The detection of antiviral antibodies is an essential process in the molecular biological diagnosis of viral disease Brasil et al., 2010, p337. The most commonly used technique for detecting these antibodies is through enzyme-linked immunosorbent assay (ELISA), as this is sensitive, inexpensive, reproducible and easily adapted for large-scale screening techniques (Winkler, 2005, p257). ELISA has been shown to assist in identifying antigens for a variety of viral infections, including measles, mumps, rubella and cytomegalovirus (Knuf et al., 2012; p464; Liu et al., 2012, p34). This may be useful in detecting active viral infection, or previous exposure to a virus through vaccination or previous infection (Knuf et al., 2012, p464). Therefore, the results of ELISA can be used for diagnostic purposes as well as to determine the effectiveness of vaccination schedules in the general population.
The aim of this experiment was to utilise ELISA in the identification of antiviral antibodies from patient sera, focusing on measles, mumps, rubella and cytomegalovirus infections. These conditions may be associated with a high level of morbidity and mortality in some patient groups and therefore accurate diagnosis is essential in clinical diagnosis (Vyse et al., 2002, p125). Furthermore, the presence of immunity to these viruses may be important in healthcare professionals and other workers who could potentially expose patients to these viruses (Ziegler et al., 2003, p400). Hence, the evaluation of indirect ELISA in diagnosis and immunity detection will be discussed based on these findings.
Materials and methods
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Antigen-coated microtitre plates were utilised in this experiment, with antigens for measles, umps, rubella and cytomegalovirus. Two samples of patient sera were used for detection of antiviral antibodies. Incubation buffer was used for diluting patient sera and diluting the conjugate, while pre-diluted positive and negative sera were used as controls. Anti-human IgG-alkaline phosphate conjugate (Sigma) was used to detect human IgG and 1mg/ml p-nitrophenyl phosphate in glycine buffer (pH10.4) was the substrate for colour detection.
Viral antigens were attached to microtitre plates by adding 200Î¼l of the diluted antigens and incubating overnight at 4Â°C. Excess antigen was removed with three washes of blocking buffer and then patient sera samples were added to the plate. Sera were diluted 1/100 through addition of 50Î¼l to 5ml of incubation buffer. Diluted sera were added to the plate (200Î¼l) and the same volume of positive and negative control sera were added to corresponding viral antigen-coated wells. Plates were incubated at 37Â°C for 45 minutes, followed by three buffer washes to remove excess serum. Following this stage, 200Î¼l of diluted conjugate was added to all wells and incubated for 30 minutes at 37Â°C. These were then washed with blocking buffer, before final addition of 200Î¼l of substrate followed by 10-15 minutes of incubation at 37Â°C until colour started to develop. Plates were then assessed for colorimetry using the positive and negative controls for reference to determine the presence of antiviral antibodies in patient sera. These steps are illustrated in Figure One.
Figure One. Indirect ELISA detection of antiviral antibodies. These steps illustrate the process of antibody detection. First, the antigen is bound to the surface of the well, followed by addition of a primary antibody and then a secondary antibody, which initiates the colorimetric reaction. Washes occur between each stage in order to remove unbound molecules (Abbas & Lichtman, 2005, p.110).
The detection of antiviral antigens was compared with control samples of colorimetry in order to identify the presence of four different viruses (measles, mumps, rubella and cytomegalovirus) in the sera of two patients. Colour reactions were clear in both experimental samples and differential positivity for antiviral antibodies was noted.
Patient one demonstrated positivity for antibodies against mumps and rubella, while testing negative for both measles and cytomegalovirus. By contrast, patient two was positive for antibodies against measles and rubella, testing negative for mumps and cytomegalovirus antibodies. These results were consistent in both samples of serum from each patient and the detection of IgG against these viruses is suggestive of exposure to viral antigens at some point, most likely indicating immunity to these diseases. Therefore, patient one is immune to mumps and rubella, either through natural exposure or vaccination, while patient two is immune to measles and rubella in the same manner. However, it is noted that without further data active or recent infection cannot be ruled out in both patients (see following section).
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The findings of this study support the role of indirect ELISA in the detection of antiviral antibodies in the laboratory setting. Antibodies against measles, mumps and rubella were detected when both samples were considered, although no patients demonstrated antibodies against cytomegalovirus. The fact that the findings were identical in both samples from each patient would indicate that the technique used was reliable, although further testing would be needed to confirm these findings. The detection of IgG antibodies is indicative of previous exposure to these viruses, either through vaccination or previous infection, suggesting that patient one is immune to mumps and rubella, while patient two is immune to measles and rubella.
Although this protocol demonstrated the presence of antibodies against specific viruses in these patients, it is difficult to interpret the significance of these findings without further information on each patient. For instance, the presence of antibodies raised against measles, mumps and rubella can be the result of successful vaccination against these diseases or it may be due to recent infection, although IgG is usually associated with a long-term response (Lambert et al., 2005, p56). However, in infections such as Epstein-Barr disease (infectious mononucleosis) IgG antibodies can emerge in the initial stages of the disease, indicating the variability of the immune mechanism (CDC, 2006). For the four viruses described in this study, active disease may have a different level of significance depending on patient age and clinical situation: health young children with measles have a low risk of complications, while disease in older adults or during pregnancy can have more complications (Fiebelkorn et al., 2010, p1521). Therefore, in order to relate these findings to specific clinical activity or course of action, further clinical details would be needed.
The use of an anti-IgM detection antibody in this experimental setting may have added value to the detection of active disease in these patients, as IgM is produced typically during active infection, while IgG against a specific disease may confer lifetime immunity and may be less indicative of current infection (Lambert et al., 2010, p55). Application of an anti-human IgM antibody may be of value in future experiments, in order to place the results of IgG detection in a clinical context (Hogrefe et al., 2004, p4647). These can be conducted as separate assays, or through the use of a polyvalent conjugate containing both anti-IgM and anti-IgG, in order to increase sensitivity for detection of early disease (Hogrefe et al., 2004, p4641).
One of the limitations of indirect ELISA in this context is the fact that viral antigen fixation to the microtiter plate is a non-specific process and therefore other proteins in the serum may adhere to the plate, resulting in competition for binding with the analyte proteins. Performing a sandwich ELISA is one way of overcoming this problem, as a specific enzyme is used to adhere the viral antigen to the plate, thereby minimising non-specific reactions (Wang & Kobayashi, 2013, p55). Competitive ELISA experiments have also been utilised, as in HIV antibody detection, which may overcome this problem (Wang & Kobayashi, 2013, p58). In this case, the specific HIV antibody in the test sample competes with an enzyme-bound antibody for antigen binding: bound antigen-antibody complexes are introduced to a coated well and unbound antibody is washed away, with more washed away if the concentration of antigen in the sample is elevated (Daskalakis, 2011, p18). As a result, the development of colour during the reaction is inversely proportional to the concentration of specific HIV antibody (Tudor et al., 2012, p12680). This principle may be applied to other viral antigen tests, resulting in high levels of specificity.
However, despite of the limitations noted, this experiment has elegantly demonstrated the ability of indirect ELISA to identify antiviral antigens in the laboratory setting, in a cheap and time-efficient manner, indicating that the technique may have clinical utility in determining previous virus exposure and mounting immune response. The significance of the findings for each patient will depend on further clinical information, including age, concurrent illness and symptomatology.
In summary, this experiment demonstrated that indirect ELISA could be used to detect antiviral antibodies in patient sera for common viruses. The technique was simple to perform and control samples were used to ensure the specificity of the reactions. However, several limitations to the methodological approach and interpretation of the findings have been noted, including the possibility of competitive binding and a lack of ability to confirm active infection. Therefore, caution should be used when placing these findings in a clinical setting without further information.
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For future studies it is recommended that sandwich or competitive ELISA techniques are used in order to reduce possible contamination of samples and lack of specificity in the results. In addition, detection of IgM should be conducted along with IgG detection in order to exclude active viral activity in these patients. These strategies will optimise the protocol and ensure optimal clinical utility of the results.