An enzyme-linked immunosorbent assay for the quantitation of human IgG subclasses using monoclonal antibodies

Journal of Immunological Methods, 93 (1986) 207-212 Elsevier

207

JIM04079

An enzyme-linked immunosorbent assay for the quantitation of human IgG subclasses using monoclonal antibodies A. Ferrante 1,2,,, B. Rowan-Kelly 1,2, L.J. Beard 1 and G.M. Maxwell 1 I Department of Paediatrics, University of Adelaide, and 2 Department of Immunology, Adelaide Children's Hospital, Adelaide, S.A. 5006, Australia (Received 11 February 1986, accepted 19 May 1986)

An enzyme-linked immunosorbent assay was established for the quantitation of human IgG1, IgG2, IgG3 and IgG4 using IgG subclass-specific monoclonal antibodies. The method could detect 1-10 ng/ml of the Ig subclasses. The technique is suitable for measuring IgG subclass concentration in sera of healthy adults and in supernatants from human lymphocytes cultured in the presence of pokeweed mitogen. Key words: IgG subclasses, human; ELISA

Introduction

Four subclasses of human IgG are currently recognized: IgG1, IgG2, IgG3, and IgG4. Acquired antibody responses to certain antigens may be limited to one or some of the IgG subclasses. In addition some of the biological activities controlled by the constant region (Fc fragment) are restricted to some of the subclasses. It is now apparent that IgG subclass deficiency may be associated with increased susceptibility to infections (Heiner, 1984; Oxelius, 1984; Beard et al., 1986). A deficiency in the IgG subclasses may not be revealed by measuring total serum IgG, since some of the subclasses are present in very low concentrations. The relative percentage of IgG1, IgG2, IgG3 and IgG4 in adults appears to be 58-72, 19-31, 5-7, and 1-5 respectively (Shakib and Stanworth, 1980). Measurement of IgG sub-

class have predominantly involved the use of radial immunodiffusion (Mancini) techniques. A more sensitive technique would be useful particularly for the measurement of IgG3 and IgG4 which are present in much lower concentrations than IgG1 and IgG2 and for measuring IgG subclasses in the paediatric population at ages where levels are often low. In addition in body fluids such as cerebral spinal fluid (CSF), tears etc. and supernatants from mitogen-stimulated lymphocytes a more sensitive technique appears to be essential. The purpose of the present study was to establish an enzyme-linked immunosorbent assay (ELISA) using IgG subclass-specific monodonal antibodies for the quantitation of IgG subclasses.

Materials and methods Sera

* Correspondence: Dr. A. Ferrante, University Department of Paediatrics and Department of Immunology, Adelaide Children's Hospital, Adelaide, South Australia 5006, Australia.

Sera were prepared from blood of healthy donors (27 females and 14 males), of age range 25-50 years. All sera were stored frozen at - 7 0 ° C in aliquots.

0022-1759/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

208

Lymphocyte culturing for immunoglobulin production Blood from healthy volunteers was drawn into lithium heparin-containing tubes. The mononuclear leucocytes (MNL) were separated by layering blood on Hypaque-Ficoll medium and low speed centrifugation as described previously (Ferrante and Thong, 1982). The MNL were harvested from the interphase, washed three times in RPMI 1640 medium and resuspended in this medium. Lymphocyte culturing for immunoglobulin production was carried out essentially as described previously (Ferrante et al., 1984). MNL were suspended in RPMI 1640 with FCS (10%) to a concentration of 1 x 106/ml and cultured in LUX tissue culture tubes (16 by 125 mm; Miles Laboratories, Naperville, IL) with or without the T-cell dependent B-cell mitogen PWM (Gibco Laboratories, Grand Island, NY) at optimal concentration (1/200 dilution). The cultures were incubated at 37°C in an atmosphere of 5% CO2-95% air and high humidity, and their supernatants collected after 7 days of incubation. The supernatants were examined for levels of IgG subclasses by the enzyme-linked immunosorbent assay.

Enzyme-finked immunosorbent assay for quantitation of IgG and IgG subclasses IgG was quantitated by the ELISA method essentially as described previously (Ferrante et al., 1984). Briefly, 2/-chain-specific antiserum (Silenus Laboratory, Victoria, Australia) was used to coat wells of microtitre plates (Dynatech laboratories, Australia) and act as a 'catching' antibody for human IgG. 5% sheep serum was added to prevent non-specific binding of the enzyme-antibody conjugate. The amount of IgG bound was quantitated by addition of horseradish peroxidaselinked sheep anti-human immunoglobulin (Amersham, Australia) followed by the substrate 2,2'-azino-di-(3-ethylbenzthiazoline sulphonate) (ABTS) (Boehringer Mannheim). The absorbance at 414 nm was measured, and the immunoglobulin concentration in each test sample was determined by comparisons with a standard curve. For the measurement of IgG subclasses, antibodies to mouse Ig were bound to surfaces of wells of microtitre plates (Dynatech Laboratories). This involved adding 200/~1 of a 1/200 dilution of

the IgG fraction of a goat anti-mouse IgG (Fc specific) (Cappel, Malvern, PA) made up in 0.1 M carbonate/bicarbonate buffer pH 9.6 to the wells. The plates were incubated at 4°C for 18-20 h and then the wells were washed with 0.1 M PBS plus 0.05% Tween 20 pH 7.4. After washing 100/11 of 10% sheep serum in PBS-Tween was added to the well, the plates incubated for 3 h at 37°C and then 50 /~1 of either a monoclonal anti-human IgG1 (Fab specific), IgG2, IgG3 or IgG4 (Miles Scientific, Victoria, Australia) was added at dilutions of 1/1000 for IgG1, IgG2 and IgG4, and 1/100 dilution for IgG3. Each IgG subclass monoclonal antibody is monospecific and does not react with the other three IgG subclasses (Chemical-Credential, Miles Scientific). Dilutions were made in PBS-Tween pH 7.4 containing 10% sheep serum..The plates were incubated for 18-20 h at 4°C and then washed with PBS-Tween. To the wells were added dilutions of the IgG subclass standard (Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, Amsterdam) or dilutions of unknown in 10% sheep serum in PBS-Tween. The plates were incubated at 37 ° C for 3 h, washed and then 50/~1 of 1/1000 dilution of horseradish peroxidase-linked sheep anti-human immunoglobulin in 10% sheep serum in PBS-Tween added to each well. After a further 18-20 h incubation at 4 ° C, the wells were washed and to each was added 100/~1 of substrate ABTS. The colour reaction was allowed to proceed at 37°C for 45 min after which the OD414n m was read as described above.

Quantitiation of IgG subclasses by radial immunodiffusion Human IgG subclasses were also quantitated by the radial immunodiffusion (RID) method using commercially available plates (Serotec, Oxon, England), as detailed in their information protocol.

Results

Initial studies conducted demonstrated quite clearly that the monoclonal antibodies with subclass specificity were not suitable for use in a

209

system where these were employed for coating of microwells. Attachment of these to solid phase was attempted by using a goat anti-mouse I g G (Fc) antibody. This antibody was successful in attaching all four I g G subclass-specific monoclonal antibodies to the polystyrene surface. Standard curves were produced using standard reference sera, and these curves are presented in Figs. 1-4. The standard curves were similar in shape and sensitivity for the four I g G subclasses. In numerous experimental runs the detection limits for IgG1 and IgG3 was approximately 1 n g / m l and 10 n g / m l for IgG2 and IgG4. Further studies demonstrated that the standard curves for each

IgG 1

~G2 2"

2.0.

~r

0

1.5-

1.0

2.5

0.5-

2-C

o

I

;

,'o

,~o

,~

,o~oo

ng / ml

Fig. 2. Standard curves showing the relationship between OD414,m and IgG2 concentration in the ELISA assay. Each curve represents the same standards run on separate plates.

1.5-

i-0-

0.5-j i

I

I0

i

I

100

i

1000

i

10000

ng / ml

Fig.

Standard curves showing the relationship between and IgG1 concentration in the ELISA assay.Each curve represents the same standards run on separate plates. 1.

OD414n m

I g G subclass did not differ significantly between plates (Figs. 1-4). Using the ELISA technique, I g G subclass levels of 41 healthy adults was determined and results are presented in Table I. The mean relative percent for IgG1, IgG2, IgG3 and IgG4 was 64.6, 22.3, 6.7 and 6.4 respectively. The summation of these values gave total I g G values essentially the same as obtained by using the ELISA to measure total I g G but slightly greater than values obtained by using the immunochemical analyser. I g G subclass concentrations in the same sera measured by R I D gave values similar to those obtained by the ELISA method. The mean ± standard deviation in g/1 (relative %) for IgG1, IgG2, IgG3 and IgG4 were 6.12 ± 1.57 (61.29_

210

19G 4

2"5

2.5

2.0

2.0 .

~r I~ 0

(~

1.5-

1-5-

1.0-

1.0-

0-5-~

0"50 I

1

10 ng / ml

I00

I000

I I0000

,w

o'.,

1

1%

~;o

,o;o

,ogoo

ng /m4

Fig. 3. Standard curves showing the relationship between OD414n m and IgG3 concentration in the ELISA assay. Each curve represents the same standards run on separate plates.

Fig. 4. Standard curves showing the relationship between and IgG4 concentration in the ELISA assay. Each curve represents the same standards run on separate plates.

10.82), 2.78 ___1.75 (26.34 + 9.76), 0.62 + 0.29 (6.30 + 3.18) and 0.61 ___0.40 (6.07 _ 3.36) respectively. The group of subjects studied consisted of 27 females and 14 males. Using both the iCS and ELISA the females had a greater mean total serum I g G than male subjects. This was reflected in the higher values for females in all four I g G subclasses using the ELISA. The most clear and significant difference was however observed only with IgG3; the mean + standard deviation for females and males was 0.86 + 0.35 and 0.60 + 0.28 g/1 ( P > 0.02; analysed by paired t-test). Measurement of I g G subclass levels in supernatants from PWM-stimulated lymphocytes was possible by the ELISA method. Their levels were quantitated in both supernatants from non-stimulated lymphocytes. The mean relative percentage

of these I g G subclasses were similar to that found in serum (Table II).

OD414n m

Discussion

An ELISA method is described for quantitating the human I g G subclasses IgG1, IgG2, IgG3 and IgG4. The technique is highly sensitive enabling the measurement of a few ng of I g G subclasses, utilizing subclass-specific monoclonal antibodies. In this procedure the monoclonal antibodies were attached to surfaces of microwells by precoating the wells with anti-mouse I g G antibodies. The system is suitable for measuring IgG1, IgG2, IgG3 and IgG4 in human serum and in supernatants from PWM-stimulated lymphocyte culture. The

211 TABLE I IgG SUBCLASS LEVELS ADULTS a

IN

SERUM

Mean ± SD

OF

HEALTHY

Range

Total IgG ICS ELISA Summation

g/1 g/1 g/l

9.48 ± 2.26 11.53 + 3.12 11.48 ± 3.48

6.55-13.60 6.67-18.85 6.31-19.28

IgG subclasses IgG1 Relative

(g/l) %

7.41 + 2.41 64.64 + 7.53

3.82-14.27 51.62-79.65

IgG2 Relative

(g/l) %

2.55 ± 0.94 22.32 ± 4.82

1.24- 4.96 12.05-34.33

IgG3 Relative

(g/l) %

0.77_+0.35 6.70 ± 2.25

0.30- 1.57 3.72-10.88

IgG4 Relative

(g/l) %

0.75±0.49 6.35 _+3.12

0.18- 2.01 1.99-15.10

a IgG subclasses of 41 healthy adults, age range 25-50 years. b Total IgG measured by Immunochemical Analyser (ICS) Becton Dickinson Palo Alto, CA), ELISA and by summation of IgG subclasses measured by ELISA.

technique could be utilized for measuring IgG subclasses in other body fluids such as CSF, tears etc. where concentration of these immunoglobulins would be small. Using the ELISA method we found the concentrations of IgG subclasses in sera of healthy adults to be in general agreement with reports by others who used either an electroimmunoassay (Oxelius, 1979) or RID (Shakib and Stanworth, 1980; French and Harrison, 1984). Although we were unable to demonstrate the relatively low IgG4 levels that others such as Oxelius (1979) and French and Harrison (1984) have observed in a proportion of the normal healthy subjects, it is possible that our small group of subjects (41) is not representative of the general population. It would appear that the higher mean IgG4 values obtained in our study compared with others can only be partly accounted for by the different techniques used, since using RID on our samples we obtained values relatively close to those obtained by ELISA; the RID values were slightly

T A B L E II IgG SUBCLASS SYNTHESIS BY P W M - S T I M U L A T E D L Y M P H O C Y T E S n g / m l m e a n ± SD Experiment 1

Experiment 2

Experiment 3

Experiment 4

Mean relative %

Resting a

IgG1 Relative %

62.7 ± 64.2±

0.4 0.3

62.3 ± 3.5 75.1± 1.9

49.7 + 3.5 61.7 ± 2.2

60.9 + 3.5 68.2 + 2.7

67.3 + 5.4

IgG2 Relative %

25.8+ 26.4±

0.9 0.6

15.1± 1.2 18.3 ± 2.0

2 1 . 6 ± 0.7 26.9 ± 1.9

19.7 + 3.7 22.0 ± 3.2

23.4 ± 4.1

IgG3 Relative %

6.1± 6.2 ±

0.3 0.3

3.5 ± 0.2 4.3 ± 0.05

5 . 7 ± 0.2 7.1 ± 0.1

5 . 3 ± 0.3 5.9 ± 0.2

5.9 + 1.0

IgG4 Relative %

3.0+ 3.1 +

0.05 0.01

1 . 9 ± 0.05 2.3 ± 0.1

3.5 + 0.2 4.3 ± 0.3

3 . 5 ± 0.05 3.9 ± 0.3

3.4 + 0.8

Stimulated a

IgG1 Relative %

1316.9 ± 173.2 79.7 ± 1.9

421.8 ± 29.1 79.3 ± 1.7

391.8 ± 33.5 7 1 . 0 ± 1.4

306.8 + 24.9 73.6 + 2.2

75.9 + 4.1

IgG2 Relative %

246.0 ± 15.0±

6.0 1.4

79.2 ± 2.6 15.0± 1.2

114.5 ± 19.8 20.6 ± 1.8

74.1 + 4.5 17.9 + 1.7

17.1 ___2.8

IgG3 Relative %

51.8 ± 3.2±

4.0 0.3

22.2± 1.6 4 . 2 ± 0.4

2 8 . 0 ± 1.6 5.1 ± 0.2

21.5 + 3.5 5.2 + 1.0

4.4 ± 1.0

IgG4 Relative %

34.0 ± 2.1 ±

3.8 0.3

8.2 ± 0.5 1.6 + 0.2

18.6 ± 0.1 3 . 4 + 0.3

13.6 4- 3.9 3.3 + 1.0

2.6 _ 0.9

a Levels synthesized in the presence (stimulated) or absence (resting) of PWM.

212 lower. Other factors such as different antibodies and calibrators, racial and aUotypic differences in subjects and geographical distribution (Shackelford et al., 1985) are also likely to have contributed to differences. French and Harrison (1984) observed I g G 4 concentrations which were significantly higher in males than females. Since our group consisted predominantly of females (27), the higher values observed b y us cannot be explained on the basis of having a higher p r o p o r t i o n of males in the group. The relative percentages of the four subclasses in sera was similar to that f o u n d in lymphocyte culture supernatants irrespective of whether the lymphocytes were stimulated with P W M .

Acknowledgements This work was supported in part b y a University Research G r a n t of the University of Adelaide.

W e are indebted to Barbara M a c G r e g o r for secretarial assistance.

References Beard, L.J., A. Ferrante, V.-A. Oxelius and G.M. Maxwell, 1986, Pediatr. Res., in press. Ferrante, A. and Y.H. Thong, 1982 J. Immunol. Methods 48, 81. Ferrante, A., B. Rowan-Kelly and J.C. Paton, 1984, Infect. Immun. 46, 585. French, M.A.H. and G. Harrison, 1984, Clin. Exp. Immunol. 56, 473. Heiner, D.C., 1984, Am. J. Med. 76, 1. Oxelius, V.-A., 1979, Acta Paediatr. Scand. 68, 23. Oxelius, V.A., 1984, Am. J. Med. 76, 7. Shackelford, P.G., D.M. Granoff, M.H. Nahm, M.G. Sc- t, B. Suarez, J.P. Pandey and S.J. Nelson, 1985, Pediatr. R~s. 19, 846. Shakib, F. and D.R. Stanworth, 1980, Ricerca Clin. Lab. 10, 561.