four different concentrations of human IgG ([Ag], [Ag'] and [Ag"]) were separately incubated with serial concentrations of each MAb. Sigmoid curves were constructed using the OD values obtained for different concentrations of each MAb. Four non-overlapping curves were selected for each MAID to calculate the affinity constant. The half maximum OD (OD-50) was assigned for all selected curves from which the corresponding antibody concentration (([Ab], [Ab'] and [Ab"]) was extrapolated.

Accordingly, [Ab] and [Ab'] are the measurable total Ab concentrations at OD-50 and OD'-50 for plates coated with [Ag] and [Ag'], respectively. The affinity constant was determined using the following equation (20): Kaff = (n-1)/2(n[Ab']t-[Ab]t), where n=[Ag]/[Ag']

Immunoblotting technique for analysis of specificity of MAbs

Specificity of MAbs was assessed by immunoblotting technique as described elsewhere (21). Briefly, affinity purified myeloma IgG subclasses, polyclonal IgG and their fragments were electrophoresed under native and denaturing conditions, in 10% polyacrylamide gel (SDS-PAGE) (Sigma) and then transferred to a nitrocellulose membrane (Schleicher & Schuell, Germany). After blocking with 2.5% skim milk (Merck, Germany), the membrane was incubated with culture supernatants containing the MAbs for 1.5 hr at 37 [degrees]C, followed by HRP-conjugated sheep antimouse lg. The bands were finally visualized with Diaminobenzidine tetrahydrochloride (DAB) (Sigma) substrate.

Results

Screening and selection of specific hybridomas

Culture supernatants from growing hybridomas were screened by ELISA using a panel of four IgG myelomas with different subclasses, including their immunogens. Results obtained for these hybridomas with different specificity profiles are illustrated in Table 1.
Table 1. Reactivity of selected MAbs with different human IgG
subclasses

                                        Antigen screening
                                           OD (492 nm)
MAb                 IgG1       IgG2           lgG3           lgC4
                  (MM 13)  (MM 12)        (MM98)         (MM147)

2F7G8                 1.4      1.9                1.7     0.25
IF8C4                 1.3      1.5                1.4      0.2
7F14F7                1.1      1.7                1.9      0.2
1F5A8                 1.6      1.8                0.3      1.4
6F11E1                 II      1.2                0.2      1.3
8F9G7                 1.5      1.8                0.3      1.7
6FI9C11               1.4      1.7                0,2      1.7
IF18A8               0.15      0.2                2.0      0.3
8F9A8                 1.8      1.7                1.6      1.4
Anti IgGl(JL512)      1.2     0.27                0.3     0.27
Anti IgG2(GOM2)       0.4      2.0                0.3     0.25
Anti IgG3(ZG4)        0.3      0.3                1.9      0.3
Anti IgG4(RJ4)        0.3      0.3                0.3      1.6

* Positive reactivity is shown m bold figures. IFI8A8 and 8F9A8
MAbs are IgG3 and Pan-IgG specific MAbs included as controls
Commercial IgG subclass monospecific MAbs. including JL512
(IgG1), GOM2 (lgG2). ZG4 (IgG3) and KJ4 (lgG4), are included
as control MAbs for each IgG subclass


Characterization of MAbs

Following cloning and subcloning, culture supernatant from the selected hybridomas was further characterized. Five MAbs belonged to IgG1, one MAb was IgG2a and the last one displayed IgG2b isotype (Table 2). Specificity of these MAbs was determined, using a panel of purified myeloma proteins, including IgG1 (n=9), IgG2 (n=4), IgG3 (n=7) and IgG4 (n=6) subclasses.
Table 2. Determination of the isotype of MAbs by ELISA

                              Isotype
MAbs             IgG1  IgG2a   IgG2b   lgG3  IgA   lgM

2F7G8             0.2    0.7     0.16  0.16  0.14  0.16
[F8C4               1   0.16     0.15  0.11  0.13  0.13
7F14F7            0.7    0.2      0.2   0.2   0.2   0.2
IF5A8             0.6   0.13     0.12  0.11   0.1  0.16
6FUE1             0.2   0.25        1  0.17  0.17  0.17
8F9G7             0.8    0.2      0.2  0.16  0.17  0.19
6F19C11          0.75    0.2      0.2   0.2  0.15  0.17
1F18A8            0.8   0.13      0.1  0.15   0.1   0.1
8F9A8             0.7    0.2      0.2  0.17  0.18   0.2
[Control.sup.-]  0.16   0.14      0.1  0.15   0.1  0.16

The results represent OD values obtained at 492 nm [Control.sup.-]:
Culture supernatant from SP2/0 myeloma cells


According to ELISA and immunoblotting studies, these seven MAbs can be categorized into two groups: 1) Four IgG1, 2, 4 specific MAbs of which three (1F5A8, 8F9G7 and 6F11E1), react with conformational epitopes located on heavy chain of IgG1, 2, 4 (Figure 1) and an unusual IgG3 myeloma protein (Goe) bearing allotypic marker of the Mongloid populations [G3m(st)] displaying similar specificity to SPA. The fourth MAb (6F19C11) reacts with a linear epitope located on heavy chain of IgG1, 2, 4 subclasses. 2) Three IgG1, 2, 3 specific MAbs of which two (2F7G8 and 1F8G4) react with linear epitopes and the third one (7F 14F7) reacts with conformational epitope located on heavy chain of IgGl, 2, 3 subclasses (Figure 2). Representative immunoblotting results are illustrated in Figures 3 and 4. All the MAbs reacted only with Fc, but not Fab fragments of their immunogens (Figure 5).

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

Cross-reactivity studies employing whole sera from a range of animal species indicated the most abundant cross-reactivity (71.4%) with monkey Igs, while no cross-reactivity was detected with hen and cat sera. While 4 MAbs displayed a restricted pattern of cross-reactivity, two MAbs [8F9G7 (IgG1, 2, 4 specific) and 7F 14F7 (IgG 1, 2, 3 specific)] displayed a broad pattern of cross-reactivity and one MAb [2F7G8 (IgG 1, 2, 3-specific)], did not bind to any of the animal sera tested (Table 3).
Table 3. Cross-reactivity of MAbs with animal sera

Animal    2F7G8       1F8G4      7F14F7     1F5A8      6F11E1
sera

Monkey      <0.1      <0.1        30        92       41)

Rabbit      <0.1      <0.1      <0.1      <0.1      <0.1

Guinea      <0.1      <0.1        19      <0.1      <0.1

Pig

Sheep       <0.1      <0.1        19      <0.1      <0.1

Goal        <0.1      <0.1        27      <0.1      <0.1

Cat         <0.1      <0.1      <0.1      <0.1      <0.1

Dog         <0.1        81        36      <0.1      <0.1

Horse       <0.1       <QJ        22      <0.1      <0.1

Hen         <0.1      <0.1      <0.l      <0.1      <0.l

Animal    8F9G7     6F19C11
sera

Monkey       102        74

Rabbit        30      <0.1

Guinea      <0.1      <0.l

Pig

Sheep         19      <0.1

Goal          17      <0.1

Cat         <0.1      <0.1

Dog         <0.l      <0.1

Horse          9        20

Hen         <0.1      <0.1

% Cross-reactivity is expressed relative to the value obtained for
human pooled serum


Determination of affinity constant of MAbs

Ascitic fluids of three MAbs were available for purification by affinity chromatography using Streptococcal protein G column. The affinity constant ([K.sub.aff]) of these MAbs was determined by ELISA. Triple serial concentrations of the antigens and MAbs were selected to construct the corresponding curves and to extrapolate the [K.sub.aff] values using the formula given in Materials and Methods. The calculated average [K.sub.aff] values are presented in Table 4.
Table 4. Affinity constants of human IgG subclass-restricted MAbs
determined by ELISA

MAbs       [Ag]           OD-50         [Ab] at      Kaff
           (ng/             *            OD-50
(ng/    (M.sup.-1)  Kafff[M.sup.-1])[+

            ml)                             ml)
1F5A8         5000                   1      760        3.1 x

                                                  [10.sup.9]
              2500                 0.9      860        3.2 x

                                                  [10.sup.9]
              1250                 0.7      900          2 x

                                                  [10.sup.8]

               625                0.55     1300

6F11E1
             20000                0.65      480         4.69

                                                 x[10.sup.8]
             10000                 0.6      560       4.54 x

                                                  [10.sup.8]
              5000                0.46      600       4.69 x

                                                  [10.sup.8]

              2500                0.28      620

7F14F7
              2000                0.97       46        4,5 x

                                                  [10.sup.9]
              1000                 0.9       56       0.94 x

                                                  [10.sup.9]
               500                0.75      190       0.75 x

                                                  [10.sup.9]
               250                 0.5      300

MAbs         Average

(ng/

             or -]SD
1F5A8

         2.8 x [10.sup.8]

6F11E1

        4.64 x [10.sup.8]

7F14F7

           2 x [10.sup.9]

* OD-50 represents the half maximum optical density obtained for a
given concentration of h-lgG ([Ag]) and the corresponding MAb
([Ab]). The affinity constant ([K.sub.aff]) for each selected
concentration of Ag and Ab was determined using the formula
described in the Materials and Methods


Discussion

Several anti-human IgG MAbs with specificity for Fc region-dependent epitopes common to three of four human IgG subclasses have been produced in the present study.

One group of our MAbs (n=4) was shown to be specific for isotypic epitopes common to IgG1, 2, 4, but not IgG3 subclasses. IgG3 subclass has different amino acid sequences at positions 276 and 291 in CH2 domain and positions 392, 422 and 435 in CH3 domain in comparison with other subclasses. In IgGl, 2 and 4 subclasses, these amino acids are Asn, Pro, Lys, Val and His, whereas in IgG3 subclass they include Lys, Leu, Asn, Ile and Arg, respectively (22). We suggest that these four MAbs recognize epitope (s) containing the differentiating amino acids located in CH2 and/ or CH3 domains common to IgGl, 2 and 4 subclasses, but not CH1 domain, because our results showed no reactivity of these MAbs with Fab fragments of IgG 1 molecules (Figures 3 and 5). MAbs with similar specificity have already been reported (HP6032, HP6033 (23); QF1, WC2, WC9 (24) and IG12 (25)).

In contrast to HP6032 MAb, HP6033 showed also reactivity with IgG3 myeloma proteins having G3m (g) allotype and with one of two IgG3 myeloma proteins having G3m (b) allotype (23). These two MAbs were evaluated only by Immunoflurometric assay (IFMA) (23) and comparative binding study with SPA has not been conducted.

IG 12 MAb reacts with a heat sensitive conformational epitope close to the hinge region of IgG and did not compete with Staphylococcus protein A (SPA) for IgG binding. Furthermore, the affinity constants of the previously produced anti IgG1, 2, 4 MAbs have not been calculated. Three of our anti IgG1, 2, 4 MAbs (1F5A8, 6F11E1 and 8F9G7), recognize conformational epitopes located to heavy chain of IgG1, 2, 4 but not IgG3 subclasses and a protein of IgG3 subclass (Goe protein) (Figure 1), a reactivity pattern similar to SPA (21).

The sequence of protein Goe resembled that of other [[gamma].sub.3] molecules, except for the presence of tyrosine at position 296, alanine at position 339 and histidine and tyrosine at positions 435 and 436 (26). The only amino acid which is common between Goe protein and IgG1, 2 and 4 subclasses and is different between Goe protein and other IgG3 molecules, is histidine at position 435. Thus, it appears that histidine 435 plays an important role in binding to 1F5A8, 6F11E1 and 8F9G7 MAbs produced in present study. Reactivity of these monoclonal antibodies with polyclonal IgG subclasses was found to be similar to SPA.

It was previously shown that histidine 435 has a major role in binding to SPA (27). In the crystal structure of the complex between IgG 1-Fe and domain B of SPA (28), it was revealed that His 435 is situated at the CH2-CH3 domain interface and is in contact with SPA. In IgG3 proteins from Caucasian populations the protein A contact residue is replaced by Arg. This lengthy side chain prevents the formation of favorable IgG-SPA contact, so that such IgG3 proteins do not bind protein A (29).

Since three of our MAbs (1F5A8, 6F11E1 and 8F9G7) displayed similar binding activity to SPA with regard to interaction with purified human myeloma proteins of IgG1, 2 and 4 and the IgG3 myeloma protein Goe, as well as human polyclonal IgG1, 2 and 4, but not IgG3 subclasses (Figure 1), the specificity of these MAbs resembled that of SPA. Our proposition is further supported by the fact that our MAbs only reacted with the unreduced form of IgGl, 2 and 4 subclasses and the Goe protein by immunoblotting (Figure 3). These results indicate that the epitopes recognized by these MAbs are conformational, depending on covalent interaction of the two heavy chains of IgG molecule.

The fourth MAb (6F19C11) in this category is completely different from the others as it does not recognize any protein of IgG3 subclass and it recognizes a linear epitope located to IgGl, 2, 4 subclasses (Figure 3).

The second group of our MAbs (three MAbs) was shown to be specific for isotypic epitopes common to IgG 1, 2, 3, but not IgG4 subclasses (Figure 2). IgG4 subclass has different amino acid sequences at positions 268, 330 and 331 in CH2 domain and positions 355, 409, 419 and 445 in CH3 domain. In IgG1, 2 and 3 subclasses, these amino acids are His, Ala, Pro, Arg, Lys, Gln and Pro, whereas in IgG4 subclasses they include Gln, Ser, Ser, Gln, Arg, Glu and Leu, respectively (23). It seems hat these three MAbs, recognize epitope (s) containing the differentiating amino acids located in CH2 and/ or CH3 domains common to IgG1, 2 and 3 subclasses, because they did not bind to the Fab fragment of IgG (Figure 5). It was shown by immunoblotting that two of these MAbs (2F7G8 and 1 F8G4) recognize linear epitopes and the third one (7F14F7) recognizes a conformational epitope located to IgG1, 2, 3, but not IgG4 subclasses. Representative data is shown in Figure 4.

A number of MAbs [(HP6034 and HP6058) (23), (Iq1, X3a8 and 3e 10) (24) and (IG14 and IG15)] (25), have been produced with specificity similar to our MAbs. HP6034 and HP6058 recognize epitopes located to hinge (23) and CH2 domains of IgG-Fc, respectively (30). The affinity constant of HP6058 has been reported to be about 2 x [10.sup.8] [M.sup.1] (30). IG14 and IG15 recognize conformational epitope (s) located to Fc region of IgG1, 2, 3 subclasses (25). The reported MAbs belonged to IgG1 subclasses. Two of our IgGl, 2, 3 specific MAbs (1 F8G4 and 7F14F7) belonged to IgG1 and one of them (2F7G8) belonged to IgG2a subclass. The affinity constant of one of our MAbs (7F14F7) was found to be 2 x [10.sup.9] [M.sup.1].

The MAbs produced in this study displayed different profiles of cross-reactivity with a variety of animal sera. The most abundant cross-reactivity (71.4%) was observed with monkey Ig, while no cross-reactivity was detected with hen and cat sera. Cross-reactivity of anti human IgG MAbs have been reported by other investigators (23), (25). Most of our MAbs displayed a restricted pattern of cross-reactivity and one of them (2F7G8) did not bind to any of the animal sera tested. This is in agreement with previous reports (23), which showed anti IgG isotype specific MAbs cross-react more commonly with IgG of other animal species, due to the amino acid sequence homology between IgG molecules of human and other animals.

In conclusion, we produced and characterized seven MAbs each recognizing triple subclasses of human IgG. These MAbs are valuable tools for purification of the non-reactive IgG subclass by negative affinity chromatography. Combination of such MAbs with different specificities may improve the sensitivity of immunoassays, as they complement each other via reactivity to different epitopes located to the same IgG. These MAbs could also be used for epitope mapping of the Fc region of IgG and for serological phylogenetic studies.

Acknowledgement

We thank Dr. Mahmood Jeddi-Tehrani, Dr. Soheila Gharagozlou, Roya Ghods, Jalal Khoshnoodi and Azarn Roohi for scientific consultations and preparation of myeloma proteins. This study was supported in part by a grant from the Research and Technology Undersecretary of the Ministry of Health, Treatment and Medical Education of Iran.

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Fatemeh Hajighasemi (1), (2) and Fazel Shokri (1), (3) *

(1.) Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran

(2.) Department of Immunology, School of Medicine, Shahed University, Tehran, Iran

(3.) Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, Iran

* Corresponding author: Fazel Shokri, Ph.D., Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, Iran, P.O. Box: 6446-14133

Tel: +98 21 22431945

Fax: +98 21 22432021

E-mail: fazshok@yahoo.com

Received: 15 Feb 2010

Accepted: 15 Mar 2010
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Title Annotation:Original Article
Author:Hajighasemi, Fatemeh; Shokri, Fazel
Publication:Avicenna Journal of Medical Biotechnology (AJMB)
Date:Jan 1, 2010
Words:4718
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