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Background <p>Autoimmune thyroid diseases (AITDs), including Hashimoto’s thyroiditis (HT) and Graves’ disease (GD), result from the immune system attacking the thyroid gland, leading to hypothyroidism or hyperthyroidism. Detection of anti-thyroid peroxidase antibodies (anti-TPO) is crucial for diagnosing autoimmune hypothyroidism. However, achieving consistent sensitivity and specificity across diagnostic platforms remains a challenge.</p> Methods <p>This study aimed to identify and validate synthetic peptide epitopes derived from immunodominant regions (IDR-A and IDR-B) of the thyroid peroxidase (TPO) enzyme to improve anti-TPO antibody detection. Seven peptides with high antigenicity scores were selected using bioinformatics tools such as Immune Epitope Database (IEDB), ABCpred, and BcePred. These biotinylated peptides were synthesized and evaluated for their ability to bind anti-TPO antibodies in patient sera using direct and competitive Enzyme-Linked Immunosorbent Assay (ELISA) assays. A total of 240 serum samples (120 anti-TPO positive and 120 negative) by standard ELISA were analyzed to assess sensitivity, specificity, and overall diagnostic performance.</p> Results <p>Of the seven peptides, P1 showed the highest sensitivity (63.64%) and the largest area under the receiver operating characteristic (ROC) curve (area under the curve (AUC)) = 0.8450. Peptides P4, P5, and P6 demonstrated superior specificity (&gt; 94%). Competitive ELISA revealed that peptide P7 significantly inhibited anti-TPO antibody binding to the TPO protein, with optimal inhibition observed at 100&#xa0;µg/mL. Statistical analysis confirmed the diagnostic relevance of these peptides, with significant differences in Optical Density (OD) between peptide-treated and untreated samples (<i>p</i> &lt; 0.0001).</p> Conclusion <p>Among the identified peptides, P1 and P7 are particularly promising as antigenic probes for detecting anti-TPO antibodies in AITDs. Their application may enhance the sensitivity and specificity of diagnostic assays, addressing current limitations in clinical testing. This peptide-based approach could facilitate the development of population-specific diagnostic kits that account for genetic variation in TPO epitopes.</p>

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Development of high-affinity peptide probes for improved diagnosis of Hashimoto’s thyroiditis

  • 期刊论文

  • 发表日期：

    2025年9月10日

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  • Azadeh Aliarab,
  • Mehdi Hedayati,
  • Mohammad Javad Rasaee,
  • Davood Khalili

摘要

Background

Autoimmune thyroid diseases (AITDs), including Hashimoto’s thyroiditis (HT) and Graves’ disease (GD), result from the immune system attacking the thyroid gland, leading to hypothyroidism or hyperthyroidism. Detection of anti-thyroid peroxidase antibodies (anti-TPO) is crucial for diagnosing autoimmune hypothyroidism. However, achieving consistent sensitivity and specificity across diagnostic platforms remains a challenge.

Methods

This study aimed to identify and validate synthetic peptide epitopes derived from immunodominant regions (IDR-A and IDR-B) of the thyroid peroxidase (TPO) enzyme to improve anti-TPO antibody detection. Seven peptides with high antigenicity scores were selected using bioinformatics tools such as Immune Epitope Database (IEDB), ABCpred, and BcePred. These biotinylated peptides were synthesized and evaluated for their ability to bind anti-TPO antibodies in patient sera using direct and competitive Enzyme-Linked Immunosorbent Assay (ELISA) assays. A total of 240 serum samples (120 anti-TPO positive and 120 negative) by standard ELISA were analyzed to assess sensitivity, specificity, and overall diagnostic performance.

Results

Of the seven peptides, P1 showed the highest sensitivity (63.64%) and the largest area under the receiver operating characteristic (ROC) curve (area under the curve (AUC)) = 0.8450. Peptides P4, P5, and P6 demonstrated superior specificity (> 94%). Competitive ELISA revealed that peptide P7 significantly inhibited anti-TPO antibody binding to the TPO protein, with optimal inhibition observed at 100 µg/mL. Statistical analysis confirmed the diagnostic relevance of these peptides, with significant differences in Optical Density (OD) between peptide-treated and untreated samples (p < 0.0001).

Conclusion

Among the identified peptides, P1 and P7 are particularly promising as antigenic probes for detecting anti-TPO antibodies in AITDs. Their application may enhance the sensitivity and specificity of diagnostic assays, addressing current limitations in clinical testing. This peptide-based approach could facilitate the development of population-specific diagnostic kits that account for genetic variation in TPO epitopes.