Objectives: Anti-TIF1γ is an important autoantibody in the diagnosis of cancer-associated dermatomyositis and the most common autoantibody in juvenile onset dermatomyositis. Its reliable detection is important to instigate further investigations into underlying malignancy in adults. We previously showed that commercial assays using line and dot blots do not reliably detect anti-TIF1γ. We aimed to test a new commercial ELISA and compare with previously obtained protein immunoprecipitation.
Methods: Radio-labelled immunoprecipitation had previously been used to determine the autoantibody status of patients with immune-mediated inflammatory myopathies and several healthy controls. ELISA was undertaken on healthy control and anti-TIF1γ sera and compared to previous immunoprecipitation data.
Results: A total of 110 serum samples were analysed: 42 myositis patients with anti- TIF1γ and 68 autoantibody negative healthy control sera. Anti-TIF1γ was detected by ELISA in 41 out of 42 of the anti-TIF1γ-positive samples by immunoprecipitation, and in none of the healthy controls, giving a sensitivity of 97.6% and specificity of 100%. The false negative rate was 2%.
Conclusion: ELISA is an affordable and time-efficient method which is accurate in detecting anti-TIF1γ.
Objective Quantitation of EGFR Protein Levels using Quantitative Dot Blot Method for the Prognosis of Gastric Cancer Patients
Purpose: An underlying factor for the failure of several clinical trials of anti-epidermal growth factor receptor (EGFR) therapies is the lack of an effective method to identify patients who overexpress EGFR protein. The quantitative dot blot method (QDB) was used to measure EGFR protein levels objectively, absolutely, and quantitatively. Its feasibility was evaluated for the prognosis of overall survival (OS) of patients with gastric cancer.
Materials and methods: Slices of 2×5 μm from formalin-fixed paraffin-embedded gastric cancer specimens were used to extract total tissue lysates for QDB measurement. Absolutely quantitated EGFR protein levels were used for the Kaplan-Meier OS analysis.
Results: EGFR protein levels ranged from 0 to 772.6 pmol/g (n=246) for all gastric cancer patients. A poor correlation was observed between quantitated EGFR levels and immunohistochemistry scores with ρ=0.024 and P=0.717 in Spearman’s correlation analysis. EGFR was identified as an independent negative prognostic biomarker for gastric cancer patients only through absolute quantitation, with a hazard ratio of 1.92 (95% confidence interval, 1.05-3.53; P=0.034) in multivariate Cox regression OS analysis. A cutoff of 208 pmol/g was proposed to stratify patients with a 3-year survival probability of 44% for patients with EGFR levels above the cutoff versus 68% for those below the cutoff based on Kaplan-Meier OS analysis (log rank test, P=0.002).
Conclusions: A QDB-based assay was developed for gastric cancer specimens to measure EGFR protein levels absolutely, quantitatively, and objectively. This assay should facilitate clinical trials aimed at evaluation of anti-EGFR therapies retrospectively and prospectively for gastric cancer.
A Cross-Sectional Study for Evaluation of KRAS and BRAF Mutations by Reverse Dot Blot, PCR-RFLP, and Allele-Specific PCR Methods Among Patients with Colorectal Cancer
Background: KRAS and BRAF genes are the biomarkers in Colorectal Cancer (CRC) which play prognostic and predictive roles in CRC treatment. Nowadays, the selection of rapid and available methods for studying KRAS and BRAF mutations in anti-EGFR therapy of patients suffering from CRC plays a significant role. In this study, the mutations of these two oncogenes were evaluated by different methods.
Methods: This study was performed on 50 Formalin-Fixed Paraffin-Embedded (FFPE) tissue blocks of patients diagnosed with colorectal cancer. After DNA extraction, KRAS and BRAF gene mutations were evaluated using reverse dot blot, and results were compared with PCR-RFLP and allele-specific PCR for KRAS and BRAF mutations, respectively.
Results: KRAS gene mutations were detected in 42% of patients, of which 30% were in codon 12 region, and 12% in codon 13. The most frequent mutations of KRAS were related to G12D and 10% of patients had BRAF mutated genes. The type of KRAS gene mutations could be evaluated by reverse dot blot method. In general, the results of PCR-RFLP and allele-specific PCR were similar to the findings by reverse dot blot method.
Conclusion: These findings suggest that PCR-RFLP and allele-specific PCR methods are suitable for screening the presence of the mutations in KRAS and BRAF oncogenes. In fact, another method with more sensitivity is needed for a more accurate assessment to determine the type of mutations. Due to higher speed of detection, reduced Turnaround Time (TAT), and possible role of some KRAS point mutations in overall survival, reverse dot blot analysis seems to be an optimal method.
A Simplified Dot–Blot Hybridization Protocol for Potato spindle tuber viroid Detection in Solanaceae
A simplified dot-blot hybridization protocol for Potato spindle tuber viroid (PSTVd) detection in Solanaceae species is described here. The protocol uses an RNA DIG-labeled probe and a simplified extraction procedure that avoids the use of hazardous chemicals. PSTVd was detected in composite tomato leaf samples in a ratio of up to 1:15 of PSTVd-infected to non-infected tissue and in composite potato tuber samples in a ratio up to 1:5 of PSTVd-infected to non-infected tissue. In Brugmansia spp., PSTVd was detected solely in the standard sample extract preparation. The method is suitable for a reliable, large-scale sample screening especially where cost is a limiting factor.
Development of a PCR-based dot blot assay for the detection of fowl adenovirus
Group-I Fowl adenovirus (FAdV) is still widespread in China’s chicken farms, leading to huge economic losses. The traditional PCR method, which can detect all serotypes at the same time, is not sensitive enough to obtain accurate results, especially in some samples containing only a low titer of virus, such as contaminated live vaccine. In order to solve this problem, this study developed a dot blot assay based on the above PCR method. A total of 6 probes targeting the conserved region of FAdV were designed and systematically optimized through sensitivity, accuracy, and stability analyses.
Results showed that it is not only suitable for 12 serotypes, but also effectively improve the sensitivity, which increased more than 100 times in comparison with PCR assay. Moreover, this sensitivity was increased 100 times when detecting contaminated live vaccine samples, showing the great prospect of this method in daily monitoring.
Dot blot manifold 48 well |
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| CSLD48 | Scientific Laboratory Supplies | EACH | 638.4 EUR |
Dot blot manifold 96 well |
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| CSLD96 | Scientific Laboratory Supplies | EACH | 750.12 EUR |
Dot blot manifold 24 well |
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| CSLS24 | Scientific Laboratory Supplies | EACH | 750.12 EUR |
DiagNano Gold Nanoparticle Passive Conjugation Kit, Dot-blot |
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| GPK-DB | Creative Diagnostics | 1 kit | 976.8 EUR |
WHATMAN MINIFOLD I 96 WELL DOT-BLOT ARRA |
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| WHA10447900 | Scientific Laboratory Supplies | EACH | 1727.1 EUR |
FLU-A DOT-ELISA Kit |
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| DEIA436 | Creative Diagnostics | 6T/30T | 696 EUR |
H5 Influenza DOT-ELISA Kit |
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| DEIA438 | Creative Diagnostics | 6T/30T | 696 EUR |
EV-71 IgM DOT-ELISA Kit |
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| DEIA435 | Creative Diagnostics | 6T/30T | 696 EUR |
mini blotter 10x10 cm |
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| EVS1100-BLOT | Consort | ea | 818.4 EUR |
mini wide blotter 20x10 cm |
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| EVS1200-BLOT | Consort | ea | 950.4 EUR |
maxi blotter 20x20 cm |
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| EVS1300-BLOT | Consort | ea | 1335.6 EUR |
mini blotter 10x10 cm |
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| EVS3100-BLOT | Consort | ea | 1183.2 EUR |
maxi blotter 20x20 cm |
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| EVS3300-BLOT | Consort | ea | 1639.2 EUR |
Western Blot Kit |
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| 20-abx098123 | Abbexa |
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ELISA Microwell Strip Reader with built-in dot matrix printer |
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| MSR-10 | Alpha Diagnostics | 1 | 5613.6 EUR |
Western Blot Enhancing Kit |
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| K3172120 | Biochain | 1200 cm2 | 171.6 EUR |
Western Blot Enhancing Kit |
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| K3172250 | Biochain | 2500 cm2 | 225.6 EUR |
BlotBot Mini Blot Tray |
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| TRAY-2MINI-2 | Next Advance | 2pack | 244.8 EUR |
BlotBot Midi Blot Tray |
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| TRAY-MIDI-2 | Next Advance | 2pack | 242.4 EUR |
Sub Blot Mini Cassette |
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| SB10C | Scientific Laboratory Supplies | EACH | 52.44 EUR |
Sub Blot Maxi Cassette |
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| SB20C | Scientific Laboratory Supplies | EACH | 87.78 EUR |
Human Cell Line II Blot |
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| 1502 | ProSci | One blot | 544.2 EUR |
Human Normal Tissue Blot II |
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| 1522 | ProSci | One blot | 544.2 EUR |
Human Normal Tissue Blot VI |
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| 1526 | ProSci | One blot | 544.2 EUR |
Human Tumor Tissue Blot I |
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| 1542 | ProSci | One blot | 638.7 EUR |
Human Tumor Tissue Blot II |
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| 1543 | ProSci | One blot | 638.7 EUR |
Human Tumor Tissue Blot III |
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| 1544 | ProSci | One blot | 638.7 EUR |
Human Tumor Tissue Blot IV |
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| 1545 | ProSci | One blot | 638.7 EUR |
Improving Prognosis of Surrogate Assay for Breast Cancer Patients by Absolute Quantitation of Ki67 Protein Levels Using Quantitative Dot Blot (QDB) Method
Background: Immunohistochemistry (IHC)-based surrogate assay is the prevailing method in daily clinical practice to determine the necessity of chemotherapy for Luminal-like breast cancer patients worldwide. It relies on Ki67 scores to separate Luminal A-like from Luminal B-like breast cancer subtypes. Yet, IHC-based Ki67 assessment is known to be plagued with subjectivity and inconsistency to undermine the performance of the surrogate assay. A novel method needs to be explored to improve the clinical utility of Ki67 in daily clinical practice.
Materials and methods: The Ki67 protein levels in a cohort of 253 specimens were assessed with IHC and quantitative dot blot (QDB) methods, respectively, and used to assign these specimens into Luminal A-like and Luminal B-like subtypes accordingly. Their performances were compared with the Kaplan-Meier, univariate, and multivariate survival analyses of the overall survival (OS) of Luminal-like patients.
Results: The surrogate assay based on absolutely quantitated Ki67 levels (cutoff at 2.31 nmol/g) subtyped the Luminal-like patients more effectively than that based on Ki67 scores (cutoff at 14%) (Log rank test, p = 0.00052 vs. p = 0.031). It is also correlated better with OS in multivariate survival analysis [hazard ratio (HR) at 6.89 (95% CI: 2.66-17.84, p = 0.0001) vs. 2.14 (95% CI: 0.89-5.11, p = 0.087)].
Conclusions: Our study showed that the performance of the surrogate assay may be improved significantly by measuring Ki67 levels absolutely, quantitatively, and objectively using the QDB method.