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A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules
Published on: March 10, 2017
A new method for screening acute/chronic lymphocytic leukemia: dual-label time-resolved fluorescence immunoassay
Zhen Zhang1, Jintao Zhang2, Shanshan Dai3
1Department of Logistics Management, Baoshan Branch, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200444, China.
Insights
A new time-resolved fluorescence immunoassay (TRFIA) method accurately detects S100 calcium binding protein A8 (S100A8) and leucine-rich alpha-2-glycoprotein 1 (LRG1) in serum. This assay shows promise for screening lymphocytic leukemia (LL) in clinical settings.
Area of Science:
- Biochemistry
- Immunology
- Clinical Diagnostics
Background:
- Lymphocytic leukemia (LL) is a significant malignant hematopoietic tissue tumor affecting children and the elderly.
- Current diagnostic methods require improvement for early and accurate LL screening.
Purpose of the Study:
- To develop and validate a novel time-resolved fluorescence immunoassay (TRFIA) for detecting S100 calcium binding protein A8 (S100A8) and leucine-rich alpha-2-glycoprotein 1 (LRG1).
- To assess the utility of this TRFIA method for screening acute and chronic LL.
Main Methods:
- A sandwich TRFIA was optimized using anti-S100A8/LRG1 capture antibodies and detection antibodies labeled with Europium(III) or Samarium(III) chelates.
- Quantitative fluorescence intensity was measured using time-resolved fluorometry.
- Assay sensitivity, dynamic range, intra- and inter-assay coefficients of variation (CVs), specificity, and affinity were evaluated in serum samples.
Main Results:
- The TRFIA demonstrated high sensitivity for S100A8 (1.15 ng/mL) and LRG1 (3.2 ng/mL) with wide dynamic ranges.
- Low intra- and inter-assay CVs (5.75–8.23% for S100A8, 5.30–9.45% for LRG1) indicated assay precision.
- Cutoff values for S100A8 and LRG1 were determined as 1849.18 ng/mL and 588.08 ng/mL, respectively.
Conclusions:
- The developed TRFIA method offers high sensitivity, accuracy, and specificity for quantifying S100A8 and LRG1 in serum.
- This TRFIA assay is a viable and effective tool for the clinical screening of lymphocytic leukemia (LL).
Background:
Lymphocytic leukemia (LL) is a primary malignant tumor of hematopoietic tissue, which seriously affects the health of children and the elderly. The study aims to establish a new detection method for screening acute/chronic LL using time-resolved fluorescence immunoassay (TRFIA) via quantitative detection of S100 calcium binding protein A8 (S100A8) and leucine-rich alpha-2-glycoprotein 1 (LRG1) in serum.
Methods:
Here a sandwich TRFIA was optimized and established: Anti-S100A8/LRG1 caputre antibodies immobilized on 96-well plates captured S100A8/LRG1, and then banded together with the anti-S100A8/LRG1 detection antibodies labeled with Europium(III) (Eu3+)/samarium(III) (Sm3+) chelates. Finally time resolved fluorometry measured the fluorescence intensity.
Results:
The sensitivity of S100A8 was 1.15 ng/mL(LogY = 3.4027 + 0.4091 × LogX, R2 = 0.9828, P < 0.001, dynamic range: 2.1-10,000 ng/mL), and 3.2 ng/mL for LRG1 (LogY = 3.3009 + 0.4082 × LogX, R2 = 0.9748, P < 0.001, dynamic range: 4.0-10,000 ng/mL). The intra-assay and inter-assay CVs were low, ranging from 5.75% to 8.23% for S100A8 and 5.30% to 9.45% for LRG1 with high specificity and affinity in serum samples. Bland-Altman plots indicated TRFIA and ELISA kits have good agreement in clinical serum samples. Additionally, the cutoff values for S100A8 and LRG1 were 1849.18 ng/mL and 588.08 ng/mL, respectively.
Conclusion:
The present TRFIA method could be used for the quantitative detection of S100A8 and LRG1 in serum, and it has high sensitivity, accuracy and specificity. Clinically, this TRFIA method could be suitable for screening of LL via the quantitative detection of S100A8 and LRG1.
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