Med Microbiol Immunol (2007) 196:151–155 DOI 10.1007/s00430-007-0037-2
O RI G I NAL IN VE S T I GA TI O N
Comparison of the neutralizing and ELISA antibody titres to measles virus in human sera and in gamma globulin preparations Holger F. Rabenau · Branko Marianov · Sabine Wicker · Regina Allwinn
Received: 12 December 2006 / Published online: 17 February 2007 © Springer-Verlag 2007
Abstract Measles virus infection as well as measles vaccination induces a long-lasting immune protection. SpeciWc antibodies have been proven to be associated with this immune protection, since measles immunity can be transferred by immune globulin application (passive immunisation). The neutralisation test (NT) is regarded as the gold standard method for measles immunity because it measures functional neutralising antibody, while with the ELISA, which is often based on cell culture grown native virus antigens, predominantly antibodies to the nucleoprotein antigen were detected. To compare the results of NT and ELISA 199 individual sera and 364 gamma globulin samples, which were made from plasma pools, were tested. Qualitative results showed that the sensitivity of the ELISA was 141/144 (97.9%) and speciWcity was 48/55 (87.3%) when compared to the NT and focused to the patient samples. For the gamma globulin samples the sensitivity and speciWcity was 100%. As expected no measles NT negative plasma pool samples were found. The present study showed that with increasing NTtitre, the ELISA-values also rise. False negative ELISA results were obtained in 1.5% of patient sera, mainly containing low levels of neutralising antibody. In both antibody tests seropositive specimens revealed a quite good to moderate correlation. Taken together, H. F. Rabenau (&) · B. Marianov · R. Allwinn Institute of Medical Virology, University Hospital, Frankfurt/Main, Paul-Ehrlich-Str. 40, 60596 Frankfurt/Main, Germany e-mail:
[email protected] S. Wicker Occupational Health Service, University Hospital of Frankfurt, Theodor Stern Kai 7, 60590 Frankfurt/Main, Germany
the measles IgG ELISA is adequately for immunity testing and identifying of seronegative individuals for vaccination. Keywords Measles virus · Immunity · Neutralisation test · ELISA antibody titres · Gamma globulin preparations
Introduction Measles is a leading cause of vaccine preventable death in children and infants worldwide [13]. The highest mortality rates are seen in developing countries but the recent measles outbreak in Germany and other industrialised countries with sub-optimal vaccination programmes shows that there is also a risk of outbreaks and resulting fatalities [2]. Measles virus infection usually induces a lifelong immunity. A long-lasting immune protection is also delivered by vaccination using live attenuated virus. Serum antibodies have been proven to be associated with this immune protection since measles immunity can be transferred by immune globulin application (passive immunisation). Public health authorities in many countries conWrm quality assurance of those immunoglobulins by determination of antibodies in a classic neutralisation test (NT). The NT is regarded as the gold standard method for measles immunity because it measures functional neutralising antibodies [5]. As an alternative, haemaglutination inhibition test (HI) might be applied in the same way, as rubella immunity is assessed. However, NT is a biological assay quite cumbersome and has not been standardized on a national or international level and does not lend
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itself readily to large-scale surveys. Furthermore NT is technically demanding and takes up to a week to complete. Measles IgG antibody detection and quantitation by ELISA has proven as a routine test widely distributed and well standardized. This test is easy and quick to perform and can be automated. Thus, this assay is preferred to routine testing of the individual immunity, although not the viral envelope (the target of NT antibodies), but the inner nucleoprotein is the predominating antigen in commercially available test kits. Similar to rubella, the borderline titre of such serum antibodies is considered to assure protection, in particular in people revealing a low level humoral immune status. Here, we present the results of a study comparing neutralising (NT) and ELISA antibody titres in specimens of individual sera and gamma globulin preparations.
Materials and methods Patient and gamma globulin samples A total of 199 serum samples from patients (99 males and 100 females, period January–November 2002) from the Institute of Medical Virology, University Hospital, Frankfurt/Main were examined Wrst by means of enzyme-linked immunosorbent assay (ELISA) and afterwards by means of neutralisation test (NT) for measles-speciWc (neutralisation-competent) antibodies. The median age was 25 years (range 2–78 years). The following patient groups were selected, control group: measles IgG ELISA negative sera (30,000 mlU/ml (1 male, 2 females). Additionally, 364 gamma globulin samples which were arranged from plasma pools from up to 10,000 plasma single donations (kindly provided by commercial manufacturers) were examined by means of measles IgG ELISA and NT. Test systems Enzymimmunoassay (ELISA) A commercially available ELISA was used for the quantitative testing of measles-speciWc antibodies
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(Dade-Behring GmbH, Liederbach, Germany, Enzygnost anti-measles virus IgG). The ELISA is based on (monkey kidney) cell culture grown native virus antigen. The test was performed according to the manufacturer’s instructions using the ELISA Processor BEP II plus (Dade-Behring). QuantiWcation took place as stepless indication of titre (in mlU/ml) according to the alpha method. Titres similar or above 200 mlU/ml were considered as anti-measles IgG positive. Measles neutralisation test (NT) The determination of neutralisation-competent antibodies was performed as already mentioned [9]. In short, serum samples were serially diluted in twofold dilution steps (1:10, 1:20... 1:1,280). Flat-bottomed 96-well microtiter plates (Nunc, Wiesbaden, Germany) were simultaneously inoculated with 25 l measles virus (4,000 TCID50/ml strain; Edmonston, Source; American type culture collection [ATCC]) and 25 l of each serum dilution. Each sample was tested in triplicate. The inoculated 96-well plates were incubated for 1 h at 37°C, afterwards 50 l of Vero cells (monkey kidney cells, Source; ATCC, concentration 300,000 cells/ml) were added. After that the plates were incubated at 37°C in a 5% CO2 atmosphere for 3 and/or 5 days before the cells were Wxed, coloured with speciWc anti-measles antibodies (Paesel and Lorei, Hanau, Germany), a biotinmarked secondary antibody and a streptavidin peroxidase conjugate and/or were evaluated by microscopic examination for virus-speciWc cytopathogenic changes (CPE). The neutralisation antibody titre corresponded to the reciprocal of the highest serum dilution showing a complete inhibition of the CPE in at least 66% of the cell cultures. A serum sample was considered positive if neutralisation antibodies were present at a titre equal or superior to 1:10; however, with questionable immune protection. Starting from a titre of 1:20 a safe immune protection was assumed. Statistic evaluation The evaluation of the results of the NT and ELISA took place by means of simple linear regression analysis (95% conWdence interval) and correlation according to Spearman (software BIAS 8,02, Epsilon Verlag Hochheim, Darmstadt) [11]. Therefore, measles IgG-ELISA values were grouped in seven categories (200–600, 601– 1,000, 1,001–2,500, 2,501–10,000, 10,001–20,000 and 20,001–>30,000 mlU/ml).
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Results For the correlation of the results in the anti-measles IgG ELISA and neutralisation test 199 patient sera were examined. To assure neutralising activity of a serum, a minimum ELISA titre of 200 mlU/ml is mandatory. Table 1 shows that 48 (94.1%) of 51 sera negative in the ELISA (
