ABSTRACT
An automatable focus-reduction neutralisation test (AFRNT) for detecting influenza neutralising antibodies in serum was developed. The assay used immunoperoxidase staining and automated foci counting with AID Diagnostika ViruSpot software. Human serum samples (n=108) were collected before and after vaccination with Pandemrix or Begrivac and were tested by AFRNT and a haemagglutination inhibition assay (HI) using seasonal and pandemic influenza vaccine strains from 2009 to 2011. Much attention has been given to the factors that influence detection of neutralising titre, such as viral quantification and the use of receptor destroying enzyme (RDE) for serum treatment. Foci counting enabled precise virus quantification and the development of a highly sensitive assay. Pre-treatment of the human sera with RDE significantly reduced the neutralising titres against all strains, with the exception of the seasonal H1N1 (2009/2010) strain. An HI titre of 1:40, which is associated with a 50% clinical protection against influenza, was equivalent to an AFRNT titre of 1:100-1:200. In conclusion, the AFRNT is rapid, highly sensitive, and fully automatable; therefore, this test is perfectly suitable for the high-throughput detection of influenza-neutralising antibodies.
Subject(s)
Antibodies, Neutralizing/blood , Antibodies, Viral/blood , High-Throughput Screening Assays , Influenza, Human/immunology , Neutralization Tests/methods , Adolescent , Adult , Automation, Laboratory/methods , Child , Female , Humans , Influenza Vaccines/administration & dosage , Influenza Vaccines/immunology , Influenza, Human/prevention & control , Male , Middle AgedABSTRACT
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.
Subject(s)
Antibodies, Viral/blood , Image Processing, Computer-Assisted/methods , Measles virus/immunology , Neutralization Tests/methods , Viral Plaque Assay/methods , Antibodies, Viral/immunology , High-Throughput Screening Assays/methods , Humans , Immunoenzyme Techniques/methods , Time FactorsABSTRACT
A convenient rapid culture assay (RCA) for the detection of enteroviruses was evaluated against RT-PCR using 576 stool and 102 cerebrospinal fluid (CSF) samples. One hundred and ninety stool samples were also tested by conventional cell culture (CCC). The RCA used immunoperoxidase staining of cell culture plates with a blend of monoclonal antibodies (mAbs) against enterovirus VP1 on the second and sixth days after inoculation. This blend was composed of 5D8/1 (Dako) and four 'in-house' mAbs. CCC was performed using fluorescence staining with the Enterovirus Screening Set (Chemicon International) for culture confirmation. Detection of enteroviruses by the RCA was more successful in colonic carcinoma (CaCo-2) and rhabdomyosarcoma (RD) cells than in human embryonic lung fibroblasts, HEp2 and A549 cells. The performance of CCC in RD cells was hindered by rapid cell degeneration and non-specific staining of cells during culture confirmation. The sensitivity of the RCA compared to RT-PCR in stool samples was found to be 71 % (115/161) on the second day and 87 % (140/161) on the sixth day. The sensitivity of the RCA in CSF samples was 38 % (22/58) after 2 days and 59 % (34/58) after 6 days. The specificity of the RCA was 100 %. All CCC-positive samples were positive by the RCA. CCC required 3-14 days for virus recovery. In conclusion, the RCA has the same sensitivity as CCC, significantly shortens the time required for the detection of enteroviruses, and prevents pitfalls associated with using RD cells for CCC. For diagnosis of aseptic meningitis in CSF samples, RT-PCR should be performed.
