67

Journal of Immunological Methods, 140 (1991) 67-78 © 1991 Elsevier Science Publishers B.V. 0022-1759/91/$03.50 ADONIS 002217599100194R

JIM 05949

ELISA procedures for the measurement of IgG subclass antibodies to bacterial antigens S. R u t h s a,3, P.C. Driedijk 1,3, R.S. W e e n i n g 2,3 a n d T.A. O u t 1,3 I Clinical Immunology Laboratory, and 2 Department of Pediatrics, Academic Medical Centre (.4 MC), 3 Laboratory for Experimental and Clinical Immunology (CLB), University of Amsterdam, Amsterdam, The Netherlands (Received 20 April 1990, revised received 17 January 1991, accepted 25 February 1991)

We have developed enzyme-linked immunosorbent assays (ELISA) of IgG subclass antibodies against whole bacteria and bacterial antigens using enzyme-labelled mouse monoclonal antibodies. The properties of different anti-subclass antibodies were compared. In sera from 18 healthy adults we measured the IgG subclass distribution of specific antibodies against Staphylococcus aureus and Haemophilus influenzae b and against distinct bacterial components: pneumococcal capsular polysaccharides, dextran and tetanus toxoid. We found that antibodies against protein (tetanus toxoid) were mainly IgG1, with some contribution of IgG4 and IgG2. Antibodies against polysaccharides (pneumococcal PS and dextran) and whole bacteria were restricted mainly to IgG1 and IgG2. Key words: ELISA, IgG subclass antibody; Monoclonal antibody; Staphylococcus aureus; Haemophilus influenzae b; Pneumococcal capsular polysaccharide; Dextran; Tetanus toxoid

Introduction

Patients with recurrent infections are more likely to have decreased concentrations of IgG subclasses in the blood, especially of IgG2 a n d / o r IgG4, than are healthy persons (Ugazio et al., 1983; Oxelius, 1984; Stanley et al., 1984; Freijd et al., 1985; Heiner, 1986; Shackelford et al., 1986). Lack of IgG2 was shown to be related to low concentrations of antibodies against polysac-

Correspondence to: T.A. Out, Clinical Immunology Laboratory, B 1 236, Academical Medical Centre, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. Abbreviations: Sta, Staphylococcus aureus; Hib, Haemophilus influenzae b; PS, polysaccharides; TT, formaldehyde inactivated tetanus toxin; MoAb, monoclonal antibody; SDB, sample dilution buffer; PBS, phosphate buffered saline; HRP, horseradish peroxidase.

charides (Siber et al., 1980), and this might explain the relation between IgG2 deficiency and clinical symptoms. However, a number of observations show that decreased concentrations of IgG2 are not strictly related to recurrent infections (Hammarstr/3m et al., 1983, 1987a; Out et al., 1986). Ideally it is desirable to measure antibodies against a range of bacterial antigens in order to obtain an insight into the immune competence of patients. This also permits a distinction to be made between specific antibody defects found in subjects with normal IgG subclass concentrations (Hammarstri3m and Smith, 1986; Ambrosino et al., 1987; Herrod et al., 1989) and IgG subclass deficiencies of a more general type (Hammarstr/3m et al., 1986, 1987a). The availability of monoclonal antibodies specific for the subclasses of human IgG has facilitated the development of the antibody de-

68

terminations and various methods have been described in a number of publications (Freijd et al., 1984; Persson et al., 1985; Barrett and Ayoub, 1986; Hussain et al., 1986; Ramadas et al., 1986; Kemeny et al., 1987). Most of these studies have been restricted to a single bacterial species or even a single antigen and there are only a few studies in which responses to several different antigens have been described. Here, we report a method for the determination of specific antibodies in the subclasses of IgG. For the purposes of the study we have chosen three bacterial antigens (pneumococcal capsular polysaccharides (Pneumovax), tetanus toxoid and dextran) and two freshly cultured bacteria (Staphylococcus aureus and Haemophilus influenzae b). Use of the latter permits a correlation of antibody activity against the intact bacteria with opsonization and phagocytosis of the same bacteria. To achieve short assay times we used enzyme-conjugated antibodies against the subclasses in order to avoid the additional step involving enzymelabelled anti-mouse reagents. The assays were used to measure IgG subclass antibodies in sera obtained from 18 healthy adults.

polysaccharides, dextran and TT) (for sources see below). Several IgG subclass antibodies bind to Sta protein A via their Fc region (Kronvall and Williams, 1969). To avoid this kind of binding we used a protein A-deficient Sta, namely, strain Wood (270581). Encapsulated Hib (strain 760705) had been passed through rats to ensure virulence. Sta was cultured in nutrient broth, and Hib in brain heart infusion broth that contained hematin and N A D ÷. The bacteria were cultured for 16 h at 37 ° C on a gyrating shaker (100 rpm). Exponential phase cells were collected by centrifugation at 3000 rpm for 5 min, washed three times and resuspended in coating buffer. Polyvalent pneumococcal capsular polysaccharide vaccine (Pneumovax 23, Merck Sharp and Dohme, Haarlem, The Netherlands) containing serotypes 1, 2, 3, 4, 5, 8, 9, 12, 14, 17, 19, 20, 22, 23, 26, 34, 43, 51, 54, 56, 57, 68 and 70 (American nomenclature) was used. 1 ml of the vaccine solution contains 50 /~g of each polysaccharide. Other antigens were dextran (Dextran T 500, Pharmacia, Uppsala, Sweden), T T (RIVM, Bilthoven, The Netherlands) and bovine serum albumin (BSA, lot number 650800, Organon Teknika, The Netherlands). Monoclonal antibodies and antisera

Materials and methods A n ligens

The antigens used were complete bacteria (Sta and Hib) provided by Dr. L. Van Alphen (Department of Microbiology, AMC), and distinct bacterial components (pneumococcal capsular

Mouse monoclonal antibodies (MoAbs) against human IgG subclasses conjugated with horseradish peroxidase were obtained from the Central Laboratory of the Dutch Red Cross Blood Transfusion Service (CLB). MoAbs with the following codenumbers were used: anti-IgG1, M H 161-l-E; anti-IgG2, MH 162-1-E (HP 6014); anti-IgG3,

TABLE 1 C O N D I T I O N S F O R ELISAS O F A N T I B A C T E R I A L A N T I B O D I E S Antigen

Sta

Coating concentration Coating volume Coating temperature Serum HRP-anti-lgG1,3,4

107/ml 5 × 107/ml 150/.tl/w b 150 / d / w RT c RT 2 ~l/w 5/xl/w 50 n g / w 50 n g / w 2 h incubation time for all antigens 100 n g / w 250 n g / w 2h overnight

HRP-anti-IgG2 a Pneumococcal PS. b Well. c R o o m temperature.

Hib

TT

PPS a

Dextran

1.5 L f / m l 150 / d / w 4oC 5/xl/w 50 n g / w

50/tg/ml 150 ~ l / w 4 oC 5 p.1/w 50 n g / w

10 ~ g / m l 150/d/w 4°C 5 ~l/w 50 n g / w

250 n g / w overnight

250 n g / w overnight

250 n g / w overnight

69 M H 163-1-E (HP 6095); anti-IgG4, M H 164-4-E (for concentrations see Table I). The specificity of these antibodies has been reported by Vlug et al. (1989). The following MoAbs were obtained from the W H O / I U I S Immunoglobulin Subcommittee ( W H O / I U I S 1987): anti-IgG1, HP 6012 and antiIgG4, HP 6011. Anti-IgG2, HP 6008 (GOM-1), was obtained from Oxoid, Hampshire, England. HP 6012, 6014, 6095, 6011 and 6008 refer to codes used by Jefferis et al. (1985) and Phillips et al. (1988). HRP-anti-human IgG (P 214, Dako, Denmark) and HRP-anti-mouse immunoglobulins (GM 17-01-E, CLB, The Netherlands) were used for the detection of human and murine IgG, respectively. Human lgG subclass proteins Human IgG1 (clone 151) and IgG3 (clone 1507), specific for T T were obtained from R.F. Tiebout and E.A.M. Stricker (CLB, Amsterdam, The Netherlands). Purified human myeloma proteins were kindly provided by Dr. A. Vlug (CLB): IgG1, (2802), IgG2, x (4691), IgG2, X (1544), IgG3, x (099292) and IgG4, x (050505). Purified human polyclonal IgG was also used. ELISA procedures Freshly cultured bacteria were coated onto microtiter plates (Immunolon M129A, Greiner, Switzerland) for 2 h at room temperature immediately before the antibody assay. The other bacterial antigens were coated for 24 h at 4 o C. All antigens were diluted in 0.05 M N a H C O 3, p H 9.6, to the concentrations shown in Table I. The coating volume was 150/~1 for all antigens. Washings were carried out first with phosphate-buffered saline (PBS) containing 0.05% T w e e n 20 (PBS/Tween), followed by three washings with PBS. Free sites were blocked with PBS containing 2% ( w / v ) BSA, 10 mM EDTA and 0.1 mM phenol, p H 7.2-7.4 (sample dilution buffer, SDB, 150 /~l/well) for 30 min at room temperature. Serial dilutions of sera in SDB were incubated for 1 or 2 h at 37 o C (1130/xl/well, in duplicate). After washing, the MoAbs to each of the subclasses were added (100 /~l/well) at optimal dilutions as determined by earlier titration. MoAbs were diluted in PBS containing 0.1% ( w / v ) gelatin and 0.02% ( w / v ) Tween 20, pH 7.3. The plates were left for 2

h at 37°C. After washing, 100 ~tl of substrate buffer were added: 0.11 M acetic acid, 0.01% ( w / v ) tetramethylbenzidine, 1% dimethylsulfoxide and 0.003% H202, pH 5.4. The reaction was stopped after a standardized time by adding 100 /~1 2 M H2SO 4. Absorbance values were read in a Titertek Multiscan MC at 450 nm. Radioimmunoassay of antibodies to tetanus toxoid Tetanus toxoid (TT) was labeled with 1251 as described by Aalberse et al. (1983). The specific activity obtained was 0.28 MBq//~g protein. The immune reactivity was higher than 70%. Mouse MoAb anti-IgG (MH 16 01, 500 /zg), anti-IgG1 and anti-IgG3 (500/~g each; as above but unconjugated) were coupled to 100 mg of CNBractivated Sepharose 4B (Pharmacia, Sweden). The Sepharose was suspended in SDB: 0.5 m g / m l for anti-IgG and anti-IgG1 and 1.0 m g / m l for antiIgG3. Serial dilutions of sera and of human monoclonal IgG1 and IgG3 anti-TT were incubated with 0.5 ml of that suspension for 16 h. After a wash lzSI-labeled TT (168 Bq/test) was added in SDB containing in addition 0.05% ( w / v ) Tween 20. After 16 h of incubation the beads were washed again and the activity bound was measured with a Packard Crystal III (Packard, The Netherlands). The amounts of antibody in the sera were expressed relative to human monoclonal IgG1 antiTT. Sera We measured specific IgG subclass antibodies in sera from 18 healthy adults (laboratory personnel, 22-58 years of age) and in two dextran responders (Hedin and Richter, 1982). At least two different dilutions of sera were used for calculation of the antibody content. Controls and standards Serial dilutions of purified polyclonal human IgG were coated onto microtiter plates and used as a positive control for the MoAbs. Coated antigen, without serum incubation, was used as a blank. As a further control several sera were preincubated with antigen to establish the specificity of the binding to the ELISA plates. For the differentiation between positive and negative sera we chose as a cut off level the mean absorbance of the blank plus twice the SD. N o coated antigen but

70 only blocking with SDB, followed by serum incubation and thereafter the MoAbs, was used as negative control for the antigenic specificity of the serum antibodies. AS antibodies against BSA may be present in some human sera (Husby et al., 1985), all samples were screened for IgG antibodies against coated SDB by comparing the response of serum samples diluted in PBS containing human serum albumin (HSA) with the response of the same samples diluted in SDB. It appeared that dilution of the samples in SDB completely inhibited any activity of anti-BSA. We used serial dilutions of several donor sera with a high content of specific antibodies as standards. In this way information about relative amounts of the specific antibodies was obtained. Specific IgG1 and IgG3 antibodies against TT could be quantified with human monoclonal IgG1 anti-TT (151) and IgG3 anti-TT (1507), respectively as standards (Tiebout et al., 1984, 1985; Hammarstr/3m et al., 1987b).

ABSORBANCE A

1.5 1.o

loI

o.s

0.5

.o ,o

q %'.5

The subclass specificity of the MoAbs used (MH 161-l-E, MH 162-1-E, MH 163-1-E and MH 164-4-E) was tested in various ways under the particular conditions of our assays. (1) Binding of MoAbs to human IgG subclasses (Figs. 1 A - 1 D ) . When testing the specificity of anti-IgG1 and anti-IgG3 the microtiter plates were coated with TT (1.5 L f / m l ) and serial dilutions of human monoclonal IgG1 (clone 151) or IgG3 (clone 1507) anti-TT were added. When testing the specificity of anti-IgG2 and anti-IgG4, the plates were coated with serial dilutions of IgG2 and IgG4 myeloma proteins. Each MoAb was incubated with each of the four subclasses of IgG in separate plates. Figs. 1A-1D demonstrate the responses of the anti-subclass reagents using increasing amounts of antibody or myeloma protein. Using all subclasses except IgG2 a high ELISA signal was obtained with about 10 ng subclass protein per well. With M H 162-1-E, M H 163-1-E and MH 164-4-E high absorbances were obtained with the corresponding IgG subclass,

~

D

MH 161-1-E MH 162-1-E MH 163-1-E MH 164-4-E

j,. i

4o

IgG1 (ng/w•ll)

iu

IgG2 (ng/welll

A

B 1.5

1.0

1.0

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0.5

o~o'.1

Specific reactions of MoAbs

2;5

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)I

Results

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1.5~

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,

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loo

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Fig. 1. Binding of MoAbs to human IgG subclasses. For anti-lgG1 and anti-IgG3 assays the plates were coated with TT followed by incubation with human IgG1 (A) and IgG3 (C) anti-TT, respectively. For IgG2 and IgG4 assays the plates were coated with myeloma IgG2 (B) and IgG4 (D), respectively. The incubations with HRP-MoAbswere as indicated in the figure. In B the test for HRP-anti-IgG1 was performed after coating the plate with anti-lgG2 (MH 162 01, 5/xg/ml), the addition of myelomaIgG2 at 10 ~g/ml and washing. In D the tests for HRP-anti-IgG2 and HRP-anti-IgG3 were performed at a coating concentration of myeloma lgG4 at 1 #tg/ml. whereas the binding onto the other subclasses did not exceed the blanks. The signal obtained with anti-IgG1 (MH-161-1-E) on IgG4 myeloma protein was due to small amounts of IgG1 present in the IgG4 preparation. This binding was inhibited by incubation of M H 161-1-E together with IgG1 (data not shown), but not with IgG4. (2) Inhibition assays. The binding of MoAbs to their corresponding human IgG subclass proteins was inhibited by an excess of that subclass protein only. Figs. 2A and 2B show representative experiments for M H 161-1-E and M H 164-4-E, respectively. The plates were coated with TT, followed by incubation with human IgG1 anti-TT, or with

71

myeloma IgG4. The MoAbs were added together with different amounts of the four IgG subclass proteins. Fig. 2 shows that the binding of M H 161-1-E was completely inhibited by IgG1 but not by other subclass proteins. The binding of M H 164-4-E was inhibited exclusively by IgG4. (3) Comparison of results with different MoAbs. Specific reactions of M H 162-1-E and M H 163-1-E have been demonstrated in the W H O study (Jefferis et al., 1985). MH 161-1-E and M H 164-4-E were not tested on that occasion. We compared the detection properties of these antibodies with those of reference MoAbs having widely accepted subclass specificity. M H 161-1-E was compared to HP 6012 (specific for IgG1) and M H 164-4-E was compared to HP 6011 (specific for IgG4) as shown in Figs. 3A and 3B. A close relationship between the IgG1 antibodies detected by M H 161-1-E (abscissa) and HP 6012 (ordinate) and also between the IgG4 antibodies detected by M H 164-4-E and HP 6011, respectively, was found (the Pearsson coefficients of correlation were both: 0.99). Fig. 3C shows the comparison between M H 162-1-E and HP 6008 (GOM-1) in the detection of IgG2 anti-dextran. In most sera the result was independent of the MoAb used although the response by HP 6008 was sometimes low (cf., Weetman et al., 1989). Differences between the two antibodies varied when other sera were used as a standard.

A

//

i

I

We have employed assay conditions (e.g., the concentration of the MoAbs, and the reaction time for HRP) such that the same absorbance value in the ELISA represents approximately similar amounts of IgG antibody for each subclass ('equipotency' of the anti-subclass reagents). The amount of IgG antibody was estimated using polyclonal anti-IgG and Fig. 4 shows a representative experiment. Plates were coated with TT, followed by the incubation with different amounts of human IgG1 (clone 151) or IgG3 (clone 1507) anti-TT; other plates were coated with various amounts of myeloma IgG2 or IgG4. The concentrations of added antibodies and of the coated subclass proteins varied from high to low, corresponding with numbers 1-5 in the figure. Each subclass protein was incubated with the subclass specific MoAb and in a separate experiment the proteins were incubated with anti-IgG. As the enzymatic reaction time is critical for the final absorbance, reaction times were standardized for each antibody in each assay. The reaction time of the HRP-anti-IgG was equal in all assays. Fig. 4 shows the results for all four IgG subclasses. The abscissa gives the results after incubation with anti-IgG and the ordinate those after incubation with the different subclass specific MoAbs. With regard to IgG3, at a particular amount of bound IgG3 anti-TT similiar absorbances were obtained

b

o" c

g

Reactivity of MoAbs

D

1.0

i

B

1¸ _

.el