Human P-Selectin IQELISA™ Kit

RayBio® Human Immunoquantitative (PCR-Based) P-Selectin ELISA Kit for cell culture supernatants, plasma, and serum samples.

Specifications

Introduction

The RayBio^® Immuno Quantitative Enzyme Linked ImumunoSorbent Assay (IQELISA„™) is an innovative new assay that combines the specificity and ease of use of an ELISA with the sensitivity of real-time PCR. This results in an assay that is simultaneously familiar and cutting edge and enables the use of lower sample volumes while also providing more sensitivity. The RayBio^® Human P-Selectin IQELISA„™ Kit is a modified ELISA assay with high sensitivity qPCR readout for the quantitative measurement of Human P-Selectin in serum, plasma, and cell culture supernatants. This assay employs an antibody specific for Human P-Selectin coated on a 96-well PCR plate. Standards and samples are pipetted into the wells and P-Selectin present in a sample is bound to the wells by the immobilized antibody. The wells are washed and a detection affinity molecule is added to the plates. After washing away unbound detection affinity molecule, primers and a PCR master mix are added to the wells and data is collected using qPCR. C_t values obtained from the qPCR are then used to calculate the amount of antigen contained in each sample, where lower C_t values indicate a higher concentration of antigen.

Comparison

Standard Curve

Typical Data

Application Notes

Kit Components

• - P-Selectin Microplate (Item A)**: 96 well PCR plate coated with anti-Human P-Selectin

• - Wash Buffer I Concentrate (20x) (Item B): 25 ml of 20x concentrated solution

• - Standards (Item C): 2 vials of recombinant Human P-Selectin

• - Assay Diluent B (Item E): 15 ml of 5x concentrated buffer

• - Detection Affinity Reagent for P-Selectin (Item F): 2 vials of a 4x concentrated solution of anti-Human P-Selectin affinity reagent

• - IQELISA„™ Detection Reagent (Item G): 1 ml of a 10x concentrated stock

• - Primer Solution (Item I): 1.5 ml vial

• - PCR Master Mix (Item J): 1.4 ml vial

• - PCR Preparation buffer (Item K): 1 ml vial of 10x concentrated buffer

• - Final Wash Buffer (Item L): 10 ml vial of 10x concentrated buffer

**The PCR plate used is a 0.2 ml, non-skirted 96-well plate (ThermoFisher, cat. no.: AB0600). Please ensure compatibility with your PCR machine prior to purchase. For additional information contact technical support (cs@medikonia.com).

Other Materials Required

• - Real-time PCR instrument, Bio-Rad recommended

• - Precision pipettes to deliver 2 µl to 1 ml volumes

• - Adjustable 1-25 ml pipettes for reagent preparation

• - 100 ml and 1 liter graduated cylinders

• - Absorbent paper

• - Distilled or deionized water

• - Log-log graph paper or computer and software for data analysis

• - Tubes to prepare standard or sample dilutions

• - Heating block or water bath capable of 80°C

Protocol Outline

• 1. Prepare all reagents, samples and standards as instructed

• 2. Add 25 µl standard or sample to each well. Incubate for 1.5 - 2.5 hours at room temperature

• 3. Add 25 µl detection antibody to each well. Incubate 1 hour at room temperature

• 4. Add 25 µl of IQELISA„™ Detection Reagent to each well. Incubate 1 hour

• 5. Add 10 µl Primer solution and 10 µl of PCR master mix to each well

• 6. Run real-time PCR

Storage/Stability

May be stored for up to 6 months at 2° to 8°C from the date of shipment. Standard (recombinant protein) should be stored at -20°C or -80°C (recommended at -80°C) after reconstitution. Opened PCR plate or reagents may be stored for up to 1 month at 2° to 8°C. Note: the kit can be used within one year if the whole kit is stored at -20°C. Avoid repeated freeze-thaw cycles.

Catalog #: IQH-PSelectin-1 -- 1 Plate Kit

Catalog #: IQH-PSelectin-2 -- 2 Plate Kit

Catalog #: IQH-PSelectin-5 -- 5 Plate Kit

Please email us at cs@medikonia.com for any enquiry. To place an order, please include the catalog number(s) of the product(s) in the email.

Manual

Safety Data Sheet