Protein A-Based ELISA: Its Evaluation in the Diagnosis of Herpes Simplex Encephalitis

Abstract

Herpes simplex encephalitis (HSE) represents one of the most severe infectious diseases of the central nervous system. As effective antiviral drugs are available, early rapid and reliable diagnosis has become important. The objective of the present study was to develop a sensitive enzyme-linked immunosorbent assay (ELISA) protocol for herpes simplex virus (HSV) antigen detection by assessing the usefulness of hyperimmune sera isolated from HSV-seropositive patients. A total of 52 cerebrospinal fluid (CSF) and 62 serum samples of HSE patients and non-HSE persons were analyzed. An in-house ELISA protocol utilizing hyperimmune sera was developed for HSV antigen detection. To improve the specificity of the method, protein A was incorporated into the protocol for ELISA. The sensitivity (70% and 90%) of antigen detection was high in CSF and serum samples, respectively, of confirmed HSE patients. However, lower specificity (52.3% and 42.3%), respectively, was obtained, which was improved by using protein A in the ELISA protocol. The modification in the method yielded good sensitivity (80% and 70%) and specificity (85.7% and 88.4%) of HSV antigen detection in the CSF and sera, respectively, of the HSE and non-HSE groups. The ELISA method utilizing hyperimmune sera along with protein A for HSV antigen detection yielded good sensitivity and specificity in both CSF and sera, and hence can be useful for the diagnosis of HSE.

Introduction

H uman herpesviruses cause various acute, subacute, and chronic disorders of the central nervous system (CNS) and peripheral nervous system in adults and children. Among herpesviruses, herpes simplex viruses (HSV) 1 and 2 cause a wide spectrum of clinical manifestations, such as meningitis, encephalitis, and myelitis in the CNS of infants as well as adults. However, clinical criteria (symptoms) are not reliable enough to differentiate between different causes of encephalitis, as numerous neurological syndromes may mimic herpes simplex encephalitis (HSE). Therefore, accurate and rapid diagnosis of HSV infections is of utmost importance for effective therapy with antiviral drugs.

The development of an HSE diagnostic test is to confirm, as rapidly as possible, the clinical impression that observed symptoms result from HSV infection. Although cell culture is considered the gold standard for HSV detection, it has several inherent limitations, including a lack of 100% sensitivity and the invasive surgical procedure that is controversial and rarely used for diagnostic purposes (1,6,7). It is also not possible to culture virus in many clinical laboratories because of a lack of the infrastructure needed to fulfill various biosafety requirements.

Molecular diagnostic assays using polymerase chain reaction (PCR) are widely used for detecting herpesvirus infections of the CNS (2,3,11). Although very sensitive, the technique has the disadvantage of being highly susceptible to PCR carry-over contamination errors, potentially leading to false-positive results (17,21).

The immunoglobulin M (IgM) enzyme-linked immunosorbent assay (ELISA) method, which provides evidence of HSV infection, is used to lend support to clinical findings in the assessment of patients with suspected HSV infection. However, the sensitivity of the IgM ELISA is low in the acute stage of illness. Additionally, IgG cannot be detected in HSV-infected patients in the acute stage (5,19,22,23). Serologic tests also need to be standardized by concomitant measurement of albumin to determine that virus-specific antibody has not passively diffused from serum into the cerebrospinal fluid (CSF), thereby yielding false-positive results (10,13,15). A rapid antigen detection test that uses ELISA for the detection of HSV infection may be a more accurate diagnostic method for patients in the acute stage of infection.

ELISA systems are specific (specificities range from 96–100%), but lack sensitivity (sensitivities range from 46–76%) in detecting HSV antigen directly from clinical specimens (14,16,20). Immunologic analyses of CSF for the presence of specific antigens have not yielded sensitive tests for the laboratory diagnosis of HSV-related CNS disease (4,8). As the ELISA technology continues to improve, better methods have been developed to capture viral antigen and therefore improve sensitivity. The objective of this study was to develop a sensitive and specific technique to detect HSV antigen by the ELISA method. The present report describes a method in which hyperimmune sera from HSV-seropositive patients was evaluated for the detection of antigen by an in-house ELISA method. The CSF and paired sera were simultaneously screened for HSV infection in patients with HSE.

Materials And Methods

Patient selection and sample collection

The Central India Institute of Medical Sciences (CIIMS), Nagpur, is a tertiary referral center. Patients with suspected cases of HSE who were admitted were prospectively were enrolled in this study. Criteria for suspicion of HSE were presence of fever, altered mental status (low level of consciousness, and behavior or personality changes), and other critical manifestations (e.g., focal neurological deficits and seizures). Neurological diagnostic investigations were performed during the first week of hospitalization; these investigations included the AFB and Gram stains; bacterial culture; determination of the protein and sugar levels and cell counts in CSF; and computed tomography scan and magnetic resonance imaging of the brain. CSF and paired serum samples were collected from 52 patients before they received any treatment. Clinical data were prospectively collected on case record forms. Informed consent was obtained from all of the patients. The Institutional Ethics Committee of CIIMS, Nagpur, India, funded and approved this study. All patients were grouped as follows.

HSE group (n = 10)

An acute case of HSV infection was defined as any case with clinical and/or MRI features consistent with HSE, and in whom HSV infection was confirmed by conventional PCR assay.

Non-HSE group

Suspected viral encephalitis (n = 24)

This group included patients with acute onset of fever and clinical features consistent with viral encephalitis. CSF findings showed a mild increase in protein and glucose, and often normal or mild pleocytosis. These patients showed a good clinical response to antiviral treatment.

Other infectious cases (n = 10)

Patients included in this group had tuberculous meningitis (TBM) or pyogenic and fungal meningitis.

Tuberculosis meningitis

A diagnosis of TBM was based on clinical features including subacute or chronic fever with features of meningeal irritation such as headache, neck stiffness, and vomiting, with or without other features of CNS involvement. CSF findings in these patients included increased protein, decreased glucose (a CSF:blood glucose ratio <0.5), and/or pleocytosis with lymphocytic predominance. All these patients showed a good clinical response to antituberculous drugs.

Non-tuberculous infectious meningitis

This group included patients having pyogenic or fungal meningitis. Pyogenic meningitis was suspected in patients who had acute high-grade fever with features of meningitis. These patients often had altered sensorium, as well as CSF findings of increased protein, very low sugar (CSF:blood glucose ratio <0.2), and pleocytosis with polymorphonuclear predominance. The response to broad-spectrum antibiotics was also considered as one of the diagnostic criteria for pyogenic meningitis. Fungal meningitis shows CSF profiles similar to TBM; however, India ink staining shows the presence of potential etiological agents such as Cryptococcus. Fungal meningitis was further confirmed by culturing on selective media.

Non-infectious neurological disorders (n = 8)

All other patients who had no evidence of CNS or extra-CNS bacterial or viral infections were grouped in the non-infectious neurological disorders group. Patients included in this group had hypertension, status epilepticus, stroke, or other disorders.

Healthy controls (n = 10)

Additionally, serum samples of healthy volunteers from the working staff of the institute with no signs or symptoms of clinical impairment were included as controls.

Anti-HSV IgG detection in sera

Anti-HSV IgG was detected in sera of HSE patients by the Platelia HSV (1+2) ELISA Kit (BioRad Laboratories, Inc., Hercules, CA), according to the manufacturer's instructions. The method uses qualitative determination of IgG class antibodies to HSV in human serum. The antigen, composed of purified and inactivated HSV types 1 and 2, is bound to the solid phase (8-well strips). The specific immunoglobulins are bound to the antigen after incubation with dilute human serum. After washing, incubation was done with the conjugate, composed of human IgG monoclonal antibodies labeled with peroxidase. After washing, the peroxidase substrate was added. The intensity of the color was read at 450 nm.

Preparation of anti-HSV

Pooled sera from patients infected with HSV [positive for anti-HSV IgG as demonstrated by the Platelia HSV (1+2) ELISA Kit] was collected, and IgG was purified by protein G affinity column chromatography (IgG purification kit; Bangalore Genei, Bengaluru, Karnataka, India), according to the manufacturer's instructions. The IgG bound to the column was eluted in different fractions of 1 mL each, and the fraction showing maximum absorbance at 280 nm was used at a dilution of 1:4000.

HSV antigen detection by ELISA

One hundred microliters of CSF (1:50) and paired serum samples (1:200) from HSV-infected patients was separately added to the microtiter wells, and then the wells were blocked with 0.5% BSA in PBS for 45 min. After the wells were washed with PBS, anti-HSV IgG was added (1:4000), and the plates were incubated at 37°C for 45 min. After incubation, the wells were washed and goat anti-human IgG–horseradish peroxidase secondary antibody (1:10,000) was added. The samples were then incubated for 45 min at 37°C. After another wash with PBS, 100 μL of the TMB-H₂O₂ substrate solution was added to the wells, which were incubated at room temperature for approximately 10 min. The reaction was then stopped with 100 μL of 2.5 N H₂SO₄. The absorbance of each well was read at 450 nm.

HSV antigen detection by ELISA with protein A

In order to improve the specificity of the method, a modification was done by incorporating protein A into the above-mentioned protocol for ELISA. The wells of ELISA plates were separately coated with 100 μL of CSF (1:50) and paired serum (1:200) samples. Protein A (1:4000) was added after blocking with 0.5% BSA in PBS for 45 min, and the plates were incubated for 45 min at 37°C. After incubation, the wells were washed and anti-HSV IgG was added (1:4000), and the plates were incubated at 37°C for 45 min. The wells were washed and goat anti-human IgG–horseradish peroxidase secondary antibody (1:10,000) was added and incubation was done for 45 min at 37°C. After another wash with PBS, 100 μL of the TMB-H₂O₂ substrate solution was added to the wells, which were incubated at room temperature for approximately 10 min. The reaction was then stopped with 100 μL of 2.5 N H₂SO₄. The absorbance of each well was read at 450 nm.

DNA extraction

The genomic DNA was extracted from 200 μL of CSF samples from patients by using a ZR Viral DNA kit (Zymo Research, Orange, CA), according to the manufacturer's protocol. The viral DNA was eluted from the Zymo-Spin IC columns in a volume of 25 μL of elution buffer, and was further used in PCR.

PCR assay

PCR was performed using a commercially available kit (CinnaGen Inc., Tehran, Iran) for the detection of HSV 1 and 2 per the manufacturer's protocol. The amplification was carried out in a 20-μL reaction volume containing 5 μL of template DNA, and the PCR reaction was performed in a thermal minicycler (Peqlab Biotechnology GmbH, Erlangen, Germany). Each cycle consisted of initial denaturation at 94°C for 1 min, followed by 50 cycles each consisting of 94°C for 1 min, 72°C for 40 sec, and further primer extension at 72°C for 7 min. The amplified DNA was detected by electrophoresing in a 2% agarose gel stained with 10 mg/mL of ethidium bromide (6 μL for 50 mL of agarose gel), and the gel was visualized on a UV light transilluminator (Biotech R & D Laboratories, Salem, Tamil Nadu, India).

Statistical analysis

The results are expressed as mean ± standard deviation (SD) together with the range. Cut-off values for the absorbance of the HSV antigen were calculated by using mean ± SD of the absorbance of the HSV antigen for the control group. The sensitivity (true-positive rate) of the test was calculated as [the number of samples in the HSV-infected group with an absorbance greater than or equal to the mean ± SD of the absorbance for the control group divided by the total number of samples for the HSV-infected group] ×100. The specificity (true-negative rate) for the test was calculated as [the number of samples in the HSV-infected group with an absorbance of less than the mean ± SD of the absorbance for the control group divided by the total number of samples for the group] × 100.

Results

CSF and paired serum samples of 52 patients admitted to the Neurological Department of CIIMS were grouped into HSE and non-HSE groups on the basis of PCR assay, clinical observations, and biochemical and pathological analyses of CSF samples.

Table 1 shows the occurrence of HSV antigen in CSF from the HSE and non-HSE groups as determined by the indirect ELISA method, along with the mean and range of the absorbance. The mean absorbance value for the HSV antigen in the HSE group was 1.26 ± 0.51 (range 0.84–2.33). The cut-off value for absorbance at 450 nm for a positive result of antigen detection was 0.87. The rate of positivity for HSV antigen with CSF for confirmed HSV infection was 70%, while the rate of positivity for patients in the non-HSE group (the viral encephalitis, other infectious, and non-infectious groups) was 47.6%. This higher rate of positivity in the non-HSE groups could possibly be explained as the presence of IgG in the samples that might be cross-reacting with goat anti-human IgG–horseradish peroxidase secondary antibody.

Table 1.

Demonstration of HSV Antigen in CSF from Subjects With and Without HSV Infection by ELISA Along with Mean Absorbance and Range

Patient group	Positivity for HSV Ag	Negativity for HSV Ag	Absorbance mean ± SD	Range
HSE (n = 10)	7 (70%)	3 (30%)	1.26 ± 0.51	0.84–2.33
Non-HSE (n = 42)	20 (47.61%)	22 (52.38%)	0.85 ± 0.33	0.32–1.55
Viral encephalitis (n = 24)	13 (54%)	11 (45.8%)	0.90 ± 0.36	0.39–1.55
Other infectious (n = 10)	5 (50%)	5 (50%)	0.88 ± 0.32	0.38–1.24
Non-infectious (n = 8)	2 (25%)	6 (75%)	0.68 ± 0.18	0.32–0.92

HSE, herpes simplex encephalitis; HSV, herpes simplex virus; CSF, cerebrospinal fluid; ELISA, enzyme-linked immunosorbent assay; SD, standard deviation; Ag, antigen.

Table 2 depicts that with the use of protein A in the ELISA protocol, there was a significant decrease in the rates of positivity for the non-HSE group (p < 0.028), thereby decreasing false-positivity and resulting in enhanced specificity. The mean absorbance value for the HSV antigen in the HSE group was 0.44 ± 0.17 (range 0.29–0.88), which was higher than that of the non-HSE group (0.26 ± 0.10, range 0.19–0.50). The cut-off value for absorbance at 450 nm for a positive result of antigen detection was 0.33.

Table 2.

Demonstration of HSV Antigen in CSF from Subjects With and Without HSV Infection by ELISA Using Protein A Along with Mean Absorbance and Range

Patient group	Positivity for HSV Ag	Negativity for HSV Ag	Absorbance mean ± SD	Range
HSE (n = 10)	8 (80%)	2 (20%)	0.44 ± 0.17	0.29–0.88
Non-HSE (n = 42)	6 (14.28%)	36 (85.71%)	0.26 ± 0.10	0.19–0.50
Viral encephalitis (n = 24)	5 (20.8%)	19 (79.1%)	0.30 ± 0.07	0.21–0.50
Other infectious (n = 10)	1 (10%)	9 (90%)	0.29 ± 0.04	0.21–0.36
Non-infectious (n = 8)	0	8 (100%)	0.25 ± 0. 03	0.19–0.29

HSE, herpes simplex encephalitis; HSV, herpes simplex virus; CSF, cerebrospinal fluid; ELISA, enzyme-linked immunosorbent assay; SD, standard deviation; Ag, antigen.

Table 3 shows the presence of HSV antigen in the paired sera samples of the HSE and non-HSE groups. The mean absorbance value for the HSV antigen in the HSE group was 1.90 ± 0.44 (range 1.0–2.47). The cut-off value for absorbance at 450 nm for a positive result of antigen detection was 1.34. The rates of positivity obtained were 70.8%, 70%, 50%, and 20% for the viral encephalitis, other infectious, non-infectious, and healthy control groups, which were still higher than that of HSV antigen detection in CSF, owing to the presence of significantly raised levels of immunoglobulin in the sera from all the subjects.

Table 3.

Demonstration of HSV Antigen in Paired Sera from Subjects With and Without HSV Infection by ELISA Along With Mean Absorbance and Range

Patient group	Positivity for HSV Ag	Negativity for HSV Ag	Absorbance mean ± SD	Range
HSE (n = 10)	9 (90%)	1 (10%)	1.90 ± 0.44	1.0–2.47
Non-HSE (n = 52)	30 (57.69%)	22 (42.30%)	1.61 ± 0.47	0.75–2.31
Viral encephalitis (n = 24)	17 (70.8%)	7 (29.1%)	1.74 ± 0.24	1.51–2.31
Other infectious (n = 10)	7 (70%)	3 (30%)	1.76 ± 0.43	1.12–2.22
Non-infectious (n = 08)	4 (50%)	4 (50%)	1.49 ± 0.48	0.99–1.74
Healthy controls (n = 10)	2 (20%)	8 (80%)	1.26 ± 0.36	0.75–1.27

HSE, herpes simplex encephalitis; HSV, herpes simplex virus; ELISA, enzyme-linked immunosorbent assay; SD, standard deviation; Ag, antigen.

However, in the protein A-based ELISA, there was a significant decrease in the rates of positivity in sera of the non-HSE group (p < 0.001; Table 4). The cut-off value for absorbance at 450 nm for a positive result of antigen detection was 0.44. The mean absorbance value for the HSV antigen in the HSE group was 0.63 ± 0.22 (range 0.41–1.13), which was higher than that of the non-HSE group (0.34 ± 0.24, range 0.16–1.08). However, the number of positive cases had declined in sera from the HSE group, giving a positivity of 70% (7/10), whereas antigen detection was positive for 80% (8/10) in the CSF of the HSE group. The HSV antigen was not found in the healthy control group (negativity 100%).

Table 4.

Demonstration of HSV Antigen in Paired Sera from Subjects With and Without HSV Infection by ELISA Using Protein A Along with Mean Absorbance and Range

Patient group	Positivity for HSV Ag	Negativity for HSV Ag	Absorbance mean ± SD	Range
HSE (n = 10)	7 (70%)	3 (30%)	0.63 ± 0.22	0.41–1.13
Non-HSE (n = 52)	6 (11.53%)	46 (88.46%)	0.34 ± 0.24	0.16–1.08
Viral encephalitis (n = 24)	3 (12.5%)	21 (87.5%)	0.36 ± 0.31	0.11–1.08
Other infectious (n = 10)	1 (10%)	9 (90%)	0.34 ± 0.21	0.22–0.89
Non-infectious (n = 08)	2 (25%)	6 (75%)	0.35 ± 0.18	0.16–0.62
Healthy controls (n = 10)	0	10 (100%)	0.26 ± 0.12	0.16–0.44

HSE, herpes simplex encephalitis; HSV, herpes simplex virus; ELISA, enzyme-linked immunosorbent assay; SD, standard deviation; Ag, antigen.

The concordance for antigen detection in sera and CSF of HSE (n = 10) patients was 70%, whereas concordance for negative ELISA results was 73.80% between CSF and sera samples of the non-HSE (n = 42) group (Table 5).

Table 5.

Concordance of ELISA Results Between Sera and CSF for the HSE and Non-HSE Groups

Category	ELISA results			Concordance (%)
		Serum positive	Serum negative
HSE (n = 10)	CSF positive (n = 8)	6	2	70
	CSF negative (n = 2)	1	1
Non-HSE (n = 42)	CSF positive (n = 6)	1	5	73.80
	CSF negative (n = 36)	6	30

HSE, herpes simplex encephalitis; CSF, cerebrospinal fluid; ELISA, enzyme-linked immunosorbent assay; Ag, antigen.

Discussion

HSV is one of the most widespread viruses known to cause acute and recurrent infections in humans. The early diagnosis remains difficult because the clinical picture of encephalitis caused by HSV is similar to that of other viral infections.

Before the advent of PCR analysis of the CSF, culture of brain tissue obtained by biopsy was considered the gold standard for the diagnosis of HSE. Brain biopsy is an invasive procedure and is rarely performed. In cases of suspected HSE, a sensitive HSV PCR test of the CSF is the most sensitive non-invasive lab test, but it requires sophisticated technology and well-trained personnel. There are certain problems with PCR too; due to its extreme sensitivity to contamination by minute amounts of DNA, the test may lead to false-positive results. Although the use of CSF serology has been studied extensively in HSE, it has little therapeutic value because of the delay in intrathecal antibody development and the requirement for immediate diagnosis of HSE for the treatment decision.

The detection of the viral antigen can be an alternative to isolation of the virus or the detection of viral nucleic acids, IgG, or IgM. In our laboratory, we have developed an antigen detection assay by utilizing the hyperimmune sera from patients with HSE. Pooled sera from HSV IgG-positive HSE patients were collected, IgG antibody was purified by protein G affinity column chromatography, and purified antibodies were used for the detection of HSV antigen. To our knowledge not much has been reported about the diagnostic significance of hyperimmune sera in HSE. However, studies have been reported, based on the evaluation of hyperimmune sera for antigen detection in clinical samples for the diagnosis of certain infections (9).

We assessed the usefulness of an antigen detection assay on the basis of an indirect ELISA method for the detection of HSV infection in sera and CSF from patients with suspected HSE. Our results indicate that the antigen detection assay had a higher sensitivity (90% and 70% in sera and CSF, respectively). However, a lower specificity was obtained with this method. The reason for low specificity was possibly due to cross-reactivity of goat anti-human IgG–horseradish peroxidase with host IgGs, which are mildly to moderately increased in response to an infection. The higher positivity obtained with the ELISA assay in other infectious cases compared to that of non-infectious disorders, also supports the above-mentioned possibility.

The so-called cross-reactivity with IgG of CSF and paired sera was ameliorated by introducing an additional step of protein A in the ELISA protocol. Protein A is a surface protein originally found in the cell wall of the bacteria Staphylococcus aureus. It binds proteins from many mammalian species, most notably the Fc portion of IgGs. When CSF or sera samples are added to microtiter wells, the host IgGs already present in the sample get bound to wells along with the antigens. These IgGs are then detected by anti-human IgG (used as a secondary antibody in the protocol). The protein A included in the ELISA protocol binds to those host IgGs, thus rendering them unavailable to be detected by anti-human IgG. The inclusion of protein A in the ELISA protocol resulted in enhancing the overall specificity for the detection of HSV infection. The higher positivity obtained with other infections was significantly lowered. The ELISA method utilizing hyperimmune sera along with protein A for antigen detection yielded good sensitivity (80% and 70%) and specificity (85.7% and 88.4%) in CSF and sera, respectively, for the diagnosis of HSV infection.

HSE can occur following primary infection or by reactivation of latent virus in the presence or absence of clinically-apparent manifestations (12,18,24). The high concordance (70%) for positive ELISA results for antigen detection in sera and CSF of HSE patients suggests a high probability for the presence of HSV in both the biological fluids in HSE. The presence of virus in both the CSF and sera of 6/10 individuals could be due to three reasons: (1) it is a part of the primary episode with the virus infecting the CNS via the olfactory route, and thus seeding the blood by crossing the blood–brain barrier; (2) the infection could be due to reactivation of endogenous latent virus; or (3) infection by an exogenous virus in the CNS in an already latently-infected individual. Of the non-HSE cases, out of 36 ELISA-negative CSF samples, HSV antigen was found to be positive in 6 serum samples, which could be due to reactivation of HSV without clinical relevance in those cases.

The findings of this study suggest that the diagnostic sensitivity of our ELISA-based system utilizing hyperimmune sera along with protein A is high when evaluated in both biological fluids (i.e., CSF and sera) of HSE patients. The results indicate that the indirect ELISA method used in this study is sensitive, specific, and cost-effective, and if confirmed further can be adopted in any clinical laboratory with minimal requirements. The test can not only be useful for initial screening purposes, but can also be repeated in serum during the course of illness, when there is a suspicion of HSV infection.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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