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Clinical and Vaccine Immunology, March 2006, p. 380-386, Vol. 13, No. 3
1071-412X/06/$08.00+0     doi:10.1128/CVI.13.3.380-386.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Humoral Immune Response to Primary Rubella Virus Infection

Kim M. Wilson,* Carlie Di Camillo, Larissa Doughty, and Elizabeth M. Dax

National Serology Reference Laboratory, St. Vincent's Institute of Medical Research, Melbourne, Australia

Received 14 November 2005/ Returned for modification 22 December 2005/ Accepted 14 January 2006


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
An assay capable of distinguishing between the immune response generated by recent exposure to rubella virus and the immune response existing as a result of past exposure or immunization is required for the diagnosis of primary rubella virus infection, especially in pregnant women. Avidity assays, which are based on the premise that chaotropic agents can be used to selectively dissociate the low-avidity antibodies generated early in the course of infection, have become routinely used in an effort to accomplish this. We have thoroughly investigated the immunological basis of an avidity assay using a viral lysate-based assay and an enzyme-linked immunosorbent assay (ELISA) based on a peptide analogue of the putative immunodominant region of the E1 glycoprotein (E1208-239). The relative affinities of the antibodies directed against E1208-239 were measured by surface plasmon resonance and were found to correlate well with the avidity index calculated from the ELISA results. We found that the immune response generated during primary rubella virus infection consists of an initial low-affinity peak of immunoglobulin M (IgM) reactivity followed by transient peaks of low-avidity IgG3 and IgA reactivity. The predominant response is an IgG1 response which increases in concentration and affinity progressively over the course of infection. Incubation with the chaotropic agent used in the avidity assay abolished the detection of the early low-affinity peaks of IgM, IgA, and IgG3 reactivity while leaving the high-affinity IgG1 response relatively unaffected. The present study supported the premise that avidity assays based on appropriate antigens can be useful to confirm primary rubella virus infection.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Rubella virus is a positive-sense, single-stranded RNA virus belonging to the Togaviridae family in the genus Rubivirus (17). Infection with rubella virus usually results in a mild disease that only rarely produces significant sequelae. However, primary rubella virus infection during the first trimester of pregnancy may result in the transmission of virus through the placenta and infection of the fetus. This may in turn result in congenital rubella syndrome (CRS), the most common manifestations of which are blindness, mental retardation, and deafness (15). The risk of the fetus developing CRS is 40 to 60% if infection occurs during first two months of pregnancy, 35 to 40% if it occurs in the third month of pregnancy, and 10% if it occurs in the fourth month of pregnancy (19).

Rubella virus infection occurs worldwide, with a seasonal peak of infections in spring in temperate climates. Globally, only 57% of countries have rubella vaccination programs, and it is estimated that more than 100,000 cases of CRS occur each year in developing countries (27).

The humoral immune response to infection commences with the production of low-affinity immunoglobulin M (IgM) molecules. Class switch recombination then results in the generation of antibody isotypes with the appropriate effector function to eliminate the invading organism. Concurrently, somatic hypermutation of the complementarity-determining regions results in the generation of antibodies of progressively increasing affinity (11).

Classically, serological diagnosis of primary rubella virus infection has relied on the detection of rubella virus-specific IgM (1) or the demonstration of a fourfold increase in the IgG titer to rubella virus antigens in sera that are taken sequentially and assayed in pairs (5). These diagnostic methods are subject to false-positive results due to nonspecific IgM reactivity (1, 5, 7, 23, 25, 28) and false-negative results when the initial sample is obtained too late to demonstrate an increasing IgG titer (5, 9). Consequently, there has been increasing interest in the use of avidity assays to distinguish recent rubella virus infection from immune responses occurring later in the course of infection (4, 10, 18, 22).

The avidity of an antibody is a combination of the number of available binding sites and the strength (affinity) with which each individual binding site can bind the antigen. Avidity assays are generally based on commercially available enzyme-linked immunosorbent assays (ELISAs) modified by the addition of a chaotropic agent (e.g., 8 M urea [18] or 100 mM diethylamine [22]) following sample application. It is assumed that this treatment will cause the dissociation of low-affinity antibody-antigen interactions, while high-affinity antibodies remain bound. The immunological interactions upon which avidity assays are based have not been thoroughly investigated, and little effort has been directed towards standardizing the results generated by the different methods. Despite the availability of commercial avidity assays for the detection of rubella virus-specific antibodies (Enzynost; Dade-Behring), many laboratories simply modify commercial ELISAs, thus developing in-house assays. Difficulties in interpreting the results can arise if careful attention is not paid to all of the variables inherent in each assay, and contradictory results have been observed with avidity assays for other viral infections, depending upon which chaotrope is used and upon which antigens the assays are based (2, 3, 24). Factors affecting the results include variations in antibody specificity, as well as changes in titer, affinity, and antibody isotype distribution at different time points during the immune response to infection.

In this study, maturation of the humoral immune response to rubella virus was followed for recently infected individuals by use of antibody isotype-specific viral lysate-based ELISAs and avidity assays. In addition, antibody isotype and avidity data generated using the putative immunodominant peptide from the E1 glycoprotein have been directly correlated with the relative affinity constants calculated from kinetic analysis on a surface plasmon resonance-based BIAcore 2000 biosensor (Biacore AB, Uppsala, Sweden).


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasma samples. Four seroconversion panels consisting of sequential samples obtained from individuals recently infected with rubella virus were used in this study. Panels 1 and 2 consisted of nine specimens taken between days 19 and 107 and between days 16 and 78, respectively, following symptom onset. These panels were collected by the National Serology Reference Laboratory, Australia. Panel 3 was a commercial seroconversion panel (PTR901, Boston Biomedica Inc., Massachusetts) which consisted of nine specimens taken between 15 days before and 31 days after symptom onset. Panel 4 consisted of four specimens taken between day 1 and day 70 after symptom onset. This panel was collected by Queensland Medical Laboratories, QLD, Australia. Anti-rubella virus antibody-positive specimens and negative control specimens were obtained from the Australian Red Cross Blood Service.

All the specimens used in the present investigation were characterized by use of Vidas IgG and Vidas IgM assays (bioMérieux, Marcy-l'Etoile, France), AxSYM IgG and AxSYM IgM assays (Abbott Laboratories, Diagnostics Division, Abbott Park, IL), and an in-house hemagglutination inhibition assay (8).

Viral lysate. K1S rubella viral lysate (PanBio, QLD, Australia) was sonicated three times for 10 seconds on ice, aliquoted, and stored frozen at –70°C.

Western blot. K1S rubella virus antigen (10 µl) was diluted in nonreducing sample buffer, and the proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis at 100 V (14). The proteins were transferred to nitrocellulose (0.45-µm pore size) for 2 h at 0.22 A. The nitrocellulose was blocked with 0.3% skim milk powder in Tris-buffered saline (TBS) (150 mM NaCl in 10 mM Tris-HCl, pH 7.4) overnight on a shaker at 4°C, cut into 3-mm vertical strips, air dried overnight, and stored at 4°C.

Plasma samples were diluted 1 in 100 in 0.1% skim milk powder in TBS and incubated with strips at room temperature for 4.5 h. Strips were washed extensively in TBS-T (TBS containing 0.05% Tween 20). The strips were then probed with biotinylated anti-human secondary isotyping antibodies (Sigma-Aldrich Inc., St. Louis, Mo.). The anti-human total IgG, IgG1, and IgG3 antibodies were diluted 1 in 1,000, anti-human IgA was diluted 1 in 10,000, and anti-human IgM was diluted 1 in 5,000 in 0.3% skim milk power in TBS and incubated with strips for 1 h at ambient temperature (approximately 20°C); subsequently, the strips were washed in TBS-T. ExtrAvidin-alkaline phosphatase (Sigma-Aldrich Inc., St. Louis, Mo) diluted 1 in 5,000 in 0.3% skim milk powder in TBS was added for 30 min at ambient temperature, and the strips were again washed extensively in TBS-T. The bands were visualized by use of a 5-bromo-4-chloro-3-indolyl-5-bromo-4-chloro-3-phosphate (BCIP)-nitroblue tetrazolium phosphatase substrate system (Kirkegaard and Perry Laboratories, Guildford, United Kingdom), and strips were then washed in water and air dried overnight.

Peptide synthesis. The synthetic peptide E1208-239 is an analogue of the immunodominant region of the rubella virus E1 glycoprotein, amino acids 208 to 239, with the sequence MNYTGNQQSRWGLGSPNCHGPDWASPVCQRHS (26). The peptide was constructed with an N-terminal amine group to facilitate uniform binding to the BIAcore sensor chip and was synthesized on an Applied Biosystems 430A automated synthesizer by the solid-phase synthesis procedure of Merrifield (16) by use of tert-butoxycarbonyl chemistry. E1208-239 was reconstituted in deionizer water to a final concentration of 2 mg/ml and stored frozen at –20°C.

ELISA and avidity assay. K1S viral lysate was diluted in coating buffer (0.2 M sodium carbonate, pH 9.6) to a final concentration of 10.0 µg/ml. The E1 peptide was diluted to a final concentration of 20 µg/ml in coating buffer, and 50 µl of each diluted antigen was added to the wells of ImmunoSorp 96-well flat-bottomed microtiter plates (Nalge-Nunc International, Denmark). The plates were incubated at 37°C overnight, washed twice in PBS-T (50 mM sodium phosphate, pH 7.4, containing 150 mM NaCl and 0.1% Tween 20), and then blocked with 150 µl of BLOTTO (50 mM Tris-HCl, pH 8.0, containing 5% skim milk powder, 2 mM calcium chloride, 150 mM NaCl, and 0.2% Nonidet P-40) for 1.5 h at 37°C. Samples were diluted 1 in 10 in BLOTTO, and three 10-fold serial dilutions were prepared. Plates were washed twice in PBS-T, and then 100 µl of each diluted plasma sample was added to the wells and the plates were incubated at 37°C for 1 h. A duplicate plate was used for the avidity assay.

For the avidity assay, the plates were washed three times in PBS-T, and then 200 µl of 8 M urea in PBS-T was added per well, while 200 µl of PBS-T was added per well to the duplicate plate for the conventional "nonavidity" assay. Both plates were incubated for 5 min at ambient temperature and then washed three times in PBS-T. Sheep polyclonal horseradish peroxidase (HRP)-conjugated anti-human IgG (Chemicon Australia Pty. Ltd., Victoria, Australia), mouse monoclonal HRP-conjugated anti-human IgG1 clone JDC-1 (Southern Biotechnology Associates Inc., Birmingham, AL), and mouse monoclonal HRP-conjugated anti-human IgG3 clone HP6050 (Southern Biotechnology Associates Inc., Birmingham, AL) were all used at a dilution of 1 in 1,000. Mouse monoclonal HRP-conjugated anti-human IgM clone SA-DA4 (Southern Biotechnology Associates Inc., Birmingham, AL) was used at a dilution of 1 in 2,000, and HRP-conjugated goat anti-human IgA (Biosource International, Camarillo, CA) was used at a dilution of 1 in 5,000. The appropriate HRP-conjugated secondary antibody was diluted in BLOTTO, 100 µl was added per well, and the plate was incubated at 37°C for 1 h and then washed three times in PBS-T.

Two mM ABTS [2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid)] in 25 mM sodium citrate buffer, pH 4.5, containing 0.3% hydrogen peroxide was used as a substrate for the HRP by adding 100 µl/well and incubating in the dark at room temperature for 20 min. The reaction was stopped by the addition of 50 µl of 5% oxalic acid per well, and the absorbance of the wells was read at 405 nm.

The avidity assays were run in parallel with the standard ELISA using the same specimens, plasma dilutions, and secondary isotyping antibodies. The mean absorbance of the negative controls for each plate was subtracted from the optical density at 405 nm for each of the specimens. An avidity index (AI), the percentage of high-avidity antibodies in a specimen, was calculated for each specimen by dividing the absorbance at 405 nm of the urea-treated specimen by the absorbance at 405 nm of the untreated specimen and multiplying by 100. In this assay, 40% was designated as the cutoff for recent infection, with an AI higher than 40% considered as highly avid and indicative of past exposure (4, 10).

BIAcore analysis. The binding kinetics of the specimens to the E1208-239 peptide were analyzed using surface plasmon resonance on a BIAcore 2000 instrument (Biacore AB, Uppsala, Sweden). The E1208-239 peptide was synthesized with an N-terminal amine group for coupling to a BIAcore CM5 sensor chip (Biacore AB, Uppsala, Sweden); this chip has a carboxymethylated dextran matrix. The CM5 chip was activated by injecting a mixture of NHS (N-hydroxysuccinimide) and EDC [1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride] over the chip in accordance with the manufacturer's instructions. This procedure introduced NHS esters onto the surface matrix by modification of the carboxymethyl groups. The E1208-239 peptide was injected over the CM5 chip at a concentration of 100 µg/ml in 10 mM sodium acetate, pH 5.0, resulting in the NHS esters reacting with the N-terminal amine on the peptide to form covalent links. Residual NHS esters were blocked by injecting 1 M ethanolamine hydrochloride, pH 8.5, over the chip. Immobilization was carried out on three of the four available flow cells, with the remaining flow cell (activated and blocked without the addition of peptide) used as the reference cell.

The specimens were diluted 1 in 10 in HBS-EP buffer (0.01 M HEPES, pH 7.4, containing 0.15 M NaCl, 3 mM EDTA, and 0.005% surfactant p20), heat inactivated for 20 min at 62°C, and then centrifuged at 13,000 rpm for 20 min. Samples were injected for 5 min at 5 µl/min and allowed to dissociate for a further 5 min. Regeneration of the chip surface after each cycle was performed with a 2-min pulse of 100 mM HCl to remove all of the bound plasma protein and return the sensorgram to baseline. Each specimen was run in duplicate with the positive and negative controls run in duplicate at the beginning and the end of each batch to detect any changes in the surface reactivity.

Binding analysis was performed using the BIAevaluation version 3.2 software. As specific antibodies bind to the antigen, the resulting change in mass concentration at the sensor surface is detected as a change in the intensity of reflected light (the resonance angle). The change in the surface plasmon resonance angle is displayed as response units, where 1,000 response units is equal to 1 ng of analyte bound per nm2 on the sensor surface. The rate of dissociation of the analyte (antibody) from the ligand (antigen) is expressed as the dissociation rate constant (KD). The KD is directly related to the stability of the complex (i.e., the fraction of the analyte-ligand complexes that dissociate per second) and is independent of concentration. Because high-affinity antibodies are expected to dissociate from the peptide more slowly than low-affinity antibodies, the KD of each specimen is a measure of the relative affinity of the antibodies in the specimen. In a polyclonal system, such as plasma, where the concentration of the analyte is unknown, the KD provides the most accurate reflection of the relative affinity of the analyte for the ligand.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Seroconversion panel alignment. Because the exact date of infection was unknown and for the purpose of comparison, the four seroconversion panels were aligned using the maximum peak of IgM reactivity. This maximum peak of reactivity was identified by using a Vidas IgM enzyme immunoassay (EIA) and was designated as day 7 after symptom onset because, in the course of a typical rubella virus infection, the IgM antibody reaches its maximum concentration at approximately 7 days after symptom onset (6). We acknowledge that using an antibody marker to set a time frame for an investigation comparing antibody responses is not ideal. However, no other method of comparing the panels was available. Following infection with rubella virus, viremia disappears prior to symptom onset, and consequently, samples are rarely collected early enough to permit the detection of nucleic acid or antigen.

Western blot analysis. Western blots were used to confirm that all three rubella virus structural proteins were present in the K1S rubella viral lysate. The reported, apparent molecular masses of the rubella virus proteins are 58 kDa for E1, 42 to 47 kDa for E2, 33 to 38 kDa for the capsid, and 70 kDa for the capsid homodimer (28), which corresponded well with the bands we observed on our Western blot probed for total IgG (Fig. 1A). Antibody isotype-specific Western blots demonstrated E1 to be the immunodominant protein, and the four antibody isotypes investigated were all found to react with this protein. IgA and IgM reacted early during the course of infection (Fig. 1D and E, respectively) while the IgG1 and IgG3 responses gradually intensified with time (Fig. 1B and C, respectively).


Figure 1
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  		FIG. 1. Antibody isotype-specific Western blots were performed on a rubella virus seroconversion panel. This seroconversion panel (panel 1) consisted of nine specimens with estimated times after symptom onset of 19, 37, 43, 50, 57, 71, 80, 88, and 107 days. Plasma specimens were diluted 1 in 100 and incubated with K1S rubella viral lysate-based Western blot strips. Biotinylated anti-human isotyping secondary antibodies were used to probe the strips for total IgG (A), IgG1 (B), IgG3 (C), IgA (D), and IgM (E); this was followed by incubation with an avidin-alkaline phosphatase conjugate. The bands were visualized by use of a BCIP-nitroblue tetrazolium phosphatase substrate system. The apparent molecular masses of three structural proteins of the rubella virus are 58 kDa for E1, 42 to 47 kDa for E2, 33 to 38 kDa for the capsid (marked as "C" to the left of the figure), and 70 kDa for the capsid homodimer (Cd) (28).

 
Rubella viral lysate ELISA. Specimens from the four seroconversion panels were analyzed by use of an antibody isotype-specific ELISA with rubella viral lysate as the antigen. The total IgG response was seen to increase gradually with time in all four seroconversion panels (Fig. 2A). The IgG1 reactivity followed this same pattern (Fig. 2B), indicating that IgG1 is the major antibody isotype contributing to this total IgG reactivity. The IgG3 (Fig. 2C), IgA (Fig. 2D), and IgM (Fig. 2E) responses all demonstrated the presence of early transient peaks of reactivity which declined with time.


Figure 2
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  		FIG. 2. An antibody isotype-specific ELISA based on rubella viral lysate was performed. Plates were coated with K1S rubella viral lysate, and specimens from the four seroconversion panels, panels 1 (•), 2 ({blacktriangledown}), 3 ({blacksquare}), and 4 ({blacklozenge}), were analyzed at a dilution of 1 in 10. The plates were probed with HRP-conjugated anti-human secondary antibodies specific for total IgG (A), IgG1 (B), IgG3 (C), IgA (D), and IgM (E). The absorbance at 405 nm is plotted against the estimated number of days postinfection.

 
K1S rubella viral lysate avidity assay. The viral lysate-based avidity assay for total IgG demonstrated rapidly increasing AI in two of the panels but a consistently high-avidity response for all samples collected over the other two panels (Fig. 3A). This response was mirrored by the IgG1-specific avidity assay (Fig. 3B), indicating that the high-avidity response observed in the total IgG assay was primarily due to a high-avidity IgG1 response. The IgG3-specific (Fig. 3C) and IgA-specific (Fig. 3D) avidity assays showed increasing avidity with time. However, the transition to a high-avidity response (>40% AI) did not occur until well after the initial peak of high-titer antibody (evident in the conventional ELISA) had disappeared. Consequently, by the time the affinity of these antibody isotypes had increased, the corresponding titer had decreased to levels where this reactivity could not significantly contribute to the overall AI observed. The IgM-specific avidity assay (Fig. 3E) demonstrated a very low avidity index over the course of infection examined, despite the presence of an initial high-titer peak of reactivity observed with the standard ELISA.


Figure 3
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  		FIG. 3. An antibody isotype-specific, rubella viral lysate-based avidity assay was performed. Plates coated with K1S rubella viral lysate were used to analyze specimens from four seroconversion panels, panels 1 (•), 2 ({blacktriangledown}), 3 ({blacksquare}), and 4 ({blacklozenge}), for the presence of different antibody isotypes. The AI was calculated by dividing the absorbance at 405 nm following treatment with 8 M urea by the absorbance at 450 nm without treatment and multiplying by 100. The dashed line at 40% AI represents the arbitrary cutoff for recent infection. Panels were probed with a polyclonal anti-human total IgG antibody (A), with an anti-human IgG1 secondary antibody (B), with an anti-human IgG3 antibody (C), with an anti-human IgA antibody (D), and with an anti-human IgM secondary antibody (E). The AI is plotted against the estimated day postinfection.

 
Reactivity to EI208-239. The major humoral immune response to the immunodominant peptide E208-239 was an IgG1 response which increased with time for all four seroconversion panels (Fig. 4A). This binding pattern was mirrored by the total binding response curves obtained with the BIAcore instrument (Fig. 4C). Application of the chaotropic agent in the avidity assay demonstrated that all samples taken following infection for each panel were of high avidity (Fig. 4B), which indicates that the immune response to this particular epitope matures in affinity very rapidly. This was confirmed by the extremely low KD values obtained with the BIAcore instrument (Fig. 4D), which indicates that the relative affinity was high for all the seroconversion samples very soon after infection.


Figure 4
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  		FIG. 4. IgG1 reactivity to the E1 immunodominant peptide (E208-239) was analyzed using EIA, avidity assay, and surface plasmon resonance on a BIAcore instrument for four seroconversion panels, panels 1 (•), 2 ({blacktriangledown}), 3 ({blacksquare}), and 4 ({blacklozenge}). (A) Immunoreactivity of IgG1 in an E208-239-based EIA plotted against the estimated number of days following the onset of symptoms. (B) Percent AI calculated for the IgG1 reactivity to E208-239 over time. The dashed line at 40% AI represents the arbitrary cutoff for recent infection. (C) Observed level of binding (response units [RU]) to the E208-239-coated BIAcore chip for samples obtained from individuals recently infected with rubella virus. This reflects the relative amount of rubella virus-specific antibody in the plasma obtained from the four individuals. (D) KD of the plasma antibodies bound to E208-239 when analyzed on the BIAcore instrument. The dissociation rate is a measure of the relative affinity of the plasma antibodies bound to the peptide. The decreasing dissociation rate correlates well with the increase in avidity (panel B). The initial high dissociation rates of the bound antibodies indicated low-affinity binding prior to infection. The dissociation rate then decreased rapidly after infection and remained low as the immune response matured and antibody affinity increased.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have demonstrated that during the primary immune response to infection by rubella virus, there is an initial peak of IgM reactivity to the viral proteins, which is closely followed by transient peaks of both IgG3 and IgA reactivity. During the course of infection, an IgG1 response develops and gradually increases in both titer and affinity until it dominates the immune response. While previous studies have reported the detection of IgM soon after rubella virus infection and prior to the appearance of significant amounts of IgG (20) and the observation of the presence of both IgG1 and IgG3, the times at which they appear following infection has been found to vary (21). By using seroconversion panels rather than single samples obtained from individuals, we have been able to demonstrate the relative times following infection that the various antibody isotypes appear.

Our data indicate that treatment with the chaotropic agent 8 M urea appears to eliminate the detection of the early low-affinity IgM, IgA, and IgG3 peaks of reactivity while apparently leaving the developing IgG1 response unaffected, explaining the principle underlying these avidity assays. IgM antibodies do not undergo affinity maturation (11, 13), which was reflected in the results we obtained with the avidity assay. Although a high-titer peak of IgM was identified in the viral lysate-based ELISA, this reactivity was completely absent in all samples from all panels in the avidity assay. Despite the presence of intense peaks of IgG3 and IgA reactivity soon after infection, the AI was not found to increase until after these peaks of reactivity had decreased significantly when the avidity assay was conducted for these antibody isotypes. Consequently, neither of these antibody isotypes could have made any significant contribution to the overall AI observed. Previous studies have also reported avidity assays to be helpful and reliable in the diagnosis of recent primary rubella virus infection (10), where IgG avidity has been found to increase with time after primary infection but not following reinfection (4, 12, 21). Analyzing seroconversion panels in an avidity assay probed with antibody isotype-specific secondary antibodies, we have been able to determine the interactions which underlie these findings.

To obtain good-quality kinetic data with the BIAcore instrument, highly purified proteins are required for immobilization. Rubella viral lysate could not be used for immobilization, since the possibility that some antigens could preferentially bind to the chip could not be discounted. For this reason, we probed viral lysate-based Western blots with antibody isotype-specific secondary antibodies to identify the antigen that would provide the most comprehensive analysis of the interactions we had observed. Not only was E1 found to be immunodominant, but it also was the only antigen recognized by all the antibody isotypes we investigated. Consequently, we used a peptide analogue of the putative immunodominant epitope (E208-239) for immobilization to the biosensor chip and subsequent kinetic analysis.

The total binding to the E208-239-coated biosensor chip was analogous to that observed in the peptide-based ELISA. The peptide-based avidity assay demonstrated that a high-avidity response to this peptide is generated rapidly following infection, and this was confirmed by the extremely low dissociation rates calculated from the sensograms. The AI measured in the avidity assay and the relative affinity measured with the BIAcore instrument indicate that the immune response to the E1 immunodominant peptide matured too rapidly to provide a means of identifying recent infection, since a high-affinity IgG1 response was already present in the samples collected immediately after infection, with only very low and quite variable reactivity observed for the other antibody isotypes.

A reliable assay to distinguish between the immune response generated by recent exposure and the immune response existing as a result of past exposure or immunization is an essential facet of the management of the health of a pregnant woman when a rubelliform rash develops or when there is suspected exposure to rubella virus. We have investigated the immunological basis of the avidity assays which are currently being used for this purpose and found that these assays do appear to discriminate between the initial peaks of low-affinity IgM, IgA, and IgG3 reactivities and the high-affinity IgG1 response indicative of past exposure.

In the absence of a more specific marker of recent infection, assays relying on the ability of chaotropic agents to dissociate low-avidity interactions provide valuable information for confirming the diagnosis of recent rubella virus infection. However, it is essential to ensure that an appropriate antigen is employed, because the immune response to each specific antigen matures at a different rate. For this reason, it is also necessary to evaluate fully each assay with large numbers of well-characterized samples.


   ACKNOWLEDGMENTS
 
We thank Frosa Katsis from St. Vincent's Institute, Melbourne, for performing peptide synthesis and Bruce Kemp from St. Vincent's Institute, Melbourne, for providing access to the BIAcore 2000 biosensor. We also thank Robin Wood at Queensland Medical Laboratories for providing seroconversion panel 4 and Wayne Dimech and Lena Panagiotopoulos from the National Serology Reference Laboratory, Australia, for providing the Vidas and AxSYM EIA results.


   FOOTNOTES
 
* Corresponding author. Mailing address: National Serology Reference Laboratory Australia, 4th Floor, Healy Building, 41 Victoria Parade, Fitzroy, Victoria, Australia 3065. Phone: 61 3 9418 1105. Fax: 61 3 9418 1155. E-mail: kim{at}nrl.gov.au. Back


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Clinical and Vaccine Immunology, March 2006, p. 380-386, Vol. 13, No. 3
1071-412X/06/$08.00+0     doi:10.1128/CVI.13.3.380-386.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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