# High-Sensitivity Protein Detection Using Due Bio TRF Immunoassay Analyzer
## Abstract
Traditional immunoassays lack the sensitivity required for early disease biomarker detection and low-abundance protein quantification. Due Bio’s TRF (Time-Resolved Fluorescence) Immunoassay Analyzer utilizes Europium Chelate technology to achieve 10–100× higher sensitivity than conventional colorimetric or fluorescent methods—without requiring expensive laboratory instrumentation. This application note demonstrates a robust protocol for quantifying infectious disease markers and hormones using Due Bio’s TRF platform.
## Introduction
Time-Resolved Fluorescence (TRF) spectroscopy has long been the method of choice for ultra-sensitive immunoassays in clinical laboratories. However, traditional TRF readers are costly, bulky, and restricted to centralized testing facilities. Due Bio’s TRF Immunoassay Analyzer brings this gold-standard technology to point-of-care and resource-limited settings through compact design and cost-effective reagents.
Europium chelate labels exhibit long fluorescence lifetimes (microseconds vs. nanoseconds for conventional fluorophores), enabling time-gated detection that eliminates background autofluorescence. The result: exceptional signal-to-noise ratios and limits of detection in the pg/mL range.
As a POCT manufacturer since 1987, Due Bio combines TRF expertise with manufacturing scalability to deliver high-performance diagnostics at accessible price points for emerging markets.
## Materials Required
– **Due Bio TRF Immunoassay Analyzer** (benchtop or portable format)
– **Due Bio IVD Reagents** (Europium-labeled antibodies, streptavidin-coated microplates)
– Target-specific antibody pairs (capture and detection)
– Sample types: serum, plasma, whole blood, or urine
– Assay buffer and wash buffer (Due Bio TRF Enhancement Solution)
– Micropipettes (10 μL, 50 μL, 100 μL)
– Plate shaker or orbital mixer
– Timer or stopwatch
– Refrigerated storage (4°C) for reagents
*Note: Europium conjugates must be protected from light. Store all reagents at 4°C. Shelf life: 12 months from manufacture date.*
## Step-by-Step Protocol
### 1. Plate Preparation
Coat a 96-well microplate with capture antibody (2 μg/mL in carbonate-bicarbonate buffer, pH 9.6, 100 μL/well). Incubate overnight at 4°C or 2 hours at 37°C with gentle shaking. Wash 3× with PBST (PBS with 0.05% Tween-20). Block with 5% BSA in PBS (200 μL/well) for 1 hour at room temperature to minimize non-specific binding.
### 2. Sample and Standard Addition
Prepare a standard curve using serial dilutions of recombinant protein in assay buffer (range: 0–10 ng/mL, 7 points in duplicate). Add 50 μL of standards or diluted samples to each well. Add 50 μL of Europium-labeled detection antibody (0.5 μg/mL in Due Bio assay buffer). Incubate with orbital shaking (300 rpm) for 2 hours at room temperature.
### 3. Washing
Wash the plate 6× with wash buffer (PBST with 0.05% Tween-20, 300 μL/well). Ensure complete removal of unbound conjugate by inverting the plate and tapping firmly on absorbent paper. Residual wash buffer can dilute the enhancement solution and reduce signal.
### 4. TRF Enhancement
Add 100 μL of Due Bio TRF Enhancement Solution to each well. Incubate with gentle orbital shaking for 10 minutes at room temperature. The enhancement solution dissociates Europium ions from the antibody-bound chelate and forms highly fluorescent micellar complexes that emit stable, long-lifetime fluorescence.
### 5. Measurement
Insert the plate into the Due Bio TRF Immunoassay Analyzer. Set measurement parameters: excitation 340 nm, emission 615 nm, delay time 400 μs, integration time 400 μs, cycle time 1000 μs. Read the plate and generate a standard curve using 4-parameter logistic (4-PL) regression. Calculate sample concentrations by interpolation.
## Troubleshooting
### Q: High background in negative controls.
**A**: Incomplete washing or non-specific binding are common causes. Increase wash cycles to 8× and ensure thorough aspiration. Add 0.1% casein or 5% normal serum to assay buffer to reduce non-specific interactions. Verify that Europium conjugate is stored protected from light and has not degraded.
### Q: Low signal in positive controls.
**A**: Antibody concentration may be suboptimal. Titrate both capture and detection antibodies to find optimal concentrations. Verify that Europium labeling efficiency is >3 Eu/molecule using standard chelation assays. Ensure enhancement solution is fresh and not expired—old enhancement solution loses activity.
### Q: Poor standard curve linearity (R² < 0.99). **A**: Pipetting errors or reagent degradation may cause non-linearity. Use calibrated pipettes and fresh tips for each dilution. Prepare standards immediately before use. Ensure plate is read within 60 minutes of enhancement—signal decays over time. --- ## Conclusion & OEM/ODM Call-to-Action Due Bio's TRF Immunoassay Analyzer delivers laboratory-grade sensitivity in a compact, cost-effective platform. The Europium Chelate technology provides 10–100× higher sensitivity than conventional colorimetric or fluorescent methods, enabling early disease detection and low-abundance biomarker quantification. **Established in 1987**, Due Bio is a global POCT manufacturer committed to advancing accessible molecular diagnostics. Beyond our standard TRF platform, we provide comprehensive **IVD Reagents OEM/ODM services** including custom antibody conjugation, sandwich pair screening, assay development, and regulatory support for CE/FDA submission. Whether you're developing an infectious disease panel, a tumor marker assay, or a hormone quantification test, Due Bio's technical team can support your project from prototype to commercialization. **Contact us today**: medtiger@foxmail.com --- *For research use only. Not for diagnostic procedures unless validated and approved according to local regulations.*