Deborah J Briggs
College of Veterinary Medicine, Kansas State University, Manhattan Kansas USA

Introduction

Conducting rabies serological testing in an appropriate and correct manner is a complicated procedure and at a minimum requires a specialized laboratory and well-trained personnel.1 Indeed, even when an adequately equipped laboratory and trained technicians are in place, the interpretation and utilization of antibody titer results obtained from rabies serological assays requires careful evaluation and interpretation. It is critical to understand that there are many different types of assays that have been (and continue to be) utilized to measure the qualitative and quantitative presence of antibodies to rabies virus but it is equally important to realize that not all rabies serological assays measure the same parameters and will not provide the same results if, for example, one serum sample is tested using different methods.2 In addition to the type of assay that is used to measure the presence of antibody to rabies virus, other criteria will significantly affect the testing results including: media components, condition of the serum sample and the cells used in the assay, dilution factor, calibration and upkeep of equipment, temperature, proficiency of the technician, strain of challenge virus used in the assay, and type of vaccine that was administered. All of these factors can be adequately monitored and controlled to provide accurate results if proper quality control and assurance measures are in place. However, even when laboratories conduct their serological assays under specific “good laboratory practices” (GLP) conditions, it should be understood that unless the investigator truly understands the strengths and weakness of the particular serological assay that he or she has used and exactly what the test was designed to measure, the results will be, at the best meaningless and at the worst incorrect. With these facts in mind, the following manuscript was prepared to serve as an introduction to the various types of rabies serological assays currently available and to provide specific background information to enable researchers and regulatory officials to choose the most appropriate serological test that will fulfill the specific requirements that he or she has in mind.

Categories of Assays

The three types of serological assays that have been utilized to measure the presence of rabies virus antibody are described below:

1. Antigen-binding assays measure the direct interaction between an antibody and antigen and are analogous to a specifically designed key (antibody) fitting into a lock (antigen). It is important to note that antigen-binding assays are not designed to specifically measure virus-neutralizing antibody, but simply measure the interaction or ‘binding’ of antibody to antigen. Examples of antigen-binding assays include: enzyme linked immunoassays (ELISA or EIA); immunofluorescence assays (IFA); and radioimmunoassay (RIA). The advantage in using antigen-binding assays is that they are usually quite sensitive. These assays can be used to measure the presence of specific classes of rabies immunoglobulins that may be present, for example, the presence of IgM that occurs early in an infection. Additionally, if purified viral antigens of specific strains of virus are available, (for example a new rabies viral variant), they can be used to detect antibodies that have been produced against this new strain. Therefore, antigen-binding assays are very useful for epidemiological studies and in some cases are used for diagnoses. One more advantage is that these assays use a killed antigen and therefore, there is no need for specialized facilities to contain and manipulate infectious live rabies virus. The disadvantages of antigen-binding assays include the fact that they do not distinguish between different types of antibodies and are therefore measure all types of antibody including antibodies that do not necessarily neutralize rabies virus. Additionally, the viral antigen that is used to coat the plates will influence the binding of the antibody and therefore, these assays will only detect antibody specific for the epitopes that are coated and present on the plates. Thus other rabies virus antibody present in the serum that is not specific for the epitopes bound to the plate will not be detected. It is important to stress once more that antigen-binding assays do not measure rabies virus neutralizing antibody and therefore should never be used to test the efficacy or immunogenicity of rabies vaccines.

2. Antibody-function assays measure a nonvirus related function that is caused as a result of the antibody binding to the antigen. Figuratively speaking, these assays are comparable to putting the specially designed key (antibody) into the lock (antigen), turning the key and opening the door (non-virus function). In this case, it is the function of opening the door that is being measured. Examples of antibody-function assays include: complement fixation (CF); immune adherence hemagglutination (IAHA); complement-mediated cell lysis (CMCL); passive hemagglutination (PHA); mixed hemadsorption (MH); and counter immuno electrophoresis (CIE). The advantage of antibody-function assays is that they generally use inactivated virus or viral proteins and therefore do not require special facilities. They are also useful for diagnostic purposes since they can detect IgM antibody, which is especially active in CF and precipitation of antigens. The disadvantage of using these types of assays is that they are less sensitive to small changes in antibody concentration. Additionally, they are not able to distinguish between the types of antibody present in sera and therefore should never be used to detect neutralizing antibody thus eliminating their usefulness in the evaluation of rabies vaccines.

3. Antigen-function assays measure the capacity of the antibody to prevent a specific viral function. Again, if one prefers a visualization of what is happening, antigen-function assays are comparable to putting the specially designed key (antibody) into the lock (antigen), which causes the lock to be malfunction. In this case, if the virus is destroyed and therefore unable to infect the cells and reproduce, it is categorized as a virus neutralization test. Examples of antigen-function assays include : neutralization tests (NT); interference inhibition tests (IIT); and hemagglutination inhibition tests (HI). Antigen-function assays measure antibodies to the rabies glycoprotein, the antibodies responsible for conferring immunity to lethal rabies infection. Although cell mediated immunity is also critical for inducing protection against infection, measuring the presence of neutralizing antibody is a simpler procedure and has come to be recognized as the means by which to evaluate the immunogenicity of a vaccine. Therefore the main advantage of NT is that they can provide a measurement of the immunogenicity of a rabies vaccine whereas the antigen-binding assays and antibody-function assays mentioned above do not. The disadvantage of NT is that they only measure antibody to the surface glycoprotein and therefore do not detect antibody to other viral proteins. Thus, they are less useful for diagnoses and surveillance. Additionally, results from NT are dependent upon the rabies virus strain used as the challenge virus in the assay and how closely related this virus is to the seed strain used in the production of the vaccine that a subject received.3 There are three types of NT used for measuring the presence of rabies virus neutralizing antibody: Mouse Neutralization Test (MNT); Rapid Fluorescent Focus Inhibition Test (RFFIT); and the Fluorescent Antibody Neutralization Test (FAVN). The MNT is heavily dependent upon several factors, including the strain of mice used, their age and the nonspecific mortality that can occur within each assay. Currently, WHO recommends both the RFFIT and FAVN as appropriate tests for the measurement of immunogenicity after vaccination and both are considered to provide equivalent results if proper quality control and assurance measures are in place.

Conclusions

Serological assays to measure the presence of antibody to rabies virus have been used for decades and represent an extremely useful tool for diagnosis, epidemiology and the measurement of an immunological response after vaccination. However, it is critical to understand that not all serological assays measure the same type of antibody and therefore should not be considered to be equivalent. The type of serological assay that is most appropriate for a research project may differ substantially from the type of assay needed to evaluate a new or existing vaccine, or the rabies serological test best suited to provide the most precise information when needed for diagnosis. Careful consideration and the proper choice as to the type of serological assay used to analyze sera are essential in order to provide the most accurate data required to help control and prevent rabies.

Literature Cited

1. Smith, J S, P A Yager, and G M Baer. A rapid fluorescent focus inhibition test (RFFIT) for determining rabies virus-neutralizing antibody. In: Laboratory techniques in rabies, 4th ed. Eds: Meslin, F X, Kaplan, M M, and Koprowski, H. WHO, Geneva. 1996. pp.181-192.

2. Smith, J S. Rabies serology. In: The natural history of rabies, 2nd ed. Ed. G M Baer. CRC Press, Boca Raton. 1991. Pp. 236-252.

3. Moore, S M, T A Ricke, R D Davis, and D J Briggs. The influence of homologous vs. heterologous challenge virus strains on the serological test results of rabies virus neutralizing assays. In press: Biologicals.

4. Briggs, D J, J S Smith, F L Mueller, J Schwenke, R D Davis, C R Gordon, K Schweitzer, L A Orciari, P A Yager and C E Rupprecht. A comparison of two serological methods for detecting the immune response after rabies vaccination in dogs and cats being exported to rabies-free areas. Biologicals. 26:347-355. 1998.

5. WHO Expert Consultation on Rabies. Technical Report Series 931. WHO, Geneva. 2004.