The immunoenzymatic test of ELISA (Enzyme-Linked ImmunoSorbent Assay)
This test was first applied for Chagas infection in 1975 in a study on filter paper.78 It is rather similar to IIF, because it has many steps, needs a well-trained technician, and has extreme sensitivity, which is one advantage, but carries the problem of a more limited specificity. On the other hand, the advantages over IIF are the objective readings (spectrophotometer) and the possibility of automatization for handling hundreds of samples at a time. The classic technique described by Voller et al.78 had several steps and the time to run a plate was 6 h. Now kits have improved the incubation time and the same plate may be ready in 2 h. As with the IIF, it is difficult to have a sample of serum from an infected individual that yields a negative result. Cutoff should be calculated as per technical instructions, but a curve with negative, low-positive, and high-positive controls is useful for the range of responses. Once the cutoff has been calculated, it is accepted that positive samples should be considered as such when Optical Density (OD) is at least 10% higher than the cutoff. In order to compare results in the follow-up of treated patients, it is useful to express results as an index, obtained through the division of the OD of the sample and the OD of the cutoff of the plate. Results below 0.9 are negative, from 0.9 to 1.2 are borderline, and above 1.2 positive.
In a recent study performed by the Ministry of Health, Brazil, 12 different brands of commercially available ELISA kits, approved by the sanitary authorities, were tested with 150 positive and negative sera, including sera of low reactivity, by four independent laboratories. Results showed that the sensitivity for all of them was high (97—100%) but specificity was low (60—98%).79 The interpretation was that kits were tailored to fulfill the needs of the main client (blood banks) and acceptable for them, but not good enough for the serological diagnosis of an individual patient (see Table 29.5).
To summarize, the ELISA test is excellent for the purpose of screening blood donors, but in order to confirm the infection, other tests of higher specificity should be employed in parallel. We will come back to these issues when the indications of serological tests are discussed.
Nonconventional methods Radio-immunoprecipitation assay (RIPA)
This test was developed in the United States by Kirchhoff et al.80 and is claimed to be the gold standard for serology.81 It is not widely available as it is only performed in few research institutions, is expensive, time-consuming, and uses radioisotopes, which make its application as a standard procedure remote. One study compared their results with conventional tests82 and, although sensitivity compared well with IFI, HA, and ELISA of several brands, the number of negative sera used in this study (n = 19) was not enough to evaluate specificity.