The RayBio^® NexaTag^® quantitative immuno-PCR ELISA (previously known as BIQ-ELISA) is an innovative protein detection method that merges the specificity of sandwich-based ELISA, the sensitivity of real-time PCR, and the easy manipulation of magnetic microspheres. In this system, the capture antibody (CAB) is biotin- labelled and pre-absorbed onto streptavidin-coated beads (CAB-Biotin-SA-Beads), while the detection antibody (DAB) is conjugated with a unique oligonucleotide acid (ONA) barcode for signal amplification (DAB-ONA).

The procedure is straightforward and a total of four hours is required to complete the assay. Part 1: The formation and purification of the immunocomplex (ONA-DAB-Antigen-CAB-Biotin-SA-Beads). Samples are incubated with DAB-ONA and CAB-Biotin-SA-Beads in a supplied 96-well plate. In the presence of the target protein, the DABONA binds to the target and attaches to the CAB-Biotin-SA-Beads via the biotin-streptavidin interaction and the unbound proteins are removed through washing with a magnetic automated washer. Part 2: PCR amplification. The bound immunocomplex will be released from the beads and transferred as the template for qPCR. Primers and PCR master mix are added to the wells and data will be collected using qPCR. Ct values obtained from the qPCR are then used to calculate the amount of antigen contained in each sample, where lower Ct values indicate a higher concentration of antigen. In a real experiment, a set of target protein standards with known concentrations will be run simultaneously to generate a standard curve for quantifying unknown samples.

The RayBio^® NexaTag^® qIPCR ELISA technology represents a significant advancement in protein detection assays, providing an ultrasensitive and highly specific method for quantifying proteins in biological samples. This technology's ability to combine multiple advantages of existing methods into a single, easy-to-use platform makes it a powerful tool for research community. The RayBio NexaTag^® qIPCR ELISA kits can be used for the quantitative measurement of proteins in various sample types including serum, plasma, and cell culture supernatants with femtogram per milliliter detection sensitivity.

• Ultra-sensitivity: Up to 1,000x more sensitive than traditional ELISA
• Ultra-low sample volume: Less than 10 µL needed
• Specificity: Sandwich-based detection
• Simplicity: Two steps processing
• Efficiency: Get data within 4 hours
• Multiplexing ready*

*Increase your throughput with RayBiotech's customizable multiplex bead-based immuno-PCR kits, designed for panels of up to 5 targets. This platform helps you save time, reduce costs, and decrease sample consumption by >2000% compared to traditional colorimetric ELISA. For more details, reach out to us at techsupport@raybiotech.com.

*The PCR plate used is a 0.2 mL, non-skirted 96-well plate (Thermo Fisher, cat. no. AB0600). Please ensure compatibility with your PCR machine prior to purchase. For additional information contact technical support (techsupport@raybiotech.com).

• Real-time PCR instrument, Bio-Rad or QuantStudio™ recommended
• Orbital 96-well plate shaker (with ability to reach 700 rpm)
• RayBio^® 96-well Magnetic Plate Separator (Catalog #: BIQ-MPS) or similar
• Optional: Automated microplate washer (BioTek 405 TS Washer recommended) with magnetic separation (RayBio^® 96-well Magnetic Plate Separator recommended)
• Precision pipettes to deliver 1 μL to 1 mL volumes
• Pipette tips (barrier tips are recommended to reduce contamination)
• Adjustable 1-25 mL pipettes for reagent preparation
• Centrifuge that can spin 96 well plates at 500 g
• Nuclease-free water or deionized water
• 1.5 mL, 2 mL, or 5 mL microcentrifuge tubes
• Absorbent paper

1. Prepare all reagents, samples and standards as instructed.
2. Add 5 μL of the bead and conjugate mix, and 5 μL of each standard and sample to each well. Incubate for 2.5 hours at room temperature while shaking.
3. Add 40 μL Assay Diluent and wash the plate using a magnetic plate separator or automated microplate washer.
4. Add 40 μL Elution buffer to each well. Incubate for 10 minutes at room temperature.
5. Prepare PCR/Primer Master Mix and pipette 15 μL into each well of the PCR plate.
6. Transfer 5 μL supernatant from V-bottom plate to the PCR plate.
7. Run real-time PCR.