Microneutralization assay for the measurement of neutralizing antibodies to human metapneumovirus

Plaque assay (PA) is a gold standard for virus titration and neutralization of various cytopathic viruses, including avian nephritis virus (ANV), the etiological agent associated with kidney disorders in chickens. In this study, as an alternative to the labor-intensive PA, we developed a spectrophotometric microplate assay (MA) for ANV titration and neutralization based on the virus cytopathicity to primary chicken kidney (CK) cells.

CK cells were infected with ANV in the presence or absence of chicken serum in a 96-well microplate, and the virus-induced cytolysis was quantified by measurement of neutral red uptake using a spectrophotometer. The absorbance values obtained were subjected to a sigmoidal four-parameter logistic regression analysis for the virus titer determination and serum neutralization assessment. Accuracy and reliability of the serum neutralization MA in comparison to the standard PA was statistically evaluated.

The ANV-MA was capable of quantifying infectious virus titers based on a virus dose-dependent cytolysis of CK cells, and serum neutralization could be assessed as an inhibition of the virus-induced cytolysis accordingly. Statistical evaluation using a 2 × 2 contingency table and receiver-operating characteristic analyses showed 82 % sensitivity, 99 % specificity and 0.97 area under the curve, supporting an overall diagnostic accuracy of the neutralization MA.

The newly developed MA using simplified experimental procedures in the microplate format and direct spectophotometric data readout is readily applicable to general laboratories for high-throughput screening of serum neutralization of ANV.

Plaque Reduction Neutralization Test (PRNT) is the standard assay used for measuring neutralizing antibody responses to Herpes simplex virus type-2 (HSV-2). The PRNT is a cumbersome, time-consuming and laborious assay. The development of a faster, high throughput microneutralization assay (MNA) for HSV-2 viruses carried out in a 96-well format will allow for rapid testing of large numbers of samples for drug and vaccine development.

We describe the generation of a MNA that utilizes a pair of anti-HSV human monoclonal antibodies (mAbs) for virus detection in HSV-2 infected Vero cells. Antibodies were generated by B-cell cloning from PBMC’s isolated from HSV-1 negative/HSV-2 positive donors. We describe the selection and characterization of the antibodies used for virus detection by ELISA with purified, recombinant anti-HSV glycoproteins, antibody binding in infected cells, and Western Blot. We determine the anti-HSV-2 neutralizing titers of immune sera from mice by MNA and PRNT and compare these results by linear regression analysis.

We show that neutralization titers for HSV-2, determined by the 96-well MNA correlate with titers determined by a PRNT completed in 24-well plates in both the absence (R² = 0.8250) and presence (R² = 0.7075) of complement.

We have successfully developed an MNA that can be used in place of the burdensome PRNT to determine anti-HSV-2 neutralizing activity in serum. This MNA has much greater throughput than the PRNT, allowing many more samples to be processed in a shorter time saving ∼90 % of the time required by the laboratory scientist to complete the task as compared to the traditional PRNT.

Human metapneumovirus (HMPV) is a common cause of respiratory diseases in persons of all ages. Because of its slow replication and weak cytopathic effect in cultured cells, conventional neutralization assays for HMPV require around one week for completion.

The purpose of this study is to establish a rapid neutralization assay based on a recombinant virus expressing Renilla luciferase (Rluc).

A recombinant HMPV expressing both Rluc and green fluorescent protein (GFP) was created by reverse genetics method. Two-fold serial dilutions of human 23 sera were made in a 96-well plate and incubated with 50 pfu/well of the recombinant virus at 4 °C for 1 h. The mixtures were then transferred to LLC-MK2 cells in a 96-well plate, incubated for 2 h, and replaced with trypsin-free fresh media. After incubation at 32 °C for 24 h, the cells were lysed and measured for Rluc activity. The neutralization titer was defined as the reciprocal of the highest serum dilution that resulted in 50% reduction of Rluc activity.

The novel assay could be completed within 24 h and eliminated the requirement of trypsin supporting multistep replication in cultured cells, as well as laborious processes including the plaque assay with immunostaining. Neutralization titers correlated well with those determined by a GFP-based assay previously developed.

The neutralization assay based on Rluc activity is the fastest and the most straightforward of all previous assays, and may be available for high throughput screening of neutralizing antibodies.

A plaque reduction neutralisation test (PRNT) is still regarded as the gold standard for the investigation of anti-measles immunity. In this study, an alternative simplified automatable focus reduction neutralisation test (AFRNT) based on the classical PRNT was developed. The AFRNT uses the conventional Edmonston strain of measles, immunoperoxidase staining with monoclonal antibodies, and automated plaque counts performed with AID ViruSpot software. The assay is performed in 96-well plates, requires 2 days, and is fully automatable. The AFRNT was evaluated in comparison with PRNT and Enzygnost anti-measles enzyme immunoassay (EIA). A total of 130 samples, which included two available WHO international anti-measles standards, sera from 90 patients, and 38 different lots of immunoglobulin products, were tested. Overall, good agreement was observed between EIA and both neutralisation tests; however, the EIA values for the immunoglobulin products and international standards were slightly but significantly higher than those of the neutralisation tests. The Bland–Altman analysis showed excellent agreement between AFRNT and PRNT. AFRNT is a fully automatable high-throughput neutralisation assay, which can be performed with measles and other types of viruses, including wild-type strains. It is perfectly suited for epidemiological and vaccine studies.

Human metapneumovirus (hMPV) is a significant cause of respiratory illness in children and adults. Presently, there are no human data regarding the role of antibody for protection against hMPV illness. Therefore, we measured serum and nasal antibody titers against hMPV by EIA and neutralization assay at baseline in hMPV infected adults compared with subjects who remained uninfected. Antibody titers were also compared in patients with mild and severe illness.

Mean serum binding and neutralizing antibody titers of hMPV infected subjects were significantly lower compared to uninfected subjects. Seventy-one percent of subjects with titers ≤10.5 (log 2) were infected compared to 36% with titers >10.5, p = 0.003. There was no difference in the mean acute antibody titers for patients with mild compared to severe illness. Serum antibody may play a role in protection from hMPV infection supporting the development of an hMPV vaccine that stimulates humoral immunity.