Serum reactivity against bacterial pyruvate dehydrogenase: Increasing the specificity of anti-mitochondrial antibodies for the diagnosis of primary biliary cirrhosis

Hiroshi Miyakawa, Atsushi Tanaka, Carlo Selmi, Naomi Hosoya, Norikazu Mataki, Kentaro Kikuchi, Takashi Kato, Junya Arai, Toshihiro Goto, M. Eric Gershwin

Research output: Contribution to journalArticle

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Abstract

Antimitochondrial antibodies (AMA) are the serum hallmark of primary biliary cirrhosis (PBC). However, AMA-positivity can be found in non-PBC sera when lower dilutions are used, thus raising issues about the specificity and sensitivity of the test. AMA reacts primarily with the lipoylated domains of pyruvate dehydrogenase-E2 (PDC-E2) which is highly conserved across species, including bacteria. We studied 77 serum samples, including 24 from patients with anti-PDC-E2-positive PBC and 53 controls (16 with autoimmune hepatitis (AIH), 10 with primary sclerosing cholangitis (PSC), and 27 healthy individuals) for their reactivities at serial dilutions (1:10, 1:20 and 1:40) against Escherichia coli DH5 alpha lysate overexpressing human PDC-E2 using immunoblotting (IB). A murine anti-human PDC-E2 monoclonal antibody (mAB) was used as control. We further studied positive sera using adsorption with a synthetic E. coli peptide sharing similarity with human PDC-E2. Finally, we verified whether a unique buffer for E. coli preparation could reduce non-specific serum reactivity. Results demonstrated that 100% of anti-PDC-E2-positive PBC and up to 38% of control sera at 1:10 dilution recognized E. coli PDC-E2 at IB while dilution tests indicated that the overall potency of PBC reactivity was 100-fold higher compared to controls. In fact, a subgroup (20-38%) of non-PBC sera were positive at low titers but lost the reactivity when absorbed with the synthetic E. coli peptide. Finally, our unique buffer reduced the reactivity of non-PBC sera as measured by ELISA. In conclusion, we demonstrated that weak cross-reactivity with E. coli PDC-E2 occurs in non-PBC sera at lower dilutions and that such reactivity is not due to AMA-positivity. The use of a specific buffer might avoid the risk of false positive AMA determinations when E. coli-expressed recombinant antigens are used.

Original languageEnglish (US)
Pages (from-to)289-294
Number of pages6
JournalClinical and Developmental Immunology
Volume13
Issue number2-4
DOIs
StatePublished - Jun 2006

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Biliary Liver Cirrhosis
Pyruvic Acid
Anti-Idiotypic Antibodies
Oxidoreductases
Escherichia coli
Serum
Antibodies
Buffers
Immunoblotting
Autoimmune Hepatitis
Sclerosing Cholangitis
Peptides
Tocopherols
Adsorption
Enzyme-Linked Immunosorbent Assay
Monoclonal Antibodies
Bacteria
Antigens
Sensitivity and Specificity

Keywords

  • Autoantibodies
  • Cross-reactivity
  • Escherichia coli
  • Specificity

ASJC Scopus subject areas

  • Immunology
  • Cell Biology
  • Developmental Biology

Cite this

Serum reactivity against bacterial pyruvate dehydrogenase : Increasing the specificity of anti-mitochondrial antibodies for the diagnosis of primary biliary cirrhosis. / Miyakawa, Hiroshi; Tanaka, Atsushi; Selmi, Carlo; Hosoya, Naomi; Mataki, Norikazu; Kikuchi, Kentaro; Kato, Takashi; Arai, Junya; Goto, Toshihiro; Gershwin, M. Eric.

In: Clinical and Developmental Immunology, Vol. 13, No. 2-4, 06.2006, p. 289-294.

Research output: Contribution to journalArticle

Miyakawa, Hiroshi ; Tanaka, Atsushi ; Selmi, Carlo ; Hosoya, Naomi ; Mataki, Norikazu ; Kikuchi, Kentaro ; Kato, Takashi ; Arai, Junya ; Goto, Toshihiro ; Gershwin, M. Eric. / Serum reactivity against bacterial pyruvate dehydrogenase : Increasing the specificity of anti-mitochondrial antibodies for the diagnosis of primary biliary cirrhosis. In: Clinical and Developmental Immunology. 2006 ; Vol. 13, No. 2-4. pp. 289-294.
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abstract = "Antimitochondrial antibodies (AMA) are the serum hallmark of primary biliary cirrhosis (PBC). However, AMA-positivity can be found in non-PBC sera when lower dilutions are used, thus raising issues about the specificity and sensitivity of the test. AMA reacts primarily with the lipoylated domains of pyruvate dehydrogenase-E2 (PDC-E2) which is highly conserved across species, including bacteria. We studied 77 serum samples, including 24 from patients with anti-PDC-E2-positive PBC and 53 controls (16 with autoimmune hepatitis (AIH), 10 with primary sclerosing cholangitis (PSC), and 27 healthy individuals) for their reactivities at serial dilutions (1:10, 1:20 and 1:40) against Escherichia coli DH5 alpha lysate overexpressing human PDC-E2 using immunoblotting (IB). A murine anti-human PDC-E2 monoclonal antibody (mAB) was used as control. We further studied positive sera using adsorption with a synthetic E. coli peptide sharing similarity with human PDC-E2. Finally, we verified whether a unique buffer for E. coli preparation could reduce non-specific serum reactivity. Results demonstrated that 100{\%} of anti-PDC-E2-positive PBC and up to 38{\%} of control sera at 1:10 dilution recognized E. coli PDC-E2 at IB while dilution tests indicated that the overall potency of PBC reactivity was 100-fold higher compared to controls. In fact, a subgroup (20-38{\%}) of non-PBC sera were positive at low titers but lost the reactivity when absorbed with the synthetic E. coli peptide. Finally, our unique buffer reduced the reactivity of non-PBC sera as measured by ELISA. In conclusion, we demonstrated that weak cross-reactivity with E. coli PDC-E2 occurs in non-PBC sera at lower dilutions and that such reactivity is not due to AMA-positivity. The use of a specific buffer might avoid the risk of false positive AMA determinations when E. coli-expressed recombinant antigens are used.",
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AU - Selmi, Carlo

AU - Hosoya, Naomi

AU - Mataki, Norikazu

AU - Kikuchi, Kentaro

AU - Kato, Takashi

AU - Arai, Junya

AU - Goto, Toshihiro

AU - Gershwin, M. Eric

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AB - Antimitochondrial antibodies (AMA) are the serum hallmark of primary biliary cirrhosis (PBC). However, AMA-positivity can be found in non-PBC sera when lower dilutions are used, thus raising issues about the specificity and sensitivity of the test. AMA reacts primarily with the lipoylated domains of pyruvate dehydrogenase-E2 (PDC-E2) which is highly conserved across species, including bacteria. We studied 77 serum samples, including 24 from patients with anti-PDC-E2-positive PBC and 53 controls (16 with autoimmune hepatitis (AIH), 10 with primary sclerosing cholangitis (PSC), and 27 healthy individuals) for their reactivities at serial dilutions (1:10, 1:20 and 1:40) against Escherichia coli DH5 alpha lysate overexpressing human PDC-E2 using immunoblotting (IB). A murine anti-human PDC-E2 monoclonal antibody (mAB) was used as control. We further studied positive sera using adsorption with a synthetic E. coli peptide sharing similarity with human PDC-E2. Finally, we verified whether a unique buffer for E. coli preparation could reduce non-specific serum reactivity. Results demonstrated that 100% of anti-PDC-E2-positive PBC and up to 38% of control sera at 1:10 dilution recognized E. coli PDC-E2 at IB while dilution tests indicated that the overall potency of PBC reactivity was 100-fold higher compared to controls. In fact, a subgroup (20-38%) of non-PBC sera were positive at low titers but lost the reactivity when absorbed with the synthetic E. coli peptide. Finally, our unique buffer reduced the reactivity of non-PBC sera as measured by ELISA. In conclusion, we demonstrated that weak cross-reactivity with E. coli PDC-E2 occurs in non-PBC sera at lower dilutions and that such reactivity is not due to AMA-positivity. The use of a specific buffer might avoid the risk of false positive AMA determinations when E. coli-expressed recombinant antigens are used.

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