A method for titration of inhibiting antibodies to bacterial immunoglobulin A1 proteases in human serum and secretions

JOUSTS OF IMMU~OLOU~CAL METHODS ELSEVIER

Journal of Immunological

Methods

191 (1996) 39-48

A method for titration of inhibiting antibodies to bacterial immunoglobulin Al proteases in human serum and secretions Jesper Reinholdt * Royal Denral College. Faculty of Health Sciences, University of Aarhus, LX-8000 Aarhus C, Denmark

Received 5 September 1995; revised 27 November

1995; accepted

14 December

1995

Abstract Bacterial IgAl proteases specifically cleave IgAI, including S-IgAl, molecules into Fab, and Fc, fragments. Hereby these enzymes interfere with the protective functions of antibodies belonging to this isotype. Antibodies inhibiting IgAl proteases have been detected in humans, but the titration of such antibodies is a matter of metrological concern. Because human serum and secretions contain inherent IgAl substrate, it is impossibie to provide uniform substrate conditions for samples of IgAl protease incubated with inhibitors differing in their origin and state of dilution. This study demonstrates that such variations in substrate are not prohibitive for a reliable titration of inhibiting antibodies. This was evident from experiments demonstrating that the variations do not interfere with the quantification of residual IgAl protease activity provided the activity is measured in terms of the proportion of IgAl substrate cleaved during incubation. Proportions of cleaved IgAl were measured by exploiting the differential reactivity of cleaved and intact IgAl molecules in an ELISA using anti-Fc, and enzyme-conjugated anti-light chain antibodies for catching and development, respectively. A protocol for the titration of IgAl protease-inhibiting antibodies based on this ELISA is described. By application of the protocol to chromatographic fractions of saliva, IgAl protease-inhibiting activity was found to co-purify with salivary S-IgA, iueywor&: IgA protease; Enzyme immunoi~ibition;

Human serum; Secretion;

1. Introduction

of this, but not other immunoglobuli~ isotypes. Hereby the molecule is left as antigen-binding Fab, fragments devoid of the Fcu, or (Fc,),.SC, portion, which is particularly responsible for the protective properties of this important immune factor of mucosal surfaces (Kilian et al., 1988). Bacteria recognized as producers of IgAl protease include not only a number of pathogens, such as Haemophilus region

IgAl proteases are bacterial endopeptidases that specifically cleave human immunoglobulin A 1 (IgAl), including secretory IgAl (S-IgAl), within a particular amino acid sequence present in the hinge

Abbreviations: Ig, imm~oglobuIin; IgA, immunoglobuiin A; IgAl, immu~oglobu1~ A, subclass 1; S-IgA, secretory immunoglobul~ A; ELISA, enzyme-linked Immunoso~nt assay; OD. ootical dens&v: K- _Michaelis constant. * Tel.: f45 89421737; Fax: +45 86196128; e-mail, [email protected]. I

ELISA

r

L‘,

0022-1759/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved SSDI 0022- 1759(95)00286-3

infiztenzae, Neisseria meningihkfis, Neisseria gonorrhoeae, and Srreptococcus ~ne~rn#niae, but also sev-

eral bacteria that are considered indigenous to the oral cavity and upper airways. Among these are S. sanguis, S. oralis, and S. mitis biovar 1, which are of

40

J. Rrinholdt/Journnl

r~f‘lmr~runok~,pic~crlMrtttotl.~

relevance to dental disease because of their ability to initiate the formation of dental plaque (Nyvad and Kilian, 1987). Besides, IgAl protease-producing S. mitis biovar 1 in the pharynx of infants may play a role in atopic sensitization (Kilian et al., 1995). IgAl protease-producing bacteria of the oral flora also include species of relevance to periodontal disease (Kilian, 1981). Early studies of the occurrence, function, and potential significance of IgAl proteases have been the subject of reviews (Plaut, 1983; Mulks. 1985; Kilian and Reinholdt, 1986). The role of IgAl proteases in host-parasite relationships is not yet clear. As an important aspect, the significance of protease-inhibiting antibodies must be clarified. Antibodies inhibiting IgAl proteases of pathogens have been detected in serum as well as in purified colostral S-IgA (Gilbert et al., 1983; Kobayashi et al., 1987; Brooks et al., 1992; Devenyi et al., 1993). and may be induced in response to invasive infection or bacterial carriage (Brooks et al.. 1992). In the case of antibodies belonging to the IgAl isotype, the inhibiting capacity is maintained by Fab, fragments resulting from cleavage by IgAl protease (Gilbert et al., 1983; Kobayashi et al., 1987). Generally, studies of immune responses to IgAl proteases are complicated by the finding of a remarkable antigenic polymorphism of these enzymes within some taxonomic groupings of bacteria (Kilian et al., 1983; Lomholt and Kilian, 1994) and extensive cross-reactions within others (Reinholdt et al., 1990; Lomholt and Kilian, 1994) Several assays have been developed for the quantification of IgAl protease activity, any of which may, in principle, be used to demonstrate the effect of inhibiting antibodies. In these assays. human IgAl, preferably in the form of purified myeloma protein, serves as indicator substrate, the specific fragments being detected by electrophoretic and/or immunochemical means (Plaut et al., 1978; Reinholdt and Kilian, 1983; Mestecky and Kilian, 1985). However, titration of IgAl protease-inhibiting antibodies in serum or secretions of humans is a matter of particular methodological concern. The inherent IgAl of human inhibitor test samples will contribute to the indicator substrate of the assay. Therefore, the molecular form (monomeric or polymeric) and the concentration of indicator substrate will depend on the origin of the inhibitor as well as on the stage of

191

f 1996)

39-48

dilution at which it is tested. Conceivably. variations in indicator substrate could influence the performance of the protease assay and. hence, the outcome of the titration. In this study, using an assay for IgAl protease activity based on ELISA technique, we have addressed this issue and established a protocol for titration of protease-inhibiting antibodies in human serum and secretions.

2. Materials

and methods

2. I. Serum and secretions Samples of serum, saliva, and colostrum (4 days post partum) were collected from adult donors upon receipt of their informed consent. All but one of the donors were healthy with no recorded history of disease due to infection with IgAl protease-producing bacteria. whereas one was convalescing from meningitis due to S. pneumoniae. To minimize microbial contamination, saliva was sampled at the orifice of the submandibular/sublingual ducts using a transfer pipet or a commercial collection device (Omnisal, Saliva Diagnostic Systems, Vancouver, WA). From one subject, also parotid saliva was collected using a plastic cup (Legler et al., 1981). Sparse debris was removed by centrifugation at 10 000 X g for 10 min at 4°C. From samples of colostrum, S-IgA was purified as described (Ah1 and Reinholdt. 1991). Samples of serum, clarified saliva, and colostral S-IgA were stored at - 70°C until used. 2.2. Purification

of myeloma IgAl

The IgAl fraction of immunoglobulins in plasma from an IgAl myeloma patient was purified by chromatography of ammonium sulphate-precipitated immunoglobulins (Harboe and Ingild, 1983) on a column of Jacalin-coupled agarose according to the recommendations of the manufacturer (Vector Laboratories. Burlingame. CA). Because total IgA in this plasma was at a level of 30 mg/ml, roughly 95% of IgAl could be expected to be of myeloma origin. Monomeric and polymeric (mainly dimeric) molecular forms of the purified IgAl were separated by size-exclusion chromatography (Ah1 and Reinholdt, 1991).

J. Rrinholdi/Jourd

2.3. Preparation

and calibration

oJ’lmmunolo~icct1

of IgAl proteases

IgAl proteases from Haemophilus injluenzae (serotype b, strain HK 3681, Neisseria meningitidis (group B, nontypeable, strain HF 161), Streptococcus sanguis (ATCC 10556), Streptococcus orufis (ATCC 10557), and Streptococcus mitis biovar 1 (strain SK 135) were prepared in crude form by methods previously described (Mestecky and Kilian, 1985). IgAl protease from Streptococcus pneumoniae (serotype 7F) and from three additional strains of N. meningitidis were prepared in the form of Todd-Hewitt broth or Levinthal bouillon culture supematant, in the case of S. pneumoniae, using the isolate from the subject convalescing from meningitis. IgAl protease preparations could be stored for two month at - 70°C or, in the case of streptococcal proteases, at 4°C without detectable loss of activity (Reinholdt et al., unpublished data). The IgAl proteases were calibrated on the basis of activity. The following protocol was adopted on the basis of experiments, results of which are presented below. Serial two-fold dilutions (25 ~1) of proteases in 0.05 M phosphate-buffered saline (pH 7.4). containing 0.05% Tween 20 (PBST) were made, in duplicate, in microplates with V-shaped wells (Nunc, Roskilde, Denmark,), using a multi-channel pipet equipped with micro volume tips. Then, 25 ~1 of monomeric or polymeric myeloma IgAl. 20 pg/ml in PBST, were added to each well. Two controls were included, in which PBST substituted for IgAl substrate and protease, respectively. The plate was sealed and incubated for 6 h at 37°C. Subsequently, IgAl protease activity was arrested by adding 240 ~1 of PBST containing 0.01 M of bathocuproine disulphonate (PBSTB) (Kilian and Reinholdt, 1986) and the proportion of IgAl cleaved in each well was measured by a modification of an ELISA previously described (Reinholdt and Kilian, 1983). Briefly, 100 ~1 aliquots were transferred to ELISA wells in which mouse monoclonal antibody specific for Fc of human IgAl (IgGl isotype, stabilized with irrelevant protein; Nordic, Tilburg, Netherlands, code MAHu/IgAI /MAb) had been immobilized via a coating layer of rabbit antibody (whole antibody molecules or F(ab’),, see results section) specific for mouse immunoglobulin (Dako, Glostrup, Denmark, code 20259). The assay was

Methods

191 (1996) 39-48

41

developed with peroxidase-conjugated antibody to human light chains of the type (K or A) carried by the myeloma IgA 1 (Dako, code PO1 29 or PO1 30), hereby labelling Fab, retained in the wells as part of uncleaved or incompletely cleaved IgAl molecules. The conjugated antibody was used at saturating concentration (1 : lo3 for anti-K as well as for anti-h chain antibodies) determined by titration in homologous ELISA wells carrying intact myeloma IgAl. Color developed by incubation for approximately 10 min with a substrate of o-phenylenediamine (0.002 M) and H,O, (0.005%) in citric acid-phosphate buffer (pH 5.0), was measured as optical density (OD) at 492 nm. In this assay, increase in cleavage of IgAl results in decrease in OD. Based on dose-response curves obtained by fitting a four parameter asymmetrical, sigmoid model (Fig. P software package, Biosoft, Cambridge. UK), to plots of OD against protease dilution, all protease preparations were diluted to a level of activity causing 50% cleavage of IgAl under the conditions of the assay. For each preparation, the dilution required was calculated as the solution to the equation: F(x)=(Cl+C2)/2, where F(x) is the fitted regression function and Cl and C2 are mean ODs for control wells in which PBST substituted for IgAl substrate and protease, respectively (cfr. results section, Fig. 2). Based on this titration protocol, a unit of IgAl protease activity, termed C,,, was defined as the activity causing 50% cleavage of IgAl under the conditions of the assay. For each protease preparation, a C,, titer was defined as the dilution required for 25 ~1 to contain one C,, unit. 2.4. Inhibition

assay

Serial, two-fold dilutions (25 ~1) of the inhibitor in PBST were made in microtiter plates with Vshaped wells on crushed ice, starting at 1 : 5 with serum, 1 : 1 with saliva, and 0.2 mg/ml with colostral S-IgA. Dilutions were made in quadruplicate. Throughout the dilution series, two of the four wells then received 25 ~1 (one C,, unit) of calibrated IgA 1 protease (duplicate experimental wells), whereas two wells received 25 ~1 of PBST and served as duplicate, intact substrate controls corre-

42

J. Reinhold

/Journd

of Immunologicd

Methods

sponding to the individual dilutions of the inhibitor. Two additional wells, in which PBST substituted for inhibitor, received protease and were destined to measure the effect of uninhibited protease. After incubation for 1 h on ice, all wells received 10 ~1 of

2 -_)

2”, 25~1

25~1 = 1 C,, unit

protease,

(Experimental

PBST, 25~1 (Intact substrate controls)

wells) Incubate

Add substrate

39-48

a 20 pg/ml solution of myeloma IgAl (monomeric or polymeric) in PBST. This myeloma IgAl was added to make sure that all wells contained IgA1 substrate sufficient for a functional detection of potentially uninhibited IgAl protease irrespective of

Test inhibitor,

IgAl

191 (1996)

on ice, lh

IgAl,

incubate

20pg/ml,

10~1

6h, 37%

Add PBSTB,

240~1

Transfer lOO@ to ELISA wells precoated sequentially with: 1. Rabbit anti-mouse lg (DAKO, code 20259) F(ab), of this antibody, 1:2000, 2h 2. MAb anti-IgAl (Fc-specific) (Nordic, code MAHu/lgAl/MAb),

Incubate,

70ng/ml,

or

2h

2h or overnight

Incubate with peroxidase-conjugated rabbit anti-human light chains (IC+~) (DAKO, code PO129, I:1000 and DAKO, code PO130, 1:lOOO). 2h

Colour development,

Calculation Fig. 1. How chart presenting

OD reading

of Cl,, titer

the sequence of the individual

steps of the inhibition assay.

J. Reinholdt/Journal

of ImmunologicalMethods

the amount of IgAl (if any) contributed by the inhibitor. Then the plate was sealed and incubated at 37°C for 6 h. After incubation, all wells received 240 ~1 of PBSTB and percent cleavage of IgAl in experimental wells was measured by analysis of experimental well contents and corresponding controls in ELISA. The ELISA was identical to that used for the calibration of IgAl proteases, except the conjugate was a mixture of anti-K and anti-A chain antibodies, both at saturating concentration. Duplicate ELISA wells in which PBST substituted for sample were included in the plate format and used for blanking of the reader. The titer of the inhibitor was defined as the dilution (prior to mixing with protease) corresponding to 50% inhibition of IgAl protease activity and termed the CI,, (i.e. 50% cleavage inhibition) titer. After fitting regression functions (usually linear for controls and 1”’ order monoexponential decay with residual for experimentals) to plots of mean OD against dilution of inhibitor (Fig.P software package), the CI,, titer was calculated as the solution to the equation:

where F,(x) and F,(x) are the fitted regression functions for experimentals and controls, respectively, and C is the mean OD for protease-containing wells in which PBST substituted for inhibitor (cfr. results section, Fig. 3). Calculated titers of less than unity were recorded as zero. A flow chart with the individual steps of the inhibition assay is presented in Fig. 1. 2.5. Saliva fractionation

experiment

2 ml of an equal mixture of submandibular/sublingual and parotid saliva (mimicking uncontaminated, whole saliva) was subjected to size-exclusion chromatography on a column (1.6 X 50 cm) of Superose 6 (Pharmacia, Uppsala, Sweden) equilibrated in PBS and attached to a high performance liquid chromatography system (Pharmacia). 1 ml eluent fractions were collected at a flow rate of 0.5 ml/min, eluted proteins being monitored spectrofotometritally at 280 nm. Eluted, salivary S-IgA was detected by ELISA (Ah1 and Reinholdt, 1991). Briefly, eluent fractions diluted lOO-fold were incubated on plates coated with affinity purified rabbit antibody to hu-

191 (1996) 39-48

43

man (Y chains (Dako). The plate was developed with enzyme-conjugated rabbit antibody specific for secretory component (Dako). Fractions containing IgAl protease-inhibiting substances were identified by the inhibition assay described above. Briefly, 25 ~1 of each fraction were incubated for 1 h on ice with 25 ~1 of calibrated IgAl protease (experimental wells) and with 25 ~1 of PBST (control wells). Subsequently, all wells received 10 ~1 of myeloma IgAl at 20 pg/ml and were incubated at 37°C for 6 h prior to analysis by ELISA. In accordance with the principle of the inhibition assay, fractions containing a protease-inhibiting substance were identified by the presence of a peak in the OD profile for experimental wells relative to that for control wells.

3. Results 3.1. Significance of variations in the molecular form and concentration of indicator IgAl substrate The protocol for titration of IgAl protease-inhibiting activity is atypical in the sense that it accepts substantial variations in the indicator substrate. Thus, the molecular form and the concentration of indicator IgAl vary with the origin as well as with the dilution of the inhibitor. Experiments were done to examine whether these variations affect the quantitation of residual IgAl protease activity and, hence, the result of the titration. To examine the significance of the molecular form of the indicator substrate, IgAl proteases prepared from three taxonomitally distant bacteria, H. influenzae, N. meningitidis, and S. oralis, were titrated twice, using monomeric and polymeric IgAl substrate, respectively. For all three protease preparations, the titration curves obtained with the two types of substrate were closely similar in terms of OD for protease-digested relative to control substrate (Fig. 2). To examine whether differences in the concentration of indicator substrate might invalidate the quantification of residual IgAl protease activity, four protease preparations were titrated four times using IgAl substrate concentrations (prior to mixing with protease) ranging from 6.25 to 400 pg/ml. 400 pg/ml is the level of IgAl to be expected in reaction wells containing human serum diluted 1:5. For all four protease preparations,

44

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0

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Protease dilution

Fig. 2. Titration of IgA I protease preparations from H. injluencwe HK 368 (A), S. sunguis ATCC 10556 (+), and N. meningitidis HF 161 (W) using myeloma IgAl (0.4 mg/ml) of either monomeric (A) or polymeric (B) molecular form as substrate. Regression curves fitted to plots of OD (mean [range] of duplicates) against dilution of protease are shown. Cl and C2 are controls with buffer substituting for IgAl substrate and protease, respectively. For each type of substrate, calculated C,, titers are inserted.

the titration results obtained with the different substrate concentrations were similar, as reflected by calculated C,, titers (Table 1). 3.2. Interpretation

of titration data

ELISA data obtained by titration of serum and saliva of a healthy subject for inhibiting activity against four IgAl proteases are presented in Fig. 3. The almost constant level of OD for intact substrate controls can be expected to reflect saturation of the monoclonal anti-IgAl catching antibody by Fab-

bearing IgAl over the entire range of dilutions, including the controls in which buffer substituted for inhibitor. In the case of the S. sanguis protease, the proportion of IgAl cleaved in experimental wells was similar to the proportion cleaved in proteasecontaining wells devoid of inhibitor, roughly 50%, irrespective of the dilution of the inhibitor (Fig. 3). Considering that a similar, constant proportion of cleaved IgAl was observed with substrates of myeloma IgAl spanning the range of concentrations expected to be represented in reaction wells (Table 1). this pattern must be interpreted as reflecting absence of inhibiting activity. Conversely, the pattern obtained with other IgAl proteases, characterized by decreasing proportions of cleaved IgA with increasing concentration of the inhibitor (Fig. 31, was interpreted as reflecting an active inhibitor. In the case of saliva tested against the protease of H. influenzae. however, 50% inhibition of cleavage was not reached (Fig. 3B) and a titer of zero was recorded. By titration of some sera, OD for intact substrate controls were not at a constant level but increased significantly with increasing concentration of serum. Studies of this phenomenon revealed that such sera produced a titratable background response in ELISA wells coated with rabbit anti-mouse immunoglobulin antibody only, but not in wells coated with this antibody in the form of F(ab’), (Fig. 4). Furthermore, substitution of F(ab’), for the intact form of the rabbit antibody in the ELISA of the inhibition assay resulted in a constant level of OD for intact substrate controls (Fig. 4). These observations indicate that the phenomenon reflects the activity of

Table I IgAl protease of substrate

titration

experiments

with different

concentrations

Origin of IgA I protease

Substrate concentration 400

loo

25

6.25

H. injlwnxe N. meniqitidi.s S. sur2pi.s

13.8 43.9 44.6 48.6

15.7 47.0 41.7 49.3

15.5 52.1 44. I 50.0

15.4 48.4 45. I 51.3

s. pneumonicrr

( pg/ml)

a

a Four IgAl protease preparations were titrated four times using IgAl substrate concentrations (prior to mixing with protease) ranging from 6.25 to 400 pg/ml. Calculated C,, titers are recorded.

.I. Reinholdt /Journal

of Immunological

serum rheumatoid factors which, in spite of broad inter-species reactivity, do not recognize Fc of mouse IgGl (J.C. Jensenius, personal communication). The F(ab’), form of the rabbit antibody code 20259 is available from Dako on request and is recommended as substitute for 20259 in titrations of sera with this activity. Lack of inhibiting activity against the S. sang& IgAl protease, as previously reported for samples of

A

45

Methods 191 (1996) 39-48

ii I to.5 8 c-4

1

0.00~ 2560

Serum

Fig. 4. Background analysis for a serum producing a non-horizontal, intact substrate control curve in the ELISA of the inhibition assay. Intact substrate control curves obtained with rabbit antimouse immunoglobulin (Dako, code 202.59) (0) or F(ab’), of this antibody (0) as first layer are shown together with titration curves for the setum in wells coated with 20259 (0) or the F(ab’), form (W), only. Data for experimental wells are not included.

0.5E c ? _ 8

0.0 ’

,

10

I

I

40

160 Serum

I

i

640

2560

/A

C

dilution

E

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P

1.9

0.0’

C

dilution

n

,

l

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10.9

,

1

2

4

4

6

Saliva

1

16

I

32

I

64

I /A

126

C

dilution

Fig. 3. ELISA data obtained by parallel titration of serum (A) and saliva (B) of a healthy subject for inhibiting activity against IgAl protease from H. influenzae HK 368 (A), S. mitis biovar I SK 135 (V ), N. menin,gitidis HF 161 (W), and S. sanguis ATCC 10556 ( + ). Regression curves fitted to plots of OD (mean [range] of duplicates) against dilution of protease are shown. Solid symbols, experimental wells. Open circles, control wells in which buffer substituted for protease. C, protease-containing controls with buffer substituting for inhibitor. Calculated Clan titers against individual IgAl proteases are inserted.

normal serum and colostral S-IgA by Gilbert et al. (19831, was a frequent, however not a general finding. By analysis of samples from other subjects, titers against this protease up to 278.8 for serum and 3.1 for saliva were recorded (unpublished data). Hence, the theoretical possibility of the S. sanguis protease having an exclusive capacity to perturb the ELISA system was excluded. The IgAl proteases used as targets of inhibition in Fig. 3 did not originate from bacteria isolated from the donor of serum and saliva. Titration of serum and saliva of a subject convalescing from pneumococcal meningitis against the IgAl protease of the strain of S. pneumoniae isolated from blood in the acute phase of the disease demonstrated the capacity of the assay to identify high titers of inhibition in saliva as well as in serum (Fig. 5). 3.3. Reproducibility

of results

The variation in OD for duplicate reactions was small (Figs. l-3). To examine the reproducibility of inhibition titer estimates, twelve sera were titrated against one of three different IgAl proteases from N. meningitidis twice on different days, the second

time after recalibration of the proteases. The mean deviation of the highest relative to the lowest of duplicate CI,, titers was 45.9% (range, 3- II9%>, 100% co~es~nding to one doubling dilution. The mean deviation of the highest relative to the lowest of duplicate C,, titers for the three IgAl proteases was 69% (range, 42-l 14%) 3.4. Salivafractionation experiment IgAI proteases by colostral S-IgA

and inhibition of I

As an application of the assay, an attempt was made to characterize IgAl pro~ase-i~ibiting substances in saliva. Column eluent fractions of saliva from a healthy subject were examined for inhibiting activity against four IgAl proteases originating from H. injluenzae, N. meningitidis, S. oralis, and S. sang&, as well as for content of S-IgA, which is the vastly predomin~t immunoglobulin isotype in salivary gland secretions. For unfractionated saliva of this subject, a CI,, titer of zero against the IgAl protease of S. sanguis had been found. Accordingly, a peak of inhibiting activity against this protease was not identified (Fig. 6). Concerning the other three proteases, which were inhibited by the unfractionated

P

, r-l 8, l

923.6 140.0

I

0.0’

I

I

:

20 a

I

I

I

80 320 128 32 Inhibitor dilution

c

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1280 512

t

I/A

5120 2048

C C

Fig. 5. Titration of serum and saliva of a subject convalescing from pneumococca1 meningitis for inhibiting activity against IgAl protease of the strain of S pntwnotziae isolated from blood in the acute phase of the disease. Solid and open circles, OD (mean [range] of duplicates) of experimental and control wells, respectively, from titration of serum. Solid and open squares, same Eype of data from titration of saliva. Lower-mos! labels on the horizontal axis are for saliva. C, protease-containing controls with buffer substituting for inhibitor. Calculated CI,, titers are inserted.

o,.

. ...... .,.. ,.

s-b@

35

45

1 ..._.

“’

_.._. _,,.”

Il/‘,,L,/II/III/

25

:..

1

.,.’

55

65

75

85

95

:

1

rf

0.0

105c

Fig. 6. Identification of IgAl protease-inhibiting activity in sizeexclusion chromatography fractions of mixed saliva of the subject studied in Fig. 2. Protein profile (OD,,, nm, dotted line) is shown together with ELISA-OD profiles of eluent fractions incubated with myeloma IgAl substrate and either buffer (0) or calibrated IgAl protease from N. mmin~iticlis HF 161 ( n ) or S. ~crn~ui,s ATCC I0556 f +). C, proteas~-containing control with buffer substituting for eluent fraction. S-IDA-containing eluent fractions. as identified by specific ELISA. are indicated. Note that inhibiting activity against the N. Nzeningitidi.s IgAl protease co-elutes with S-IgA.

saliva at CI,, titers ranging from 8.3 to 16.5, inhibitory substances co-eluted with S-IgA as shown for the N. ~zenjn~j~~~js protease (Fig. 6). These results suggest that in normal saliva no substances other than S-IgA interfere with IgAl protease activity. However, the possibility remains that antibodies of other isotypes may mediate this activity in saliva of individuals with IgA deficiency or other conditions affecting the balance of immunoglobulin isotypes in saliva. As another application of the assay, we examined the IgAl protease-i~ibiting capacity of purified colostral S-IgA, as noted by others (Gilbert et al., 1983: Kobayashi et al., 1987). By titrating colostral S-IgA preparations (0.2 mg/ml) from two healthy donors against five IgAl proteases of different bacterial origin, the results were essentially as for samples of saliva in terms of patterns of curves and overall level of CI,, titers (results not shown).

4. Discussion The relationship of IgAl proteases to the immune system has been studied mainly by animal immu-

nization experiments (Kilian and Reinholdt, 1986; Lomholt and Kilian, 1994). Because IgA of animals, except hominoid primates, is not substrate to IgAl proteases (Kornfeld and Plain, 19811, such studies have not presented me~odolog~cal problems. Possibty for metrological reasons, only few data exist regarding the enzyme-specific immune response of humans in relation to infection with IgAl proteaseproducing bacteria (Brooks et al., 1992; Devenyi et al., 1993). In this study, a protocol has been developed for the titration of IgAl protease-inhibiting activity in human serum and secretions. The potential problems caused by the inherent IgAl of human samples has been previously mentioned by Gilbert et al. (1983). To titrate protease-inhibiting activity, they incubated serial dilutions of serum or purified, colostral S-IgA with a selected quantity of IgAI protease and then identified persisting protease activity, if present, by its effect on a small quantity of subsequently added, radiolabeled myeloma IgAl acting as indicator substrate. The authors speculated that this assay was at risk of overestimating i~ibiting activity because the inherent IgAl of the inhibitor might inhibit cleavage of indicator Ighl by a competitive mechanism independent of the activity of enzyme-neutralizing antibodies. As for the present assay, the observation that the proportion of IgAl cleaved by IgAl protease was unaffected by variation in the concentration of substrate within the range to be expected in reaction wells, makes this possibility irrelevant, with the possible exception of titration of sera abnormally rich in IgAl. In terms of enzyme kinetics, this property of the assay may reflect the fact that, by titration of normal serum or saliva, the IgAl protease operate at substrate concentrations well below the level of K, values calculated for IgAl proteases (approximately 1 mg/ml of monomeric IgAl) (Labib et al., 1978; Kilian and Reinholdt, 1986). Furthermore, it was shown that the proportion of IgAl cleaved by protease was independent of the molecular form of the substrate being monomeric or polymeric. This result corroborate previous observations (Plaut et al., 1985) that cy chains of IgAl molecules are cleaved in a non-cooperative way. The conclusion from these experiments that populations of IgAl molecules differing with respect to concentration and molecular form make equivalent

indicator substrates in the assay, has two implications of advantage. First, myeloma IgAl of arbitrary molecular form and of low concentration can be used for calibration of IgAl proteases. Secondly, comparison of titers measured for inhibitors that differ in the level and molecular form of inherent IgAl, such as serum and saliva, is justified. In spite of a low variation in OD for duplicate reactions analyzed on the same ELISA plate, a substantial day-to-day variation of inhibition titers was observed. considering that a similar variation was observed by duplicate titration of IgAl protease preparations, the limited reproducibility of inhibition titers may be explained partly as a result of inexact protease calibration. In support of this notion, the proportion of IgAl cleaved by calibrated proteases in the absence of an active i~ibitor varied signific~tly (Figs. 2 and 3). Taking into account the protease-diluting effect of the myeloma IgAl added in the inhibition assay, a proportion of cleaved IgAl slightly below 50% was the common effect expected for uninhibited proteases. Reaction volumes matching the wells of standard microtiter plates were used for reasons of convenience, low cost, and limited spending of human test samples. On the other hand, the adoption of the microplate format may be partly responsible for the limited reproducibility of titers. Even when performed by a skilled technician, serial two-fold dilutions of 25 ~1 volumes over ten or more steps may be unreliable. More laborious protocols for the calibration of proteases and the inhibition assay, involving serial dilution at a larger volume scale and subsequent transfer of microvolumes to reaction wells. can be expected to improve the accuracy of dilutions and, hence, the reproducibility of titers measured. Whereas scarce volumes of human test samples can make such protocols irrelevant, consumption of IgAl protease is not a problem. With most IgAl protease-producing bacteria, su~mat~t from a 10 ml overnight culture provide protease sufficient for a large number of tests. A considerable amount of indirect evidence exist to indicate that IgAl proteases of pathogenic bacteria are important virulence factors. The pronounced antigenie diversity of these enzymes (Kilian et al., 1983; Lomholt and Kilian, 1994) along with the response of the human host with inhibiting antibodies in serum

48

J. Rrinholdt/Journuf

c~j’lmmunologicai

and secretions, support this notion. The present assay may help clarify the significance of enzyme-inhibiting antibodies in the control of IgAl protease-producing bacteria by gritting studies of antibody responses in relation to bacterial colonization as well as development and remission of disease. Such studies are underway in our laboratory.

Acknowledgements This work was supported by the Danish Medical Research Council grant no. 12-1615. I am grateful to Mogens Kilian, Knud Poulsen, and Michael W. Russell for valuable discussions and critical review of the manuscript.

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