Heat serum inactivation as a mandatory procedure for antiactin antibody detection in cell culture
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E L Cançado1, L S Vilas-Boas, C P Abrantes-Lemos
1Department of Gastroenterology, School of Medicine, University of São Paulo, Brazil.
Abstract:
In autoimmune hepatitis (AIH), the smooth-muscle antibody is specific for polymerized actin. Detection of antiactin antibody (AAA) has been hampered by technical problems. We have investigated AAA in 30 sera from patients with liver diseases and smooth-muscle antibody. AAA was detected by indirect immunofluorescence in 1:40, 1:80, and 1:160 dilutions. Five techniques were performed using fibroblasts: with vinblastine (A); without drugs (B); with sodium citrate (C); without drugs but with heat serum inactivation (D); and with sodium citrate and heat serum inactivation (E). For comparative analysis, we considered: the total number of AAA-positive sera regardless of the dilution in which reactivity was observed, as well as in each dilution separately; and the comparison of AAA intensity between 1:40 x 1:80, 1:40 x 1:160, and 1:80 x 1:160 dilutions. AAA was more positive in techniques B, C, D, and E than in A (P < .001) in general, and in each dilution separately. AAA was more positive in technique D than in B in 1:40 (P = .0005) and 1:80 dilutions (P = .03), as well as in E than in C (P = .0001) in 1:40 dilution. Techniques B and D yielded results similar to C and E, respectively. AAA staining was significantly more intense in 1:80 and 1:160 than in 1:40 dilution in A, B, and C; it was both significantly less intense in 1:80 and 1:160 than in 1:40 dilution and in 1:80 than in 1:160 in techniques D and E. We concluded that heat inactivation increased AAA seropositivity/intensity in 1:40 and 1:80 dilutions, preventing false-negative results; actin polymerization with sodium citrate did not enhanced AAA seropositivity/intensity. The technique with vinblastine was the least effective.
Insights
Detecting antiactin antibody (AAA) in autoimmune hepatitis (AIH) is crucial. Heat inactivation significantly improved AAA detection, preventing false negatives, while vinblastine was ineffective.
Area of Science:
- Immunology
- Hepatology
- Biochemistry
Background:
- Autoimmune hepatitis (AIH) diagnosis involves identifying specific autoantibodies.
- Smooth-muscle antibody (SMA) in AIH targets polymerized actin.
- Detection of antiactin antibody (AAA) has faced technical challenges.
Purpose of the Study:
- To investigate and optimize the detection of antiactin antibody (AAA).
- To compare the efficacy of different techniques for AAA detection in patient sera.
Main Methods:
- Indirect immunofluorescence was used to detect AAA in 30 sera from patients with liver disease and SMA.
- Five techniques were compared using fibroblasts: with vinblastine (A), without drugs (B), with sodium citrate (C), without drugs but with heat serum inactivation (D), and with sodium citrate and heat serum inactivation (E).
- Comparative analysis included overall positivity, positivity at specific dilutions (1:40, 1:80, 1:160), and staining intensity.
Main Results:
- Techniques B, C, D, and E showed significantly higher AAA positivity than technique A (P < .001).
- Heat inactivation (technique D vs. B; E vs. C) significantly increased AAA positivity, especially at 1:40 and 1:80 dilutions.
- Techniques D and E demonstrated reduced staining intensity at higher dilutions (1:80, 1:160) compared to 1:40, suggesting improved specificity.
Conclusions:
- Heat inactivation of serum is crucial for enhancing AAA seropositivity and intensity, thereby preventing false-negative results.
- Actin polymerization methods, including sodium citrate, did not improve AAA detection.
- The technique involving vinblastine was found to be the least effective for AAA detection.