A rapid enzyme immunoassay for the determination of antibodies to rabies virus
Rabies is world-wide in distribution. Despite significant scientific advances in its prevention and control, the disease has been spreading in most parts of the world where it continues to persist as a major public health problem. The disease is endemic in Kenya where the dog plays a major role in its transmission to man. In view of the almost 100% mortality rate of human rabies, prevention becomes essential. Also in animals, rabies is a disease approaching a 100% case fatality rate, but it has been known since the days of Pasteur that some animals survive both natural and experimental infection, and that a healthy and infective carrier state may occur. Since efficient vaccines are now available, vaccination may be performed on a large scale in relevant animals and in high-risk human populations. The emergence of highly efficient vaccines has to a large extent obviated the need to confirm adequate immune responses of vaccinated individuals by serum antibody determinations. Nevertheless, a rapid, specific and reproducible assay for the detection and accurate quantitation of antibodies to rabies virus would be highly desirable for the following purposes : (i) evaluation of potency of vaccines, (ii) evaluation of individual immune responses, (iii) standardization of hyperimmune antiserum for therapeutic/prophylactic use, (iv) determination of efficacy of large-scale vaccination programmes, (v) aid in the diagnosis of rabies in suspected non-vaccinated cases, (vi) comparative studies of rhabdoviruses. The results obtained by the mouse neutralization test (MNT) and the rapid fluorescent focus inhibition test (RFFlT) have been recognized as true reflections of the protective potency of a serum, and can be expressed as international units per millilitre (I.U./ml) in comparison with an internationally recognized reference serum. Both tests suffer from poor reproducibility and demands for highly skilled man-power. The absolute requirements for live infective challenge virus, a large number of animals or tissue culture capability as well as special facilities and equipment render these two neutralization tests unsuitable for routine or large-scale use in most countries. Although promising, most of the replacement tests have been found to be inadequate largely because of poor correlation with the MNT and the RFFlT. In this study, an inhibition enzyme immunoassay (INH-EIA) for the detection and quantitation of rabies antibodies was developed and evaluated. In this system, the interaction of specific enzyme-labelled antibodies with their antigen is inhibited by non-labelled antibodies of the same specificity. The antibody titre is expressed as that dilution of a serum sample which gives 50% inhibition. This can subsequently be converted to equivalents of I.U./ml calculated on the basis of an antirabies reference serum whose potency has been determined by the MNT or the.RFFIT. The INH-EIA was carried out by coating microtitre plates with a predetermined dilution of a rabies virus preparation. The IgG fraction of absorbed serum from a goat immunized with human diploid cell rabies vaccine from InstitutMerieux, France, was conjugated with horseradish peroxidase and shown to be specific for rabies virus components. It was shown that the conjugate possessed antibody activities to both the glycoprotein and the ribonucleoprotein components of the virus when compared with known antiglycoprotein and antiribonucleoprotein sera obtained from other laboratories. No inhibition was observed when dilutions of tissue culture homogenates and known negative sera from 7 different animal species were assayed. A high ionic strength salt (1M KCl) buffer containing 2% polyethylene glycol 6000 was used as test serum diluent. The composition of the diluent would enhance antigen-antibody interactions and might consolidate these reactions, thus presumably allowing the estimation of even low affinity antibodies. The INH-EIA was performed sequentially allowing antibodies of the test serum to interact with antigen prior to the addition of conjugate. It is expected that such an assay would be capable of detecting both low and high affinity antibodies, and also low levels of antibody. The ability of the INH-EIA to detect low levels of antibody was evident when low but definite antibody activity (equivalent to 0.25 - 1.95 I.U./ml) was detected in 5 human sera obtained 7 days after primary intradermal vaccination with human diploid cell rabies vaccine. The assay gave a mean titre equivalent to 0.06 I.U./ml (range 0.04 - 0.08 I.U./ml) in 14 sera from non-vaccinated humans. The ability of the INH-EIA to detect low levels of antibodies should render the test suitable for the intra-vitam diagnosis of rabies. This became evident when a serum sample from a previously unvaccinated rabid goat, bled one week before death, gave an antibody titre corresponding to 2.0 I.U./ml, i.e. ten times the highest titre obtained in serum samples from non-vaccinated humans and animals. The reproducibility of the INH-EIA was assessed by performing 15 complete titrations of each of two antirabies sera obtained from the World Health Organization (WHO) and the Institut Herieux. The WhO serum which had a potency of 10 I.U./ml as determined by the MNT, gave a mean titre correspondng to 8.7 I.U./ml (range 7.2 - 9.9, coefficient of variation + 10%) calculated on the basis of the Institut Merieux serum. The Institut Merieux serum gave a mean titre corresponding to 174 I.U./ml (range 151.6 - 205.4 I.U./ml, coeff. var. + 10%) when the WHO serum was used as a standard. The Institut Merieux serum had been titrated against an international standard by using the RFFIT. A mean titre equivalent to 194 I.U./ml (range 183 - 209, coeff. var. + 6%) was found. Thus, excellent agreement was found between the INH-EIA and both the MNT ana the RFFIT. In the INH-EIA, duplicate single serum dilutions of 1:2 could be used to accurately quantitate rabies antibodies as long as the inhibition was within 20-75%. This procedure proved valuable in the screening of large numbers of serum samples from any animal species within a short time. The highest antibody titre obtained in sera from non-vaccinated persons and animals was 0.2 I.U./ml. Consequently, this titre was chosen as a cut-off point for the differentiation of rabies antibody positive and negative sera. Some of the sera from man and different animal species gave antibody titres above the equivalent of 0.2 I.U./ml. This was notable with sera obtained from goats, cattle, elephants and hyenas. Since no antirabies vaccination is carried out in these animals in Kenya, it is surmised that the animals may have been exposed to the rabies virus or to one of the rabies-related viruses. It is concluded that INH-EIA is a highly reproducible, sensitive and specific method for the detection and quantitation of antibodies to rabies virus and correlates well with the internationally accepted MNT and RFFIT. The method allows the screening at a single serum dilution of large numbers of specimens from different animal species. It is therefore likely that the method will find considerable application in seroepidemiological studies of rabies. The INH-EIA appears to be an attractive substitute for both the hNT and the RFFIT because of its high sensitivity and specificity, high reproducibility, ease of performance and interpretation, rapidity and low cost. Further investigations are needed to confirm whether the antibody titres obtained in the INH-EIA represent true reflections of various levels of protective immunity.