A Method to Determine Significant Levels of Immunoglobulin G toAspergillus FumigatusAntigens in an Elisa System and a Comparison with Counterimmunoelectrophoresis and Double Diffusion Techniques

Abstract

A method is described which assesses results obtained from an ELISA system for the determination of human serum levels of IgG class antibodies to Aspergillus fumigatus. The method is used to discriminate positive from negative samples, and significant antibody activity may be reported to the clinician, relative to a reference positive control serum monitored simultaneously under the same test conditions. Antibody content is expressed as the absorbance of a certain dilution of serum. Duplicate samples were analysed at a single serum dilution and their absorbtion values obtained from a semi-automated ELISA microplate reader. These were entered into a computer programmed to convert the data into units on a logarithmic scale. In parallel experiments, ELISA results were compared with those obtained by the techniques of counterimmunoelectrophoresis and double diffusion which measure precipitating antibody of all classes. A relatively good degree of correlation between tests was found only among sera with a high level of antibody.

Detection of Antibodies against <i>Aspergillus fumigatus</i>: Comparison between Double Immunodiffusion, ELISA and Immunoblot Analysis

“…In agreement with previously described results [3,7,8,10] I found a good correlation between DID and ELISA among sera that contain a high level of anti body to A. fumigatus. Antibodies in these sera react with several (up to 20) antigens as detected by immunoblot analyses.…”

“…The distribution of antibody titers is a continuous one and any discrimination point is more or less arbitrary [10]. In this study, I compared the results obtained in ELISA with those of immunoblot analyses, irrespec tive whether or not involvement of A.fumigatus in the patients' diseases was proven.…”

“…Discrimination between normal and positive re sults in ELISA is a well-known problem [5,9,10]. The distribution of antibody titers is a continuous one and any discrimination point is more or less arbitrary [10].…”

“…Most widely used is the en zyme-linked immunosorbent assay (ELISA) [3][4][5][6]. Numerous studies have been performed in which the results obtained in DID were compared with IgG ti ters measured in ELISA [3][4][5][7][8][9][10]. Although there is a correlation between these methods, precipitin-nega tive sera may contain increased ELISA titers for IgG whereas, on the other hand, there are precipitin-posi tive sera that are negative for IgG in ELISA.…”

“…Although there is a correlation between these methods, precipitin-nega tive sera may contain increased ELISA titers for IgG whereas, on the other hand, there are precipitin-posi tive sera that are negative for IgG in ELISA. False negative results in ELISA for IgG may be due to poor binding of the relevant antigens to microtiter plates or to the presence in patient sera of antibodies that be long to the IgM class instead of IgG [10]. Therefore, precipitin-positive sera were assayed in this study for both A.fumigatus-specific IgG and IgM by means of ELISA.…”