An ELISA for the detection of maternal anti-trophoblast antibodies in human pregnancy

Journal oflmmunologicalMethods, 77 (1985) 109-118

109

Elsevier JIM03392

An ELISA for the Detection of Maternal Anti-Trophoblast Antibodies in Human Pregnancy Martin Davies Reproductive Immunology Research Group, Department of Pathology, University of Bristol, Bristol BS8 1 TD, U.K.

(Received 13 August 1984, accepted 30 October 1984)

An ELISA has been developed which is able to detect antibodies directed against determinants present on the plasma membrane of the outer layer, the syncytiotrophoblast, of term placentae. The IgG and IgM anti-trophoblast antibodies are present in the sera of women during the course of a normal pregnancy. The ELISA was based on the use of syncytiotrophoblast plasma membranes as the antigenic targets and utilised a developing antiserum conjugated to the bacterial enzyme urease. Although the assay was simple and reproducible, its viability was dependent on several factors including the stability of the antibodies and the proper preparative techniques for the production of the plasma membranes. Failure to adhere to the correct procedures resulted in an inferior non-viable assay. Key words: E L I S A - anti-trophoblast antibodies - membrane vesicles - syncytiotrophoblast

Introduction D u r i n g the course of a n o r m a l h u m a n p r e g n a n c y the m o t h e r generates a n t i b o d y responses d i r e c t e d against p a t e r n a l l y derived alloantigens i n c l u d i n g the H L A a n d A B O systems ( W o o d r o w , 1970; W i n c h e s t e r et al., 1975; D o u g h t y a n d G e l s t h o r p e , 1976; Vives et al., 1976; T h o m p s o n , 1977). These responses are elicited following e x p o s u r e of the m o t h e r to an alloantigenic stimulus a p p a r e n t l y occurring either following a p l a c e n t a l h a e m o r r h a g e d u r i n g gestation a n d the s u b s e q u e n t transfer of cells or as a result of the t r a u m a t i c events s u r r o u n d i n g the process of p a r t u r i t i o n . However, since the outer layer o f the placenta, the s y n c y t i o t r o p h o b l a s t , which is in direct c o n t a c t with the m a t e r n a l circulation, is d e v o i d of intact H L A a n d A B O antigens (Szulman, 1973; G o t o et al., 1976; Davies et al., 1982a) the a l l o a n t i b o d y responses d o not a p p e a r to b e d i r e c t e d against the p l a c e n t a l barrier. In the c u r r e n t report, however, an E L I S A is described which is a b l e to detect the presence of m a t e r n a l I g M a n d I g G a n t i - t r o p h o b l a s t a n t i b o d i e s which interact with d e t e r m i n a n t s p r e s e n t on the p l a s m a m e m b r a n e of the s y n c y t i o t r o p h o b l a s t . A l t h o u g h the assay described is simple a n d r e p r o d u c i b l e , certain criteria have to b e satisfied in o r d e r to ensure success. 0022-1759/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

110 Materials and Methods

Preparation of placental plasma membrane vesicles Plasma membrane vesicles were prepared from the syncytiotrophoblast layer of fresh term placentae by the method of Smith et al. (1974) and described by Davies et al. (1981). The membrane vesicles were suspended in phosphate-buffered saline (PBS), pH 7.4, aliquoted and stored at - 2 0 ° C within 4 h of delivery. Each preparation was assigned a code number, e.g. 1805, 1707. Variations from this procedure and how they affected the ELISA will be discussed in the Results section.

Sera Maternal sera were prepared from routine blood samples obtained from pregnant women at various times throughout gestation. Normal sera were obtained from healthy volunteers who were either males or nulliparous non-pregnant females.

Enzyme-linked immunosorbent assay (ELISA ) Fifty microlitres of a placental plasma membrane vesicles suspension (50 /~g protein, ml-~) in 0.05 M carbonate-bicarbonate buffer, pH 9.6, containing 0.02% (w/v) sodium azide, were added to the wells of a microtitre plate (Linbro, Flow Laboratories, Scotland) and incubated at room temperature for 60 min. The unbound membrane vesicles were removed by aspiration and the wells treated with 0.1% glutaraldehyde in the carbonate-bicarbonate buffer at room temperature for 3 min. After thorough washing in 0.01 M PBS, pH 7.4, containing 0.05% (v/v) Tween 20, the wells were incubated at 37°C for 30 min with 50/xl of a serially diluted serum sample (test serum). After washing in PBS, each well was incubated at 37°C for 30 min with 50/~1 of a 1/100 diluted urease-conjugated sheep anti-human IgG or IgM antiserum (Sera-Lab, Crawley Down, England). Finally the wells were washed 3 times each with PBS and distilled water and incubated with 50 t~l of the urease substrate (weakly buffered urea solution containing the pH indicator bromocresol purple; Sera-Lab) at 37°C for 30-60 min. The development of the colour reaction was arrested by the addition of 20/~1 1% (w/v) thiomersal and the activity estimated either spectrophotometrically at 588 nm or by visual assessment. The end-point of the titration (titre) was defined as the lowest concentration of the test serum able to elicit a positive reaction after incubation at 37°C. A positive control which was an immunoglobulin preparation from a positive maternal serum, was incorporated into each assay to enable the comparison of data obtained at different times. In some experiments the plastic immobilised membranes were incubated for 30 min at 37°C with various amounts of isolated human IgG Fc fragments (Wellcome Reagents, England) before the addition of the test serum.

Determination of serum IgG concentrations Serum IgG concentration was estimated by single radial immunodiffusion (SRID) (Mancini et al., 1965) with reference to purified human IgG (Sigma Chemical Co., St. Louis, MO).

111

Absorption studies Two hundred microlitres serum were absorbed with either 108 erythrocytes (RBC) or 5 × 107 peripheral blood lymphocytes (PBL) or 120 ~tg protein of the placental plasma membrane vesicles, at room temperature for 60 min. The RBC and PBL populations were prepared from citrated whole blood using Lymphocyte Separation Medium (Flow Laboratories, U.K.). Statistics The correlation between serum IgG concentrations and anti-trophoblast antibody titres was determined and expressed as the Spearman rank correlation coefficient (r,).

Results

Practical application of the ELISA Sera from males and pregnant and non-pregnant females were screened for the presence of IgG and IgM anti-trophoblast antibodies. The pregnancy serum samples cited in Table I, selected from primigravid women in their first trimester were positive for both IgG and IgM antibodies using visual and spectrophotometric assessment. The IgG levels were greater than the IgM levels, although a strict comparison is not feasible due to the possible differential binding of the different antibody classes. The end-point of the titration, estimated visually was always positive by spectrophotometric measurement and in general the dilution after the end-point exhibited an absorption at 588 nm that was comparable to those obtained for the controls. For the screening of large numbers of samples the visual assessment method was adopted. In a preliminary study 113, sera obtained from primiparous women at various stages throughout pregnancy and 43 control sera were screened for the antitrophoblast antibody activity. The data (Table II) demonstrated that on a population basis pregnancy sera contained higher levels of activity than control sera, especially early in gestation. The IgG concentrations of some of the sera were estimated by SRID. There was no correlation between IgG anti-trophoblast antibody activity and serum IgG concentration either on a population basis (data presented in Table II) or on an individual basis (data not presented in detail: rs = +0.26). Further, preincubation of the target membranes with a commercial source of human IgG did not significantly affect the titration of the maternal sera (Table III). The commercial sample of IgG was devoid of significant levels of the anti-trophoblast antibody activity. The specificity of the anti-trophoblast antibody was determined by absorption of the sera with either erythrocytes (RBC), lymphocytes (PBL) or placental plasma membrane vesicles (Table IV) before performing the ELISA. The antibody activity was unaffected by absorption with either RBC or PBL but was significantly reduced by treatment with the placental plasma membrane. These initial experiments also demonstrated that the antibodies were not allospe-

112 TABLE 1 P R A C T I C A L A P P L I C A T I O N OF T H E ELISA Serum sample

IgM a

Maternal serum (MS 2848) b Maternal serum (MS 2844) Maternal serum (MS 2835) Normal male serum Normal female serum

IgG a

Esssnm

Titre

E588nm at 1 / 2

E588nm at end-point

Titre

E588nm at 1 / 2

E588nm at end-point

128 128 32 8 8

1.612 0.958 0.676 0.791 0.703

0.490 0.412 0.535 0.454 0.567

512 1024 256 32 16

1.806 1.277 1.419 0.800 0.967

0.423 0.484 0.523 0.503 0.567

Controls c Without membrane target Without test serum Without developing antiserum Substrate alone

0.381 0.379 0.336 0.254

The IgM or IgG anti-trophoblast antibody activity is represented either as the reciprocal of the lowest dilution to elicit a positive response (titre; visual assessment) or spectrophotometrically as the absorbance at 588 n m either at 1 / 2 dilution of the test serum or at the end-point. The activity is measured against membrane target code number 1805. b Maternal sera were from primigravidae at the 9th week of gestation. c The E588nm was estimated for controls which represented various incomplete assays. a

c i f i c SO t h a t a s i n g l e m e m b r a n e of women,

i.e., t h e p l a c e n t a

preparation

and the serum

could serve to screen sera from a variety sample

did not have to come

from

the

same individual.

T A B L E II ANALYSIS OF P R I M I P A R O U S P R E G N A N C Y SERA A N D C O N T R O L SERA BY T H E ELISA Serum source

Anti-trophoblast antibody titre a n

Pregnant, 5-12 weeks Pregnant, 20-27 weeks Pregnant, 33-40 weeks Normal male Normal female

IgM

IgG

Serum IgG content b

Mean+SEM

Range

Mean+SEM

Range

n

Mean_+SEM

Range

45

5.4+0.45

3-9

8.7+0.21

4-11

5

10.2+1.1

7-13.8

36

3.5 + 0.21

3-7

6.1 + 0.30

4-9

5

11.5 5-1.3

6.6-14

32 22 21

3.65-0.22 2.5+0.14 2.35-0.09

2-6 2-4 2-3

6.05-0.28 4.2+0.15 3.25-0.22

4-7 3-7 2-6

4 5 5

10.4±1.4 10.5+1.5 10.4+1.3

6-13 7-14 7-12.8

The antibody activity presented as titres (log2) was measured against membrane target code number 1805. b The IgG concentration (rag. m l - l ) in the sera was estimated by SRID. a

113 TABLE III EFFECT OF PREINCUBATION WITH HUMAN IgG AND ALBUMIN IgG anti-trophoblast antibody titre

Target membranes incubated with a

Assay buffer 400/~g albumin 400/~g IgG 200 #g IgG 50/~g IgG

Maternal serum absent

Maternal serum present

1/512 1/512 1/512 1/512 1/1024

1/16 1/32 1/64 1/64 1/32

The immobilised target membranes (code number 1805) were incubated with either albumin, IgG or the assay buffer at 37°C for 30 rnin before the assay proper. The preincubated targets were then incubated with a maternal serum source (first pregnancy, 9th week of gestation). a

TABLE IV SPECIFICITY OF THE ANTI-TROPHOBLAST ANTIBODY RESPONSE a Serum source

Unabsorbed

Pregnant, 9-12 weeks of gestation Normal female

RBC-absorbed

PBL-absorbed

PPMV-absorbed

IgM

IgG

IgM

IgG

IgM

IgG

IgM

IgG

5.0

7.4

4.8

7.4

5.0

7.4

3.0

4.0

3.0

5.0

3.0

5.0

3.0

4.8

3.0

4.0

a The IgM and IgG antibody titre (log2) measured against membrane target code number 1805 is represented as the means obtained from sera (n = 5) after absorption with either pooled erythrocytes (RBC), or pooled peripheral blood lymphocytes (PBL) or placental plasma membrane vesicles (PPMV).

TABLE V EFFECT OF ISOLATED Fc FRAGMENTS ON THE ELISA a Amount of Fc

Dilution of antiserum (MS 832809)

fragment (t'g)

1/8

1/32

1/128

1/256

No serum added

0 10 100 1000

1.36 1.29 1.24 1.30

1.07 1.08 1.10 1.13

0.43 0.37 0.31 0.55

0.29 0.29 0.25 0.27

0.18 0.29 0.27 0.25

a The binding of antibody was measured at E588nm after the membrane targets were preincubated with various amounts of isolated IgG Fc fragments.

114

The role of Fc receptors in the E L I S A Since the placental plasma membranes have been reported to possess active Fc receptors (FcR) for both IgG and IgM (Davies et al., 1981), experiments were set up to determine whether these FcR interferred with the estimation of antibody activity. The FcR on the target membranes were blocked by prior incubation with isolated Fc fragments which had no demonstrable effect on the subsequent titration of the antiserum (Table V). However, there was a slight increase in the background reading at 588 nm, from 0.18 to 0.27, which indicated that some Fc fragments had bound to the membranes and were recognised by the developing antiserum. This increase in background reading was not comparable with the maximal absorption obtained with the maternal serum and represented only 8% of this maximum value.

Effect of serum storage on the antibody titre The sera were stored at - 2 0 ° C and in general the antibody activity was estimated within 14-21 days of removal from the patient. By remeasuring the antibody activity up to 8 months after the initial estimation, it was found that the activity was stable for at least 6 months (Table V1). This data also provided evidence for the reproducibility and precision of the assay. For maternal sera (n = 34) over the initial 6 month period the mean difference from the original estimation (as a measure of reproducibility)iSEM (as a measure of precision) was 0.11 _+0.12, which represents less than a 'one-well' deviation. The positive antiserum control used in all assays, under the same conditions, produced values of 0.2 _+ 0.1.

Effect of the membrane preparation on the antibody titre Initial observations demonstrated that the membrane preparations stored at 20°C deteriorated and became unsuitable as targets in the ELISA with the loss of the ability to bind the anti-trophoblast antibodies (Table VII). A similar effect could be achieved if the membranes were incubated at temperatures of 4°C and above, and indicated that proper membrane handling was required. Hence the membranes had to be prepared and stored at - 2 0 ° C as soon after delivery as possible. The handling of the placenta, prior to membrane preparation, was also investi-

TABLE VI EFFECT OF STORAGEON ANTIBODYTITRE a Time of storage (months) 1-2 3-4 5-6 7-8

Variation in antiserum titre b 0,0, -1, +1,0,0,0, 0, 0, 0. +1, +1,0,0, +1, -1,0, -1, -1,0,0, +1,0,0, +1. -2,0,0,0,0, +1, +1, +1, +1. -3, -3, -2, -1, -3, -3,0,0, +1,0, -1.

a lgG anti-trophoblast antibody titres were measured in the ELISA within 20 days of venipuncture and then remeasured at various intervals followingstorage at -20°C. b The variation in titre was calculated as (t 2 - tl), where t1= the titre (log2) 20 days after venipuncture and t 2 = the titre after storage.

115 T A B L E VII T H E E F F E C T OF V A R I O U S T R E A T M E N T S OF T H E A N T I G E N T A R G E T ON T H E M E A S U R E M E N T OF T H E IgG RESPONSE Treatment of placenta a

Treatment of membrane targets b

IgG antibody titre Normal serum Maternal serum

None None None None None None None None None Incubation Incubation Incubation Incubation Incubation Incubation

None, fresh membranes Storage at - 2 0 ° C , 268 days Storage at - 20°C, 329 days Incubation 4 ° C / 5 h Incubation 4 ° C / 1 8 h Incubation room temperature/5 h Incubation room temperature/18 h Incubation 3 7 ° C / 5 h Incubation 3 7 ° C / 1 8 h 4°C/5 h None 4°C/18 h None room temperature/5 h None room temperature/18 h None 37°C/5 h None 37°C/18 h None

64 32 16 64 32 16 16 8 8 64 64 64 32 32 16

256 256 64 128 128 64 64 64 32 256 128 128 32 32 32

a Fresh term placentae were subjected to a variety of incubation conditions before the membrane vesicles were prepared. b Membrane vesicles, prepared from fresh term placentae within 4 h of delivery, were subjected to a variety of incubation conditions prior to the assay.

gated. Exposing the placenta to various incubation conditions had the effect of lowering the detectable antibody titre (Table VII). Hence for viable membrane targets attention to proper handling and storage conditions of the placentae and prepared membranes had to be observed. Further, the effect on the antibody titre of using freshly prepared membrane targets from different placentae was investigated. In all the experiments higher antibody levels could be detected in maternal sera (primigravid, first trimester) T A B L E VIII VARIABLE N A T U R E OF T H E P L A C E N T A L P L A S M A M E M B R A N E T A R G E T S Antibody source

Placental plasma membrane target (code number) 2709

1508

0307

1607

1707

2802

0510

0912

1805

Maternal s e r u m 1 2

64 64

64 128

128 128

128 128

128 128

256 128

256 256

512 512

512 1024

N o r m ~ serum 1 2

32 32

16 32

64 64

16 32

32 32

128 64

16 16

64 64

64 64

a

a Antibody titres were measured against a variety of plasma membrane targets which were prepared from fresh term placentae within 4 h of delivery.

116 compared with control sera (nulliparous, non-pregnant) regardless of the membrane target. However, from the representative data (Table VIII) the difference between the maternal and control sera titres and the absolute values of these titres was dependent on the membrane target used in the ELISA. Hence whereas some membrane preparations, e.g. 0912 and 1805, were suitable for the assay, other preparations, e.g. 2709 and 0307, were not ideal. Although the latter could be used in the ELISA, the difference between maternal and normal sera titres was so small that large numbers of sera would have to be screened in order to obtain statistically significant data. A large number of sera (18 maternal and 7 control) have been tested on this panel of membrane targets and the overall pattern of reactivity was the same as that determined for the representative samples (Table VIII). Since the membranes were prepared under identical conditions, the differential binding of the antitrophoblast antibody appeared to be an inherent property of the membranes. Hence for a successful ELISA, the membrane targets have to be prepared from fresh placentae, stored for a limited period and selected on the basis of their inherent binding properties.

Discussion

Although the potentially alloantigenic foeto-placental unit is tolerated in normal uncomplicated pregnancies, it is recognised by the maternal immune system which generates antibody responses to a variety of paternally derived alloantigenic systems (Woodrow, 1970; Winchester et al., 1975; Doughty and Gelsthorpe, 1976; Vives et al., 1976; Thompson, 1977). These responses do not appear to be directed against the placental barrier, whose outer layer, the syncytiotrophoblast, is in direct contact with the maternal circulation and is devoid of the alloantigenic systems (Szulman, 1973; Goto et al., 1976; Davies et al., 1982a). The absence of these alloantigens on the syncytiotrophoblast has been proposed as one of the protective mechanisms which ensure the survival of the foetus. However, there are antigen systems apparently present on the syncytiotrophoblast plasma membrane and these have been detected either by mouse monoclonal antibodies (Johnson et al., 1981; Sunderland et al., 1981) or by heterologous rabbit antisera (Faulk et al., 1979); whether the mother recognises these xenogeneically identified antigens is unknown. However, the ELISA currently described indicates that there are maternally recognised antigens present on the plasma membrane of this outer layer, which elicit the production of IgM and IgG antibodies during the course of normal pregnancies. The ELISA uses plasma membranes prepared from the syncytiotrophoblast of term placentae as the antigenic targets and attention to the proper preparation of the membranes is necessary for a viable assay. Firstly, the suitability of the membranes is achieved by utilising a developing antiserum which is conjugated to the bacterial enzyme urease, which is absent from mammalian tissue and hence ensures a low background to the assay. The main enzymes currently used in enzyme-linked assays are alkaline phosphatase and peroxidase which would produce unacceptably high background levels to the assay due to the presence of endogenous enzymes in the

117

membrane preparations. Secondly, although the membranes possess active Fc receptors for both IgG and IgM (Davies et al., 1981), they do not appear to interfere with the assay (Table V). Finally, the handling and storage of both the placentae and the prepared membranes is critical for the production of viable targets. The placentae have to be obtained fresh and the membranes prepared within 3-4 h of delivery. Failure to observe these procedures resulted in a decrease in the antibody binding capacity of the prepared membranes. This effect could be reproduced artificially by preincubating the fresh placentae before membrane preparation or by incubating the prepared membranes (Table VII). Although both these incubation procedures reduced the antibody binding capacity of the membranes, the mechanisms involved were thought to be different. Since the incubation of the fresh placentae, even at 4°C, resulted in an increased yield of the membrane fraction (unpublished observation), the decrease in binding capacity was interpreted as being due to contamination with non-binding cellular components other than plasma membrane. However, if the membranes were prepared in the correct manner and were either stored at - 20°C for a prolonged period or incubated at various temperatures to accelerate the process, then the decrease in binding capacity was possibly a result of endogenous proteolytic enzyme activity which denatured the antigenic structures on the plasma membrane. A similar storage effect of syncytiotrophoblast plasma membranes used in other assays has been previously reported (Davies et al., 1982b). Finally even under the proper preparative conditions, the membranes obtained from different placentae exhibited an apparent inherent variability in their antibody binding capacity, which resulted not only in different antibody titres which could be obtained for the same serum using the various targets, but also the differential titre between maternal serum and normal serum varied (Table VIII). Hence an antibody titre for a particular serum has to be defined by reference to the membrane target used in the ELISA. Further, for a practical assay, a large differential titre was desirable even though all the membranes tested demonstrated higher levels of the anti-trophoblast antibody in maternal sera (primigravid, first trimester) compared with the control sera (nulliparous, non-pregnant female). Therefore, the major factor contributing to a successful ELISA was the membrane target. However, a further consideration was the serum antibodies which were relatively stable at -20°C, but did show some evidence of loss of activity after a prolonged period of storage (Table VI). The main advantage of this ELISA is that a membrane target prepared from a single term placenta may be used to detect anti-trophoblast antibodies in first trimester sera and in sera obtained from a variety of individuals (Table II). By this technique, it is possible to determine the kinetics of anti-trophoblast antibody production in both normal and pathological pregnancies and to assess their biological relevance. Initial experiments have detected the anti-trophoblast antibodies in the maternal sera both in a 'free' form and in the form of immune complexes (M. Davies: unpublished observations). Hence the antibodies could function either as blocking factors or in the control of anti-foetal-placental responses to ensure foetal survival.

118

Acknowledgements This work was supported by a grant from the Medical Research Council. I wish to thank Mrs. C.M. Browne for her excellent technical assistance.

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