A screening assay to detect antigen-specific antibodies within cerebrospinal fluid

Journal of Immunological Methods 311 (2006) 81 – 86 www.elsevier.com/locate/jim

Research paper

A screening assay to detect antigen-specific antibodies within cerebrospinal fluid Patricia Morris a , Nicholas W.S. Davies b , Geoffrey Keir a,⁎ a

Department of Neuroimmunology, National Hospital for Neurology and Neurosurgery, Queen Square, London, WC1N 3BG, UK b Department of Clinical Neurosciences, Guy's, King's and St Thomas' School of Medicine, London, UK Received 15 September 2005; received in revised form 16 December 2005; accepted 11 January 2006 Available online 24 February 2006

Abstract Identification of the aetiology of central nervous system infections requires the detection of either the organism or a microbespecific immune response within the brain or cerebrospinal fluid. We describe a screening assay to detect herpes simplex virus, varicella zoster virus, cytomegalovirus, measles and Toxoplasma gondii specific antibodies in cerebrospinal fluid. Antigen-specific immunoblotting of oligoclonal IgG and IgM was used to confirm the presence of antibody. Of 51 consecutive cerebrospinal fluid samples received by the laboratory from patients with suspected central nervous system infection 18 (35%) were screen positive for one or more antigen. In only 7 of these were antigen-specific oligoclonal IgG or IgM bands confirmed. The assay provides a simple, cheap assay to screen for microbial-specific antibody in the cerebrospinal fluid samples of patients with suspected neurological infections. © 2006 Elsevier B.V. All rights reserved. Keywords: Antigen-specific; Screen; Oligoclonal bands; Infection; Cerebrospinal fluid

1. Introduction We describe an assay to screen cerebrospinal fluid (CSF) for antigen-specific antibodies, which is tailored to assist diagnosis of neurological infections prevalent within the United Kingdom. Laboratory confirmation of central nervous system (CNS) infections requires the detection in tissue or CSF of either the aetiological

Abbreviations: CNS, central nervous system; CSF, cerebrospinal fluid; CMV, cytomegalovirus; HSV, herpes simplex virus; VZV, varicella zoster. ⁎ Corresponding author. Tel.: +44 20 7837 3611; fax: +44 20 7837 8553. E-mail address: [email protected] (G. Keir). 0022-1759/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2006.01.015

microbe or microbe-specific antibody. Whilst molecular techniques, such as PCR, are routinely used to detect a variety of neurotropic organisms in CSF, the diagnostic sensitivity of these techniques is dependent upon the time of CSF sampling within the natural history of the disease (Davies et al., 2005). Thus to diagnose CNS infections such as herpes simplex virus encephalitis and Toxoplasma gondii encephalitis, the combination of both PCR and antibody assays applied to CSF is recommended (Cinque et al., 1996; Luft and Sivadas, 2004). For other infections, such as subacute sclerosing panencephalitis, detection of a microbe-specific antibody in CSF is the test of choice. Detection of microbe-specific IgM within CSF alone is sufficient to confirm the diagnosis of

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infections such as Japanese encephalitis, where CNS involvement occurs at the time of primary infection (Solomon et al., 1998). However, many encephalitides are caused by microbes, such as herpes simplex virus, which establish latency and cause CNS disease through reactivation or re-infection. Reactivation or re-infection causes a poor IgM response that is not reliable for diagnostic purposes, therefore microbe-specific IgG assays are utilised. However, in contrast to IgM assays, detection of microbe-specific IgG in CSF alone is not sufficient to confirm involvement of the central nervous system, as high levels of IgG in CSF can result from passive transfer from serum. Localisation of the immune response to the CNS can be confirmed by showing that intrathecal synthesis of microbespecific IgG is present. Demonstration of intrathecal production of IgG requires the comparison of IgG in CSF with that in a homologous serum sample. Two techniques are commonly used to detect antigenspecific IgG in CSF: calculation of specific-antibody indices and antigen-specific immunoblotting of oligoclonal IgG (Moyle et al., 1984; Reiber and Lange, 1991). Both methods have similar sensitivity and specificity for the diagnosis of herpes simplex encephalitis (Monteyne et al., 1997). Whilst these assays are useful in confirming antigen-specific antibody within CSF they are labour intensive, require replication with multiple different antigens, and are time consuming. Thus a simple and sensitive assay with a quick turnaround time would be helpful to screen for the presence of antigen-specific antibodies in CSF. 2. Methods Development and use of the assay utilised sample incubation manifolds obtained from Hoefer (Amersham Biosciences, UK). These are precision-machined Perspex modules comprising a solid base and a matching lid with milled slots to contain liquid samples. Cross-contamination between adjacent slots is prevented by a pair of silicon rubber gaskets, one of which is similarly slotted. Freeze dried pooled herpes simplex virus (HSV) types 1 and 2, varicella zoster (VZV), cytomegalovirus (CMV), measles and T. gondii complement fixation grade antigens (all Virion, Switzerland) were used throughout development of the assay. Each was reconstituted in 1 mL of deionised water and allowed to stand for 30 min, after which they were centrifuged to remove particulates. These solutions formed the stock antigen solutions. For each antigen solution the

total protein concentration was determined by a modified Lowry (Bio-Rad DC Protein Assay) after reconstitution. The microbial and control antigens were initially studied by Western immunoblot. CSF samples obtained from patients with diagnosed CNS viral infections, that were known to possess antigen-specific oligoclonal IgG to the causal microbe, were used to demonstrate specificity of binding to the microbial but not control antigen. A common method was used to prepare and develop the nitrocellulose membrane. A 10 by 10 cm sheet of nitrocellulose (Transblot Membrane, Bio-Rad Laboratories CA, USA) was cut to fit the Hoefer incubation chamber. Using the slotted silicon rubber gasket, the centre of each slot was marked on the membrane top and bottom. This allowed identification of the positions of each antigen on the membrane. The membrane was then wetted in 0.9% saline, placed on the bottom gasket and the incubation chamber was re-assembled. A watertight seal to the manifold chamber was confirmed by allowing it to stand for 10min after adding 2 mL of 0.9% saline to each slot. Subsequently the wells were emptied and 2 mL each of a dilution of antigen was then added to the slots. The chamber was then incubated overnight at 4°C on a rocker. The following day the antigen solutions were decanted and all the wells were thoroughly washed with 20 changes of tap water. The incubation chamber was then disassembled and the membrane removed. After a brief wash with 0.9% saline, the membrane was blocked for 2h in 2% dried skimmed milk in saline, and then rinsed in 3 changes of 0.9% saline only. Subsequently, the incubation chamber was re-assembled but with the membrane rotated through 90°. Dilutions of CSFs could now be added and incubation overnight at 4 °C on a rocker repeated. Following incubation the CSF solution was decanted and the membrane washed 20 times with tap water, followed by two changes in 0.9% saline and finally for 5min in 2.5mL of 0.2% dried skimmed milk. After removal of the milk solution 2.5 mL of 1 / 1000 dilution of horse-radish peroxidase conjugated rabbit anti-human pan immunoglobulins (Dako Cytomation, Denmark) was added and incubated for 2h at room temperature. The antibody solution was then discarded and the strips washed in 20 changes of tap water, followed by 3 rinses in 0.9% saline then 3rinses in deionised water. The strips were then developed using 25mL 4-chloro-1-naphthol colour reagent (10 mg dissolved in 10 mL ethanol then added to 50 mL acetate buffer pH 5.1, with 50 μL hydrogen peroxide added immediately before use) for 15min at room temperature. The colour development

P. Morris et al. / Journal of Immunological Methods 311 (2006) 81–86 Dilution of antigen (in this case measles)

Titration studies were performed to ascertain the optimum antigen concentration for coating the nitrocellulose membrane as well as CSF dilution for use in the assay. Dilutions of CSF were made with saline containing 0.2% milk and covered the range 1 / 25 down to 1 / 1280. Once the optimal antigen dilutions had been determined it was then tested against a range of CSF samples diluted at a constant value of 1 in 100. This led to the selection of 1 / 100 dilution for all antigens as acceptable. One lane acted as an assay control and consisted of a lane coated with a 1 / 4000 dilution of normal human serum. The screening assay required the detection of this internal control before being read. Positive results were identified by eye. The assay was assessed using consecutive CSFs from patients with suspected CNS infections received by the laboratory. The laboratory performs all CSF analysis on samples obtained within the hospital. All test samples showing a positive result for any antigen were further investigated by antigen-specific immunoblotting to demonstrate microbe-specific oligoclonal bands as described by Moyle et al. (1984). After isoelectric focusing the CSFs were blotted with membranes previously coated with either the microbial antigen of interest or control antigen. Antigen-specific IgG or IgM bands were visualised by a colour reaction after incubation with either goat anti-human IgG or IgM, followed by HRP-conjugated polyclonal rabbit anti-goat antibody. Detection of antigen-specific oligoclonal IgG or IgM bands was recorded when one or more bands were identified that were not present on the control membrane.

1 in… .

800 400 200 100 50 1 in 25

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50 100 200 400 800 1600 3200

dilution of CSF

Fig. 1. A typical development matrix used to optimise the amount of antigen, and the optimum dilution of CSF to use in the screening assay. Varying amounts (expressed as dilutions) of stock antigen solution (Yaxis) and CSF (X-axis) were sequentially applied to a membrane (see text for details). In the example shown here, using measles antigen, it can be seen that using antigen at a dilution of 1 / 50 allows CSF to be tested down to a dilution of 1 in 1600. The decision on which dilutions to choose is based upon getting a reasonable strength signal, yet conserving the amount of CSF used for screening. In this example we chose to work with an antigen preparation at a dilution of 1 / 100 from stock, and CSF at a dilution of 1 in 100. As discussed in the text we found a dilution of CSF of 1 / 100 worked well for all antigens, and this was chosen as the dilution for use throughout. As the working volume for the screen is 25mL, so using CSF at a dilution of 1 / 100 is equivalent to applying 25μL of neat CSF. Each screen allows us to test for up to 9 different antigens.

step required accurate timing. After colour development the membrane was washed thoroughly in several changes of tap water, and then dried with warm air.

HSV (1+2) Measles VZV CMV T. carinii control

Positive:

1 CMV

Weak positive: HSV

2 HSV

3

HSV

4 5 HSV HSV

6 M

7

VZV

8

9 VZV

10 VZV

VZV

HSV

HSV

11

T. carinii

Fig. 2. Example set of screens for 11individual CSF samples; against 5microbial species (HSV: Herpes simplex virus; M: measles virus; VZV: varicella zoster virus; CMV: cytomegalovirus; T. carinii: Toxoplasma carinii). Each vertical strip (1–11) corresponds to individual CSF samples (we do not routinely screen serum; only CSF). Each horizontal track corresponds to a specific antigen. Results are divided into positive (dark square) and weak positive (lighter grey square). CSF samples 1, 9 and 10 show reactions against more than one antigen, whilst samples 2, 3, 5, 6, 7, 8, and 11 show reactions to only one antigen. The weak positive result against VZV on lane 8 does not show up in the figure due to limitations in sensitivity of scanned image, but is more clearly visible by eye on the original strip. Samples that show a positive result on this screen are then tested for speciesspecific oligoclonal IgG (for examples, see Fig. 3).

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3. Results The titrations of dilutions of both coating antigen and CSF were assessed using checkerboards as illustrated in Fig. 1. From checkerboards for each antigen optimum dilutions of antigen were chosen to detect known antibody-positive CSFs diluted 1 / 100. Examples of screenpositive CSFs that were also positive to the same antigen by antigen-specific immunoblotting are illustrated in Fig. 2. To check the false negative rate, 38 unselected CSF samples were tested. The screen revealed a total of 9 positive spots. All 38 CSF were also tested for oligoclonal antigen-specific bands by immunoblotting

S CSF S CSF Patient:

1

Antigen:

Measles

2

S CSF S CSF 3

4 VZV

(Moyle et al., 1984) against all antigens. Oligoclonal antigen-specific immunoblotting showed weak banding (either a mirror pattern or one with more bands in CSF compared to serum) in only 6 instances. In the other three screen-positive spots no antigen-specific bands were seen. The screening test therefore gave no false negative results, but had a false positive rate of 3 / 38 (∼10%), which is well tolerable when using oligoclonal antigen-specific immunoblotting as the final arbiter (Fig. 3). Table 1 shows the results of the prospective screen of CSFs from patients with presumed CNS viral infections. Of 51 CSF samples screened 18 were positive for 1 or more antigen. Seven CSF were shown to have antigen-

S CSF S CSF 5

6 VZV

S CSF S CSF 7

8 HSV

Fig. 3. Representative examples of screen and follow-up antigen-specific oligoclonal IgG immunoblots. Screening immunoblots (top) CSF only, whilst antigen-specific oligoclonal IgG immunoblots (underneath) are CSF and paired serum. Patient 1: negative for measles on screening and also negative measles-specific oligoclonal IgG; Patient 2: positive for measles on screening and showing positive measles-specific oligoclonal IgG, showing an identical pattern of measles-specific bands in both CSF and serum; Patients 3 and 4: both positive against VZVon screening, and showing VZV-specific oligoclonal bands in CSF and serum. The serum of patient 3 shows a pattern typical of a monoclonal IgG antibody to VZV (which is also present in CSF). Patients 5 and 6: both negative for VZV screening, and also negative for VZV-specific oligoclonal IgG; Patients 7 and 8: weak positive for HSV on screening, and showing mirrored HSV-specific IgG bands in both CSF and serum.

P. Morris et al. / Journal of Immunological Methods 311 (2006) 81–86 Table 1 Results of CSF microbe-specific antibody assay compared with immunoblotting results HSV VZV CMV Measles Toxoplasma gondii Screen positive a 16 Immunoblotting b: IgG positive 5 IgM positive 2c Negative 9

3

3

2

2

1 0 2

0 0 3

1 0 1

1 0 1

a

Of 51CSF samples screened in total 18CSFs were positive for one or more antigen. b Two CSF samples were oligoclonal IgG band positive for two antigens (one against HSV and VZV, the other for HSV and measles). c One positive and one negative for IgG HSV-specific oligoclonal bands.

specific IgG or IgM bands. Only 2 CSF had IgM antigen-specific bands, of which one had accompanying IgG bands to the same antigen. 4. Discussion Microbes have varying neurotropic properties that result in syndromes of CNS infection ranging from encephalitis to poliomyelitis and meningitis. In the UK the aetiology of presumed CNS viral infections has not be well studied (Davison et al., 2003) but world-wide HSV is thought to be the commonest cause of acute sporadic necrotising encephalitis (Whitley and Roizman, 2001). Amongst the immunosuppressed, particularly in HIV/AIDS, CMV and the protozoan T. gondii are frequent causes of opportunistic CNS infection. More recently VZV has been highlighted as causing a greater proportion of CNS viral infections than previously suspected (Koskiniemi et al., 2001). Thus our screening assay seeks antibody specific to aetiologies common to our target population. A screening assay should have high sensitivity resulting in a low false negative rate and a false positive rate higher than that of the “gold standard” to which it is compared. The sensitivity of the assay described in this paper was established by titration with known microbespecific antibody positive and negative CSFs. Antigenspecific immunoblotting of oligoclonal IgG and IgM was used as the standard to which the screening assay was compared. The screen does not incorporate control (cells without virus) and so is prone to false positive results. This is acceptable as all samples positive by the screen were then tested for antigen-specific oligoclonal bands, which include a paired control lane. Hence the screen has more positive results than the antigenspecific immunoblots.

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The assay was further studied by its use on a cohort of CSFs samples from patients with suspected viral CNS infections. The sensitivity of the screening assay was shown to be higher than the standard as the majority of screen positive samples were shown to be antigen-specific oligoclonal IgG and IgM negative. Few of the CSFs were positive for antigen-specific oligoclonal IgM as the CNS infections caused by herpesviruses and T. gondii are most frequently through reactivation, or, in the case of measles, as a remote complication of primary infection. The study could be criticised as the screen was capable of detecting microbe-specific IgA antibodies, which were not sought by antigen-mediated immunoblot. However, the screening assay was designed to identify viral and protozoal specific antibody. CNS infections in which the intrathecal humoral response is characterised by microbe-specific IgA predominating over IgG or IgM are typically bacterial (Reiber and Peter, 2001). Such aetiologies include Mycobacterium tuberculosis, Neisseria meningitidis, and Streptococcus pneumoniae — microbes not sought by the screen. The assay is simple, inexpensive and has a turnaround time of 24h. An internal control ensures that addition of the appropriate anti-sera and colour developer has been undertaken. Furthermore, by cutting a coated membrane into strips 5mm wide perpendicular to the direction of coating, individual CSFs may be tested. This reduces assay turnaround time, as samples no longer need to be batched into groups of 10. We have found that the coated membrane strips after being airdried and stored at − 20 °C in a sealed plastic bag are stable for at least 6months. The assay could be adapted to screen for other antibody, specific to microbes endemic in the area of use, such as flaviviruses in North America or Asia. From the cohort of CSFs from patients with suspected CNS infection 65% were screen negative. Thus the assay can limit resource expenditure by reducing the number of target antigens for which antigenspecific immunoblotting of oligoclonal IgG and IgM is performed. Whilst a positive result cannot be used with certainty to predict the presence of the screened for antigen-specific oligoclonal IgG or IgM, the assay can provide a quick negative result. A timely negative result, combined with clinical information regarding the time of CSF sampling, could aid the physician in guiding the diagnostic process when a CNS infection is suspected. A positive result will inform choice of antigen for definitive CSF specific-antibody studies.

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