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    Home > Active Ingredient News > Endocrine System > JCLA: Comparison of ELISA and Dot Blot for the Detection of Graves' Disease TSH Receptor Antibodies

    JCLA: Comparison of ELISA and Dot Blot for the Detection of Graves' Disease TSH Receptor Antibodies

    • Last Update: 2022-04-16
    • Source: Internet
    • Author: User
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    Background: Graves' disease (GD) is an autoimmune disease, accounting for about 0.
    5% of the total population, with a higher incidence in women than in men

    .
    Clinically, GD is characterized by suppression of thyroid-stimulating hormone (TSH) levels, hyperstimulation of thyroid hormones, and production of antithyroid antibodies

    .
    Thyroid-stimulating hormone receptor autoantibodies (TRAb) are currently recognized as a serological marker of GD and are often helpful in differentiating GD from other causes of hyperthyroidism

    .
    Furthermore, the role of TRAbs has potential not only in confirming the diagnosis of GD, but also in predicting the clinical course, risk of recurrence and response to treatment of GD
    .
    It is the main cause of hyperthyroidism (hyperthyroidism)

    .
    Anti-thyrotropin receptor/TSHR antibody (TRAb) is associated with hyperthyroidism (hyperthyroidism) and is considered a diagnostic marker for GD

    .
    Therefore, we developed a recombinant human TSHR-169 protein (hTSHR-169), which can specifically recognize TRAb in sera of GD patients, and compared the diagnostic ability of TRAb detected by ELISA and dot blot for GD

    .

    Graves' disease (GD) is an autoimmune disease, accounting for about 0.
    5% of the total population, and the incidence of women is higher than that of men

    .
    Clinically, GD is characterized by suppression of thyroid-stimulating hormone (TSH) levels, hyperstimulation of thyroid hormones, and production of antithyroid antibodies

    .
    Thyroid-stimulating hormone receptor autoantibodies (TRAb) are currently recognized as a serological marker of GD and are often helpful in differentiating GD from other causes of hyperthyroidism

    .
    The role of TRAbs has potential not only in confirming the diagnosis of GD, but also in predicting the clinical course, risk of recurrence, and response to treatment of GD
    .
    It is the main cause of hyperthyroidism (hyperthyroidism)

    .
    Anti-thyrotropin receptor/TSHR antibody (TRAb) is associated with hyperthyroidism (hyperthyroidism) and is considered a diagnostic marker for GD

    .
    Therefore, we developed a recombinant human TSHR-169 protein (hTSHR-169), which can specifically recognize TRAb in sera of GD patients, and compared the diagnostic ability of TRAb detected by ELISA and dot blot for GD

    .
    diagnosis

    Methods: Twenty GD patients and 20 healthy people were selected from the Indonesian population as the research subjects
    .
    Quantitative determination of TRAb concentration and density

    .
    Receiver-operator curve (ROC) analysis was used for comparative analysis

    .

    Results: The lowest concentration of TRAb detected by dot blot was 100 ng, and the dilution of antiserum was 1:60
    .
    Compared with dot blot, the AUC for GD was higher by ELISA (0.
    95 and 0.
    85, respectively)

    .
    The efficacy of the two assays was examined by comparing their specificity and sensitivity for clinical diagnosis using the recommended cut-off values

    .
    The sensitivity and specificity of the ELISA assay were 80% and 95%, respectively, and the sensitivity and specificity of the dot blot assay were 70% and 95%, respectively

    .

    Figure 1 FIGURE 1 cloning of HTSHR-169 in pET28a(+) vector
    .
    (A) Schematic representation of the pET28a(+) expression vector encoding the hTSHR-169 protein

    .
    (B) PCR products of recombinant clones (amplified fragment size 689 bp); M: DNA marker; NC, negative control; Lanes 1-5: recombinant clones, replicated 1-5

    .
    (C) Restriction endonuclease analysis of recombinant pET28a-hTSHR-169 expression vector

    .
    M: DNA marker; Lane 1: pET28a(+) vector that did not disappear; Lane 2: digested pET28a(+) vector; Lane 3: recombinant pET28a(+) vector that was not digested; Lane 4: with BamHI and The recombinant pET28a(+) vector was digested with XhoI, wherein 5.
    32kb refers to the fragment of pET28a(+) vector (arrow), and the 411bp band refers to the cDNA insert of hTSHR-169 (arrow)

    .
    (D) Alignment between cDNA inserts against hTSHR sequences

    Figure 1 FIGURE 1 cloning of HTSHR-169 in pET28a(+) vector
    .
    (A) Schematic representation of the pET28a(+) expression vector encoding the hTSHR-169 protein

    .
    (B) PCR products of recombinant clones (amplified fragment size 689 bp); M: DNA marker; NC, negative control; Lanes 1-5: recombinant clones, replicated 1-5

    .
    (C) Restriction endonuclease analysis of recombinant pET28a-hTSHR-169 expression vector

    .
    M: DNA marker; Lane 1: pET28a(+) vector that did not disappear; Lane 2: digested pET28a(+) vector; Lane 3: recombinant pET28a(+) vector that was not digested; Lane 4: with BamHI and The recombinant pET28a(+) vector was digested with XhoI, wherein 5.
    32kb refers to the fragment of pET28a(+) vector (arrow), and the 411bp band refers to the cDNA insert of hTSHR-169 (arrow)

    .
    (D) Alignment between cDNA inserts against hTSHR sequences

    Figure 2 (A) The expression profile of hTSHR-169 fusion protein induced by 0.
    1 mM IPTG

    .
    Samples were separated by 12% SDS-PAGE gel

    .
    Coomassie blue staining showed protein bands

    .
    M: protein marker; NI: without IPTG; I: with IPTG

    .
    (B) Western blot detection of the antigenicity of the purified hTSHR-169 fusion protein

    .
    When purified hTSHR-169 fragment was incubated with anti-TSHR monoclonal antibody and GD patient serum, a single band with the expected size of 16.
    8 kDa was detected, but not in healthy controls

    .
    M: protein marker

    .
    (C) Optimizing the dot blot method, recombinant hTSHR-169 was spotted on NCM (different concentrations 10-1000 ng) and incubated with GD patient or healthy human serum (2-fold dilution 1:20~1:120)

    .
    Ag(−) or Ab(−) indicate that NCM can only be probed with recombinant TSHR-169 or TRAb, respectively

    .
    Arrows indicate the minimum concentration required to detect TRAB in GD samples compared to controls

    Figure 2 (A) The expression profile of hTSHR-169 fusion protein induced by 0.
    1 mM IPTG

    .
    Samples were separated by 12% SDS-PAGE gel

    .
    Coomassie blue staining showed protein bands

    .
    M: protein marker; NI: without IPTG; I: with IPTG

    .
    (B) Western blot detection of the antigenicity of the purified hTSHR-169 fusion protein

    .
    When purified hTSHR-169 fragment was incubated with anti-TSHR monoclonal antibody and GD patient serum, a single band with the expected size of 16.
    8 kDa was detected, but not in healthy controls

    .
    M: protein marker

    .
    (C) Optimizing the dot blot method, recombinant hTSHR-169 was spotted on NCM (different concentrations 10-1000 ng) and incubated with GD patient or healthy human serum (2-fold dilution 1:20~1:120)

    .
    Ag(−) or Ab(−) indicate that NCM can only be probed with recombinant TSHR-169 or TRAb, respectively

    .
    Arrows indicate the minimum concentration required to detect TRAB in GD samples compared to controls

    Figure 3 (A) Immunoreactivity of 20 known GD and control samples
    .
    Each sample was in duplicate; Ag(−) or Ab(−) denote NCMs with recombinant TSHR-169 or TRAb probes alone, respectively

    .
    (B) TRAb levels in GD and control samples were determined by enzyme-linked immunosorbent assay (ELISA)

    .
    (C) TRAb density was detected by dot blot

    .
    (D) Using 20 GD patients as the disease variable and 20 normal subjects as the control variable, the ROC results of TRAb-ELISA and dot blot analysis were compared

    .
    GD, Graves disease; HC, healthy controls

    Figure 3 (A) Immunoreactivity of 20 known GD and control samples
    .
    Each sample was in duplicate; Ag(−) or Ab(−) denote NCMs with recombinant TSHR-169 or TRAb probes alone, respectively

    .
    (B) TRAb levels in GD and control samples were determined by enzyme-linked immunosorbent assay (ELISA)

    .
    (C) TRAb density was detected by dot blot

    .
    (D) Using 20 GD patients as the disease variable and 20 normal subjects as the control variable, the ROC results of TRAb-ELISA and dot blot analysis were compared

    .
    GD, Graves disease; HC, healthy controls

    Table 1 Comparison of detection of TRAb by ELISA and dot blot

    Table 1 Comparison of detection of TRAb by ELISA and dot blot

    Conclusion: Although the detection effect of dot blot method is lower than that of ELISA method, dot blot method is a simple and rapid diagnostic method, which is suitable for the diagnosis of GD in rural areas that are sometimes difficult for medical institutions to access
    .

    Although the detection effect of dot blot method is lower than that of ELISA method, dot blot method is a simple and rapid diagnostic method suitable for the diagnosis of GD in rural areas that are sometimes difficult for medical institutions to access
    .

    Original source: Zulkarnain Z, Ulhaq ZS, Sujuti H, et al.
    Comparative performance of ELISA and dot blot assay for TSH-receptor antibody detection in Graves' disease.
    J Clin Lab Anal 2022 Feb 20

    Comparative performance of ELISA and dot blot assay for TSH-receptor antibody detection in Graves' disease.


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