An ELISA-based method for detection of rabies virus nucleoprotein-specific antibodies in human antemortem samples

Abstract

Rabies is a fatal encephalitic disease in humans and animals caused by lyssaviruses, most commonly rabies virus (RABV). Human antemortem diagnosis of rabies is a complex process involving multiple sample types and tests for the detection of antibodies, antigen (protein), and nucleic acids (genomic RNA). Serological diagnosis of human rabies includes the detection of either neutralizing or binding antibodies in the cerebrospinal fluid (CSF) or serum samples from unimmunized individuals without prior rabies vaccination or passive immunization with purified immunoglobulins. While neutralizing antibodies are targeted against the surface-expressed glycoprotein (G protein), binding antibodies to viral antigens are predominantly against the nucleoprotein (N protein), although there can be antibodies against all RABV-expressed proteins. To determine N protein-specific antibody responses in the CSF and serum during RABV infection, we developed an enzyme-linked immunosorbent assay (ELISA) with purified recombinant N protein expressed in E. coli. N protein-specific immunoglobulin (Ig) subtypes IgG and IgM were detected in the CSF or serum of previously diagnosed human rabies cases. In addition, anti-N protein seroconversion was demonstrated over the course of illness in individual rabies cases. We compared the N protein ELISA results to those of an indirect fluorescent antibody (IFA) test, the current binding antibody assay used in diagnosis, and show that our ELISA is consistent with the IFA test. Sensitivity and specificity of the N protein ELISA ranged from 78.38-100% and 75.76-96.77% with respect to the IFA results. Our data provide evidence for the use of an N protein ELISA as an additional option for the detection of RABV-specific IgG or IgM antibodies in human CSF or serum specimens.

Fig 1. Antibodies in patient serum are able to detect RABV N protein.

MNA cells grown on glass cover slips were infected with RABV CSV-11 strain for 20 hours. Antibodies present in a human serum samples from a rabies positive patient was used to detect rabies antigen (green, top left panel) and serum from a non-rabies case was used as a control (bottom left panel), followed by FITC conjugated anti-human IgG for visualization. RABV N protein (red) was visualized using anti-N protein mAbs (middle top and bottom), followed by Alexa 594 conjugated secondary antibody. Merged image of rabies serum with N protein is shown on the top right and merged non-rabies serum with N protein is shown on the bottom right. Merged images include the nucleus (blue), which was visualized using DAPI. Scale bar = 10μm. Original magnification: x40.

Fig 2. N protein expression purity and confirmation of native epitopes.

(A) Recombinant N protein was separated on SDS-PAGE gel at indicated concentration and stained with Coomassie stain for visualization of all proteins in the N protein preparation. Protein ladder indicates estimated size of protein bands. (B) OD absorbance value of N protein detected with primary mAbs against N protein conformation epitopes at different antibody concentrations in an ELISA format. Mouse IgG isotype was used a control. Secondary antibody against mouse IgG conjugated with HRP was used.

Fig 3. N protein IgG and IgM ELISA values in CSF relative to IFA result.

ELISA values were determined by subtracting the COV from the OD values for each sample. (A) ELISA values for each IFA positive and negative CSF samples tested for IgM are shown. Values >0.02 are N positive. Statistics reported are with the exclusion of outlier data point. (B) CSF samples tested for IgG are shown. Values >0.00 are positive. IFA and ELISA results used to calculate sensitivity and specificity for (C) IgM and (D) IgG. The 95% confidence interval (CI) for each is shown. Mean and standard error are indicated by the error bars. *, P ≤ 0.01; **, P ≤ 0.001; ***, P ≤ 0.0001.

Fig 4. N protein IgG and IgM ELISA values in serum relative to IFA result.

ELISA values were determined by subtracting the COV from the OD values for each sample. ELISA values for each samples for (A) IgM and (B) IgG are shown. Values >0.00 are ELISA positive. IFA and ELISA results are shown and used to calculate sensitivity and specificity for (C) IgM and (D) IgG. The 95% confidence interval (CI) for each is shown. Mean and standard error are indicated by the error bars. *, P ≤ 0.01; **, P ≤ 0.001; ***, P ≤ 0.0001.

Fig 5. Detection of antibodies in CSF and serum of rabies positive cases.

(A) A series of CSF and serum specimens from 16 diagnosed rabies cases (numbered 1–16) were tested for IgG and IgM N protein binding by ELISA and results were compared to IFA results. ‘P’ indicates the test result was positive, ‘N’ indicates the test was negative, and ‘−’ indicates no result could be obtained. (B) The total number of rabies cases with IFA positive or IFA negative results are listed for each IgM or IgG IFA test and the breakdown of how many of those were ELISA negative, positive, or had no data available due to sample availability are indicated.

Fig 6. N protein antibodies over the course of illness in the CSF of rabies cases.

N ELISA values were determined by subtracting the COV from the OD values for each sample. N protein IgG (blue circles) and IgM (red circles) ELISA values for a series of specimens from a single rabies case corresponding to Table 1A are shown. The corresponding IFA result for that particular sample is indicated by the (+) symbol for a positive result, (-) for a negative result, and no symbol indicates no IFA data was available for that sample. The color of the symbol corresponds to the IFA result for IgG (blue) or IgM (red). The x-axis indicates days from illness onset that the specimen was collected and the y-axis shows the ELISA value. (A) Rabies case 3 IgG and IgM N ELISA values. (B) Rabies case 4 IgG and IgM N ELISA values. (C) Rabies case 14 IgG and IgM N ELISA values. (D) Rabies case 15 IgG and IgM N ELISA values. (E) Rabies case 16 IgG and IgM N ELISA values.

Fig 7. N protein antibodies over the course of illness in the serum of rabies cases.

N ELISA values were determined by subtracting the COV from the OD values for each sample. N protein IgG (blue circle) and IgM (red circles) ELISA values for a series of specimens from a single rabies case corresponding to Table 1A are shown. The corresponding IFA result for that particular sample is indicated by the (+) symbol for a positive result, (-) for a negative result, and no symbol indicates no IFA data was available for that sample. The color of the symbol corresponds to the IFA result for IgG (blue) or IgM (red). The x-axis indicates days from illness onset that the specimen was collected and the y-axis shows the ELISA value. (A) Rabies case 3 IgG and IgM N ELISA values. (B) Rabies case 4 IgG and IgM N ELISA values. (C) Rabies case 8 IgG and IgM N ELISA values. (D) Rabies case 10 IgG and IgM N ELISA values. (E) Rabies case 15 IgG and IgM N ELISA values. (F) Rabies case 16 IgG and IgM N ELISA values.