An immunoassay for the determination of total IgA subclass antibodies in human serum

Immunology Letters, 26 (1990) 3 7 - 4 4 Elsevier IMLET 01470

An immunoassay for the determination of total IgA subclass antibodies in human serum Mathias H a u n and Safia Wasi The Canadian Red Cross Society, National Reference Laboratory, Protein Chemistry Section, Ottawa, Ontario, Canada (Received 12 April 1990; accepted 23 May 1990)

1. Summary An enzyme immunoassay capable of determining total IgAl and IgA2 concentrations in human serum has been developed. Subclass-specific monoclonal antibodies are bound to polyacrylamide bead-conjugated anti-mouse immunoglobulin antibodies. Bound IgA is detected with an anti-IgA peroxidase conjugate and the standard curve is linear in the region 0.25- 2.0/zg/ml. Coefficient of variation values range from 0.24-5.77°7o for the IgAl standard curve and from 0.86-5.92°70 for the IgA2 standard curve. Inter-assay variation for the IgA1 and IgA2 control sample values were 8.20/o and 13.4°/o, respectively. 2. Introduction Human immunoglobulin A (IgA) consists of two subclasses, IgA1 and IgA2, as well as two allotypes, IgA2m(1) and IgA2m(2). IgA is present in serum primarily as a monomer and in secretions (saliva, tears, colostrum, etc.)as a dimer [1]. Circulating IgA is comprised of approximately 80-90% IgA1 and 10-20070 IgA2 in normal individuals. Serum IgA concentrations have been observed to change in a wide range of disease states [2]. In some populations the incidence of selective deficiency of only one of the two IgA subclasses is much higher than that of total IgA deficiency [3]. In Ig,~-deficient Key words: Immunoassay; Immunoglobulin A; Subclass Correspondence to." Mathias Haun, The Canadian Red Cross Society, National Reference Laboratory, Protein Chemistry Section, 1800 Alta Vista Drive, Ottawa, Ontario, Canada, KIG 4J5.

individuals serum IgA levels are known to fluctuate widely [4] and the ratios of IgAl and IgA2 have been shown to shift significantly [5]. More recently it has been shown that serum IgA levels in human immunodeficiency virus (HIV)-infected individuals increase with the progression of the disease [6]. Evaluation of the subclass coml,osition of the increased IgA levels may offer further insight into the immunoregulatory disorders associated with acquired immunodeficiency syndrome (AIDS). Previous attempts to measure concentrations of IgA subclasses have been hindered by the lack of suitable reagents. Polyclonal antibodies specific for IgA1 or IgA2 were often difficult to prepare due to the relatively minor structural differences between the subclasses. Extensive adsorption with myeloma proteins was required to ensure specificity of such antibody preparations [7]. The recent development of monoclonal antibodies (mAbs) specific for the IgA subclasses has greatly improved immunological detection systems. Where suitable subclass-specific antibodies have been prepared, assay systems utilized radioisotopes [5, 8] or coated red blood cells [3]. Radioimmunoassays (RIAs) require radiolabeling steps which may alter the immunological activity of antigens or antibodies. Hemagglutination inhibition assays require tedious red blood cell conjugation procedures and are more subjective. Enzyme immunoassays utilizing monoclonal antibodies have been developed which are able to quantitate antigen-specific IgA1 and IgA2 antibodies [9-12]. This paper describes enzyme immunoassays which measure IgA1 and IgA2 in serum utilizing subclass specific monoclonal antibodies linked to

0165-2478 / 90 / $ 3.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

37

rabbit anti-mouse immunoglobulin Immunobeads. The assays have a linear dose-response in the region between 0.25 and 2.0/zg/ml. With the exception of the standard proteins, all the reagents are commercially available. The assays obviate the use of radioisotopes and offer a simple and rapid approach to measuring IgA subclass concentrations in human serum samples. 3. Materials and Methods

3.1. Materials

Rabbit anti-mouse immunoglobulin Immunobeads were obtained from Bio-Rad Laboratories (Canada) (Mississauga, Ont.); monoclonal antiIgA1 and anti-lgA2 antibodies were from Nordic Immunologicals (Cedarlane Labs, Hornby, Ont.); horseradish peroxidase-conjugated goat anti-mouse IgG (Fc-specific) was obtained from Jackson Immunologicals (Bio/Can Scientific, Mississauga, Ont.); horseradish peroxidase-conjugated goat F(Ab')2 anti-human IgA was purchased from Tago Immunologicals (Inter Medico, Markham, Ont.). WHO Immunoglobulin reference (lot 67/86) was obtained from the World Health Organization International Laboratory for Biological Standards and Control (Potter's Bar, Herts., U.K.). CAP Reference Preparation for Serum Proteins (lot 3) was obtained from the College of American Pathologists (Skokie, IL). Behring Control Serum (lot 048817E) was purchased from Behring Diagnostics (Montreal, Que.). Jacalinagarose was obtained from Vector Labs (Dimension Labs, Mississauga, Ont.). D-Galactose, o-melibiose, RIA grade bovine serum albumin (BSA) and o-phenylenediamine dihydrochloride were purchased from Sigma Chemicals (St. Louis, MO). Dynatech Immulon 96-well microtitre plates were obtained from Dynatech Laboratories (Fisher Scientific, Ottawa, Ont.). All other reagents were of analytical grade. IgA-deficient sera were obtained from The Canadian Red Cross Society Blood Services Centres. 3.2. Evaluation o f monoclonal antibodies

Previously purified and characterized IgA myeloma proteins [13] were subjected to jacalin-agarose chromatography to remove any residual subclass im38

purities. This methodology has previously been described [14]. Briefly, IgA1 proteins were dialyzed against 20 mM D-galactose in Tris-buffered saline (175 mM Tris/150 mM NaC1, pH 6.8 (TBS)) and chromatographed on a jacalin-agarose affinity column equilibrated in the same buffer. Any unbound material was discarded and the bound protein was eluted with 200 mM melibiose in TBS and concentrated. IgA2m(1) and IgA2m(2) proteins were dialyzed against TBS and chromatographed on jacalinagarose. Unbound protein was collected and concentrated and bound material was eluted as above and discarded. Solutions (2/zg/ml) of each protein (IgA1, IgA2m(1), IgA2m(2)) were serially diluted (× ½) to 0.015/~g/ml in carbonate buffer (100 mM Na2CO 3, pH 9.6) and coated onto 96-well microtitre plates for 2 h at 37 °C. The plates were then washed 3 times with phosphate-buffered saline-Tween (30 mM phosphate/150 mM NaC1, pH 7.4/0.05°70 Tween (PBSTween)) and non-specific binding sites were blocked with 100 mM NHnC1 for 45 min at ambient temperatures. The plates were washed 3 times again and solutions of monoclonal antibodies (mAbs) (diluted 1/1000 in PBS-Tween) were added (0.200 ml/well). Monoclonal anti-IgA1 and anti-IgA2 antibodies were both incubated with each of the three IgA proteins coated. The plates were incubated for 1.5 h at 37 °C, washed 3 times with PBS-Tween and 0.200 ml goat anti-mouse IgG horseradish peroxidase conjugate (diluted 1/5000 in PBS-Tween) was added to each well. The plates were incubated for 1 h at 37 °C, washed 3 times and substrate (0.200 ml) added to each well (10 mg o-phenylenediamine dihydrochloride (OPD) in 50 ml 200 mM citrate-phosphate buffer, pH 5.5/0.05 ml 30070 hydrogen peroxide). Color development was quenced with 0.025 ml 6M HC1 after approximately 25 min and absorbance at 492 nm measured. 3.3. Antibody-bead binding curve To establish the optimal concentration of mouse monoclonal antibody for binding to the rabbit antimouse immunoglobulin Immunobeads, a mAb solution was serially diluted (× ½) from 8.0 ~zg/ml to • 0.007/zg/ml. Triplicate samples (0.100 ml) of each solution were mixed with 100 tzg of Immunobeads and 0.200 ml phosphate-buffered gelatin (150 mM

phosphate/l mM EDTA/0.1OT0 gelatin, pH 7.4 (PBG)) in 12 × 75 mm borosilicate tubes. The tubes were incubated at ambient temperature for 1 h and then centrifuged at 1500×g for l0 min (Beckman T J-6, TH-4 rotor). The supernatant was decanted and 3 ml PBG added to each tube. The tubes were vortexed and centrifuged again. This wash cycle was repeated once, followed by the addition of 3 ml of goat anti-mouse IgG horseradish peroxidase conjugate (1/10000 dilution). The tubes were incubated at ambient temperature for 1 h, followed by two wash cycles as described above and briefly drained onto filter paper. One ml of substrate solution (5 mg OPD in 25 ml 200 mM citrate-phosphate buffer, pH 5.5/0.025 ml 30°7o hydrogen peroxide) was added to each tube and vortexed. Color development was allowed to proceed for 3 min and then quenched with 0.100 ml 6M HCI. Aliquots (0.200 ml) from the tubes were transferred to blank microtiter plates to facilitate rapid spectrophotometric measurement at 492 nm on a Biomek 1000 Automated Workstation (Beckman Instruments, Mississauga, Ont.). Data analysis was performed with Immunofit EIA/RIA software (Beckman Instruments, Mississauga, Ont.).

IgA1 and IgA2 standards of 4.0/~g/ml were prepared in PBG and serially diluted (xV2) to 0.031 #g/ml. PBG (0.200 ml), mAb-beads (0.100 ml) and standard solution (0.100 ml) were mixed in 12×75 mm glass tubes, in triplicate. The tubes were incubated for 1 h at ambient temperature. The beads were washed twice with 3 ml volumes of PBG, as described above, and 3 ml of goat antihuman IgA (F(ab')2 fragment) horseradish peroxidase conjugate (diluted 1/8000 in PBG, 1% BSA) were added. The beads were re-suspended by vortexing and incubated for 1 h at ambient temperatures. The beads were washed twice again and briefly drained onto filter paper. One ml of substrate solution (10 mg OPD in 100 ml 200 mM citratephosphate buffer, pH 5.5/0.050 ml 30°7o hydrogen peroxide) was added to each tube and the color al20-

A

/°

15-

E

04

10-

05-

3.4. Preparation of lgAl and IgA2 standards IgA1 and IgA2 from normal human serum were purified as previously described [14]. Each subclass fraction was quantitated by an enzyme immunoassay [15], utilizing a World Health Organization immunoglobulin reference preparation as an internal control. 3.5. IgA subclass enzyme immunoassays These assays measure the amount of IgA1 or IgA2 which binds to subclass-specific monoclonal antibodies linked to rabbit anti-mouse immunoglobulin Immunobeads. The bead matrix was first prepared by incubating 4 /~g of either anti-IgA1 or antiIgA2 monoclonal antibody per mg of beads (bead suspension, 1 mg/ml) in a 16×100 mm glass tube for 1 h at 37 °C. The beads were then centrifuged, the buffer decanted and the beads re-suspended in l0 ml PBG. This wash cycle was repeated twice, as described above, and then re-suspended to a working concentration of 1 mg/ml in PBG.

i 0.10

001

i" 10

,ug/ml 20-

B

15-

g

10-

05-

o~ 0.01

~ i 0.10

10

pg/ml

Fig. 1. Determination of subclass specificity of monoclonal antibodies. Concentration of purified IgA myeloma protein ( o , IgA1; •, IgA2m(1); o, lgA2m(2)) coated onto microtitre plate versus absorbance at 492 nm. (A) Proteins incubated with antiIgAl mAb. (B) Proteins incubated with anti-IgA2 mAb. Each point is mean of triplicate values and blanks have been subtracted.

39

lowed to develop. The tubes comprising the IgA1 standard dilutions were quenched after 5.0 min while the IgA2 standards were quenched after 10.0 min with 0.100 ml 6M HC1. Aliquots (0.200 ml) from each tube were transferred to a blank microtiter plate and the absorbances at 492 nm measured as described above. 4. Results

4.1. Evaluation of monoclonal antibodies Figs. 1A and B indicate that the commercially available monoclonal antibodies to IgA1 and IgA2 were subclass-specific and did not cross-react to any significant extent. Furthermore, the ability of the anti-IgA2 mAb to recognize both IgA2m(1) and IgA2m(2) allotypes was confirmed (Fig. 1B). 4.2. Optimization of immunobead-monoclonal an-

prepared at a minimum of 4/~g/ml mouse IgG. 4.3. Standardization Utilizing the previously purified and quantitated normal IgA1 and IgA2 serum proteins, standard curves were generated for each subclass (Fig. 3). Graphs of protein concentration versus absorbance at 492 nm produced sigmoidal curves in the region between 0.015 izg/ml and 8.0 t~g/ml. These curves both displayed linearity between 0.25 ~g/ml and 2.0 #g/ml. Coefficient of variation values ranged from 0 . 2 4 - 5 . 7 7 % for the IgA1 standard curve and 0 . 8 6 - 5 . 9 2 % for the IgA2 standard curve (Table 1). Reference sera from WHO, C A P and Behring were assayed for the IgA1 and IgA2 content and the sum o f these two values compared to the stated total IgA concentration in the preparations. A human serum standard prepared in-house was also evaluated in this manner (Table 2).

tibody binding 4.4. Interference of serum proteins A graph of mouse IgG (mAb) concentration versus absorbance at 492 nm indicated that the amount of mAb bound to the rabbit rabbit anti-mouse immunoglobulin beads reached a m a x i m u m at approximately 4 # g / m l IgG (Fig. 2). The anti-IgA1 and anti-IgA2 mAbs behaved similarly and all Immunobeads utilized in the assays were subsequently

25-

°/e''/

IgA1 and IgA2 standards were normally prepared by dilution in PBG. To confirm that there were no components in serum that could interfere with the determination of IgA1 and IgA2 concentrations, standards were prepared in IgA-deficient serum. The serum utilized was diluted 1:100, and had been tested for anti-IgA antibodies [13] and quantitated to conf i r m an IgA level of less than 25 ng/ml [15]. The as-

/ 20-

/

cE 15-

? /

30-

/o-

/°~

2.0-

3

0

/

10lO.

/° 010

10 /Jg/ml

10

Fig. 2. Binding of mouse lgO to Immunobeads. Concentration of monoclonal antibody incubated with rabbit anti-mouse immunoglobulin beads versus absorbance at 492 nm. Each lboint is mean of triplicate values and blanks have been subtracted. 40

OOl

olb

,ug/ml

1.'o

~b

Fig. 3. IgAl and lgA2 standard curves. Concentration of IgA subclass standard versus absorbance at 492 nm. o, IgA1; 4, IgA2.Each point is mean of triplicate valuesand blanks havebeen subtracted (IgA1, A =0.047; IgA2, A =0.047).

TABLE 1 Coefficient of variation values for data in standard curve. Concentration of standard (#g/ml)

8.0 4.0 2~0 1.0 0.50 0.25 0.125 0.0625 0.03125 Blank

Absorbance 492 n m a

Coefficient of variation (%)

IgAl

IgA2

IgA1

lgA2

2.386 2.241 1.934 1.482 0.973 0.573 0.327 0.172 0.084 0.038

1.926 1.686 1.412 1.044 0.670 0.388 0.207 0.111 0.059 0.048

0.46 3.52 0.24 0.87 2.07 1.50 5.77 2.52 3.98 12.37

0.86 1.96 1.05 2.25 2.54 4.07 3.55 1.58 5.92 11.02

a Mean of triplicate values.

say values for the IgA1 and IgA2 in serum and PBG did not differ significantly, and the coefficients of variation for the values were similar (Table 3). 5. Discussion

Assays which are capable of quantitating IgA subclasses have previously been described. Several of these measure IgA1 or IgA2 antibodies directed at specific food, bacterial or viral antigens [10, 11, 12, 9] or measure the subclass distribution of plasma cells in tissues [16]. Those assays which have been used to measure total IgA1 and IgA2 have been radioimmunoassays [8, 5] or hemagglutination inhi-

bition assays [3]. Radioimmunoassays involve extra radiolabeling steps which may alter the biological activity of the protein, and also require long incubation periods. Hemagglutination inhibition assays necessitate red blood cell coating procedures and offer a more subjective methodology for quantitation. An ELISA which utilizes monoclonal antibodies specific for the IgA subclasses has been used to measure IgAl and IgA2 concentrations in cell culture supernatants as well as in serum [17]. The nature of the standards used in the assay was not indicated and the monoclonal antibodies were prepared in-house by the investigators. Polyacrylamide beads have been used to assay IgE [18] and low levels of IgA in human serum [15]. The assays described in this paper utilize commercially available beads to which rabbit anti-mouse immunoglobulin antibodies have been covalently linked. This allows any mAb preparation to be bound with relative ease, requiring only a 1 h incubation. The optimal level of mAb binding to the antimouse immunoglobulin was established initially by determining the saturation level of the beads (Fig. 2). This procedure should be carried out with every new lot of Immunobeads or mAb to maximize the amount of antibody bound per mg of beads while avoiding the waste of unbound reagent. MAbs from different commercial sources were compared with respect to their affinities for purified IgA1 and IgA2 protein standards. This was done subjectively on the basis of signal strength with a range of standard concentrations (results not

TABLE 2 Evaluation of assay utilizing IgA reference standards. IgA standard

Assayed concentrations a (mg/ml) IgA'l

Behring control serum C A P reference preparation W H O immunoglobulin reference H u m a n serum b

2.06 2.16 1.45 1.60

Ratio total IgA, (IgAl + IgA2) measured:actual IgA2

(2.74) (3.11) (2.98) (3.33)

0.19 0.20 0.17 0.16

(1.96) (2.41) (2.71) (4.00)

2.25:2.36 2.36:2.34 1.62:1.68 1.76:1.82

= = = =

0.953 1.008 0.964 0.967

a Mean of 25 values. Mean coefficient of variation values (%) from four separate determinations in parentheses, b Pooled sera from hundreds of blood donors. IgA level calculated as m e a n of multiple radial immunodiffusion determinations.

41

TABLE 3 Effect of components in serum on the measurement of IgA subclass concentrations. Concentration of IgA (#g/ml)

Absorbance a 492 nm

Coefficient of variation (%)

IgA1

lgA2

lgAl

lgA2

1.989 1.747 1.286 0.870 0.056

1.421 1.137 0.858 0.570 0.047

2.56 2.63 1.94 2.53 1.78

1.50 1.81 2.14 4.16 2.03

0.78 1.36 0.85 1.20 1.69

2.16 0.88 0.59 5.15 9.65

Phosphate buffer 2.0 1.0 0.50 0.25 Blank

lgA-deficient serum (1:100 in PBG) 2.0 1.0 0.50 0.25 Blank

1.907 1.687 1.291 0.836 0.059

1.396 1.091 0.830 0.539 0.052

a Mean of n = 4 values.

shown). It has previously been reported that IgA subclass-specific mAbs can vary widely in their affinities [19] and the evaluation performed in this study agrees with this observation. The mAbs which displayed a significantly higher affinity for the standards were studied further to confirm that the subclass specificity was satisfactory and also to verify that the anti-IgA2 mAb recognized both the A2m(1) and A2m(2) allotypes (Fig. 1). Further evaluation of the specificity of the mAbs was performed to examine the possibility that other components in serum could interact with the antigen-binding site of the molecules, either in a cross-reactive manner or by non-specific binding. Table 3 indicates that IgA1 and IgA2 standards prepared in 1:100 (minimum dilution of a normal serum sample under assay conditions) IgA-deficient serum did not assay significantly differently from standards prepared in PBG. The IgA1 and IgA2 standard proteins were purified from a pool of sera samples collected from normal, healthy blood donors. These standards most closely resemble the unknown proteins in serum samples by reflecting the heterogeneity of the IgA1 or IgA2 molecular population. It has been shown that purified myeloma proteins of the same subclass can be sufficiently distinctive from each other that 42

they may bias results if used as standards [9]. The IgA1 and IgA2 content of the reference standards employed to assess the accuracy of the assay are shown in Table 2. Although the concentrations of each subclass were not previously known, the sum of the two subclasses was within 5 % of the manufacturer's stated total IgA concentration. Coefficient of variation (CV) values for the standard curves ranged from 0 . 2 4 - 5 . 7 7 % for the IgA1 standards and from 0 . 8 6 - 5 . 9 2 % for the IgA2 standards. CV values for the blanks were higher, but this was not considered significant. Interassay variations for the measured IgA1 and IgA2 concentrations of control samples were 8.2% and 13.4%, respectively. The assays described in this paper measure total concentrations of IgA2 and IgA2 in serum, which are present in predominantly monomeric forms [20]. Preliminary studies with secretory IgA suggest that the ability of the monoclonal anti-IgA1 and antiIgA2 antibodies to bind to the dimeric form of IgA may be altered. Since sIgA consists of an IgA dimer linked by a J chain (molecular weight 15 600) and secretory component (molecular weight 72000) [21], the quaternary structure of the molecule is more complex than that of monomeric IgA. Although the J chain is thought to be buried under the secretory component [22] it is conceivable that the presence of these proteins in sIgA could sterically interfere with the ability of a subclass-specific mAb to bind to its epitope. It has already been shown that assay systems utilizing polyclonal anti-IgA antibodies cannot be used to accurately quantitate polymeric forms of IgA without calculating correction factors [23, 24]. Utilizing sIgA1 and sIgA2 as standards may permit more accurate measurement of dimeric IgA in secretions such as saliva or tears. This approach is currently being studied further. The methodology described above for measuring IgA1 and IgA2 in serum provides a simple and rapid alternative to other types of assays, and can be completed in 4 - 5 h. Converting centrifuge test tube holders to allow simultaneous decanting and mixing of all the tubes reduces this time considerably. Efforts were made to utilize as many commercially available reagents as possible. The IgA subclass standards used in this assay are prepared in-house, but once one of the reference controls has been adequately characterized for IgA1 and IgA2 content, it could be used to prepare standard solutions.

T h e assays d e s c r i b e d a b o v e are c u r r e n t l y b e i n g utilized to e x a m i n e t h e levels o f IgA1 a n d I g A 2 in b l o o d d o n o r s w h o s e total I g A c o n c e n t r a t i o n s are lower t h a n t h e n o r m a l r a n g e values. Similarly, q u a n t i t a t i o n o f IgA1 a n d I g A 2 i n s e r u m s a m p l e s f r o m d o n o r s w h o possess a n t i - I g A a n t i b o d i e s directed at o n l y o n e o f the two subclasses is also b e i n g u n d e r taken. F u t u r e a p p l i c a t i o n s o f the assays will i n c l u d e q u a n t i t a t i o n o f I g A subclasses in s e r u m s a m p l e s f r o m H I V - i n f e c t e d i n d i v i d u a l s . T h i s m a y lead to a n i m p r o v e d u n d e r s t a n d i n g o f the i m m u n o r e g u l a t o r y m e c h a n i s m s i n v o l v i n g I g A in this disease.

Acknowledgements T h e a u t h o r s w o u l d like to t h a n k Ms. D e b b i e A d a m s o n for excellent secretarial assistance.

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