Human Betacellulin ELISA Development Kit

Human Betacellulin ELISA development kit contains the key components required for the quantitative measurement of natural and/or recombinant betacellulin in a sandwich ELISA format. Using the ELISA protocol described below, this kit provides sufficient reagents to assay Betacellulin in approximately 1500 ELISA plate wells.

1.) Capture Antibody
2.) Detection Antibody
3.) Standard
4.) StreptAvidin-HRP
5.) TMB Liquid Substrate "Ready to Use"

Store the unopened product at 2 - 8 °C. Refer to the lot-specific datasheet (C of A) for details on each component.

Additional Required Materials
ELISA microplates (Thermo Fisher Cat. # 456529)
BSA (Sigma Cat. # A-7030)
Dulbecco’s PBS [10x] (Leinco Part No.: D388)
Stop Solution 2 M Sulfuric Acid (Leinco Part No.: T125)

Required Solutions
PBS: dilute 10xPBS to 1xPBS, pH 7.2, in sterile water
Wash Buffer: 0.05% Tween-20 in PBS (Leinco Part No.: W101)
Reagent Diluent: 1.0% BSA in PBS*
Blocking Buffer 1.0% BSA in PBS (Leinco Part No.: B395)
Note: Other acceptable blocking buffers like Leinco's Ultra-FISH Block (Leinco Part No.: B396) or Ultra-Sythetic Block (Leinco Part No.: B397) may be used for optimization of assay.

Some of the required components may contain acid cause allergic reactions. Wear protective gloves, clothing, eye, and face protection. Wash hands thoroughly after handling. Please refer to the MSDS on our website prior to use.

Mouse anti-Human Betacellulin capture antibody: Centrifuge vial prior to opening. Reconstitute in 1.0 mL sterile PBS. Refer to the lot-specific datasheet for amount supplied and dilute in PBS without carrier protein to the working concentration indicated on the C of A. Following reconstitution the capture antibody may be stored at 2 – 8°C for up to 6 months. For long term storage, it is recommended to aliquot into working volumes and store at -70°C in a manual defrost freezer. Avoid repeated freeze and thaw cycles.

Biotinylated goat anti-human Betacellulin: Refer to the lot-specific datasheet for amount supplied. Centrifuge vial prior to opening. Reconstitute with 1.0 mL of reagent diluent. Dilute in Reagent Diluent to the working concentration indicated on the C of A shipped with product. Detection antibodies may be stored at 2 – 8°C for up to 6 months. For long term storage, it is recommended to aliquot into working volumes and store at -70°C in a manual defrost the freezer. Avoid repeated freeze and thaw cycles.

E. coli-expressed Recombinant Human Betacellulin: Centrifuge vial prior to opening. Reconstitute each vial with 0.5 mL of Reagent Diluent. Refer to the lot-specific datasheet for amount supplied. The rProtein may be stored at 2 – 8°C for one (1) month or aliquoted and stored at -70°C for up to three months in a manual defrost freezer. Avoid repeated freeze and thaw cycles.

Streptavidin-HRP: Each vial contains approximately 1.0 mL of streptavidin horseradish-peroxidase (HRP). Dilute to the working concentration specified on the vial label using Reagent Diluent. Upon receipt, SteptAvidin-HRP conjugate should be stored at 2 – 8°C, <b>DO NOT FREEZE.</b>

TMB Liquid Substrate "Ready to Use" (TMB Substrate should be at ambient temperature prior to use): 60.0 mL of TMB HRP Microwell Substrate Standard Kinetic One Component "Ready Use" (Leinco Prod. T118) is provided. The high quality of the substrate can be preserved by storing at temperatures between 2 – 8ºC. When properly stored, TMB Microwell Substrate is stable for a minimum of 48 months from the manufactured date.

1. Dilute the capture antibody to the working concentration in PBS without carrier protein and immediately add 100 μL to each ELISA plate well. Seal the plate and incubate overnight at room temperature.
2. Aspirate the wells to remove liquid and wash the plate 4 times using 300-400 μL of wash buffer per well. Note: We recommend using an autowasher although a squirt bottle or manifold dispenser would suffice.
3. After the last wash invert plate to remove residual buffer and blot on paper towel.
4. Add 300 μL block buffer to each well and incubate for at least 1 hour at room temperature.
5. Aspirate and wash plate 4 times.
NOTE: Complete removal of the liquid at each step is essential for good performance and sensitivity of assay.

Standard/Sample: Add 100 μL of the working dilution with reagent dilution standard or sample to each well (Duplicate Recommended) and incubate at room temperature for at least 2 hours.

Detection: Aspirate and wash plate 4 times. Add 100 μL of the diluted in Reagent Diluent detection antibody per well and incubate at room temperature for 2 hours.

StreptAvidin-HRP Conjugate: Aspirate and wash plate 4 times. Add 100 μL of the working dilution (this dilution factor may require some optimization) to each well. Cover & Incubate at room temperature for 20-30 minutes.

TMB Liquid Substrate: Aspirate and wash plate 4 times. Add 100 μL of TMB HRP Microwell Substrate Standard Kinetic One Component "Ready Use" (Leinco Prod. T118) to each well. Incubate at room temperature for 20-30 minutes and monitor color development, but Avoid placing plates in direct light.

Stop Solution: Add 50-100 μL of stop solution (Leincio Part No.: T135) to each well. Monitor color development with an ELISA plate reader at 450 nm with wavelength correction set at 540 nm or 570 nm.

Betacellulin (BTC) is a new member of the EGF family of cytokines that is comprised of at least ten proteins including EGF, TGF-α, amphiregulin, HB-EGF, and the various heregulins. All of these cytokines are synthesized as transmembrane precursors and are characterized by the presence of one or more EGF structural units in their extracellular domain. The soluble forms of these cytokines are released by proteolytic cleavage. BTC, a heparin-binding protein, was originally isolated from the conditioned media of mouse pancreatic beta tumor cells. The cDNA encoding human BTC was cloned from a human breast adenocarcinoma cell line (MCF-7) cDNA library. At the amino acid sequence level, human BTC precursor protein exhibits 79% identity with that of the mouse BTC precursor.

BTC is produced in several tissues, including the pancreas, small intestine, and in certain tumor cells. Betacellulin is a potent mitogen for retinal pigment epithelial cells and vascular smooth muscle cells.