ELISA/assay

Human Activin R2B/ACVR2B ELISA Kit

DEIA6918

96T

Human Activin R2B/ACVR2B ELISA Kit

ACVR2B

sELISA

ACVR2B

Human

Quantitative

HEK293 Cell culture, EDTA plasma, serum

96T

Unopened Kit: Store at 2-8°C for up to 4 months. For longer storage for up to 10 months, unopened Standard, Positive Control, Detection Antibody Concentrate, Dilution Buffer and Antibody & HRP Diluent Solution should be stored at -20°C or -70°C. Streptavidin-HRP Conjugate and TMB Substrate Solution should be stored only at 2-8°C. Do not use kit past expiration date.

The research pooled mouse serum or EDTA plasma, and rat serum or EDTA plasma samples do not show any cross-reactivity with this ELISA Kit. The heated and filtered fetal bovine serum samples does not show any cross-reactivity.

ACVR2B

Human

ACVR2B; activin A receptor, type IIB; activin receptor type-2B; ActR IIB; HTX4; ACTRIIB; ActR-IIB; bimagrumab; 1356922-05-8

For the quantitative determination of human Soluble ACVR2B or ACVR2B concentrations in cell cultures or circulating samples.

This ELISA Kit contains the necessary components required for the quantitative measurement of recombinant and/or natural soluble ACVR2B from gene transfected human cell cultures, circulating samples contains human ACVR2B in a sandwich ELISA format. Other sample types need to be validated with this assay. This immunoassay contains recombinant human sACVR2B (HEK293 derived) and antibodies raised against human soluble ACVR2B. Results from this immunoassay have shown to accurately quantify recombinant and natural human soluble ACVR2B or recombinant in soluble ACVR2B samples.

This assay employs the quantitative sandwich enzyme immunoassay technique. The plate is precoated with a monoclonal antibody specific for human sACVR2B. The capture antibody can bind to the sACVR2B in the standard and samples. After washing the plate of any unbound substances, a biotinylated antibody against human sACVR2B is added to the wells. After another washing of the plate, a Streptavidin-HRP conjugate is added. After the last wash to remove any unbound enzyme, a substrate solution is added to the wells and color develops in direct proportion to the amount of sACVR2B bound in the standard solutions or samples. A standard curve can be established and sample values can be read off the standard curve.

1. ACVR2B Microplate , 96 well polystyrene microplate (12 strips of 8 wells) coated with a monoclonal antibody against human soluble ACTRIIB. 1 plate. 2. ACVR2B-Fc Standard , 25 ng/vial of recombinant human soluble ACVR2B (HEK293) in a buffered protein base with preservative; lyophilized. 1 vial. 3. Detection Antibody Concentrate , 1.2 mL/vial, 10× concentrate of a purified biotinylated antibody against human soluble ACVR2B with preservative; lyophilized. 1 vial. 4. Positive Control , one vial of recombinant human soluble ActR-IIB; lyophilized. 1 vial. 5. Streptavidin-HRP Conjugate , 100 μL/vial, 100× concentrated solution of Streptavidin conjugate to HRP. 1 vial. 6. Dilution Buffer , 40 mL of buffered protein based solution with preservative. 1 bottle. 7. Antibody & HRP Diluent Solution , 25 mL of buffered protein based solution with preservative. 1 bottle. 8. Wash Buffer , 25 mL of 20× concentrated buffered surfactant, with preservative. 1 bottle. 9. TMB Substrate Solution , 11 mL of TMB substrate solution. 1 bottle. 10. Stop Solution , 11 mL of 0.25M HCl solution. 1 bottle. 11. Plate Sealer , 1 12. Plastic Pouch , 1

1. Microplate reader capable of absorbance measurement at 450 nm. 2. Microplate shaker (350-450 rpm). 3. Microplate washer or manifold dispenser. 4. 100 mL and 500 mL graduated cylinders. 5. Multi-channel pipette, pipettes and pipette tips. 6. Deionized or distilled water. 7. 500mM TCEP (fresh preparation) 8. Sample Buffer Solution: 20 mL of 0.1% SDS in PBS, PH 7.4 (Warm it in 37°C water bath before use).

Note: Due endogenous ligands were binding to soluble ACVR2B, the natural human serum or EDTA plasma samples may require pretreated before being added to the microplate. Standard and Positive Control DO NOT NEED to be treated. 1. Add 240 μl of 500 mM TCEP to 11.76 mL of Sample Buffer Solution to prepare the Pretreatment Solution (10mM TCEP, 0.1% SDS in PBS, pH 7.4) (12 mL for 50 samples pretreatment). 2. Add 70 μl of sample to 210 μl of Pretreatment Solution in a polypropylene tube or vial. Note: This pretreatment dilution (4-fold dilution) may require optimization. 3. Vortex gently and incubate for 30 minutes at room temperature. Assay immediately and discard any excess pretreated samples. Optimal dilutions should be determined by each laboratory for each application with a pretest. Use polypropylene test tubes.

Note: Bring all reagents to room temperature before use. 1. Wash Buffer: If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have completely dissolved. Dilute 25 mL of Wash Buffer Concentrate into deionized or distilled water (475 mL) to prepare 500 mL of 1× Wash Buffer. 2. ACVR2B Standard: Reconstitute the human soluble ACVR2B standard with 1.0 mL of Dilution Buffer. This reconstitution produces a stock solution of 25 ng/mL. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making dilutions. Pipette 500 μL of Dilution Buffer into tubes #1 to #7. Use the stock solution to produce a dilution series (below). Mix each tube thoroughly before the next transfer. The 12.5 ng/mL standard serves as the high standard. The Dilution Buffer serves as the zero standard (0 ng/mL). 3. Positive Control: Reconstitute the Positive Control with 1 mL of Dilution Buffer. 4. Detection Antibody Concentrate: : Reconstitute the Detection Antibody Concentrate with 1.2 mL of Antibody & HRP Diluent Solution to produce a 10-fold concentrated stock solution. Pipette 9.45 mL of Antibody & HRP Diluent Solution into a 15 mL centrifuge tube and transfer 1.05 mL of 10-fold concentrated stock solution to prepare working solution. 5. Streptavidin-HRP Conjugate: Pipette 10.89 mL of Antibody & HRP Diluent Solution into a 15 mL centrifuge tube and transfer 110 μL of 100-fold concentrated stock solution to prepare working solution ( protect from light ). The working solution of Streptavidin-HRP Conjugate should be used within a few hours.

Note: Bring all reagents and samples to room temperature before the start of the assay. Blank, standard dilutions, positive control and samples should be assayed in duplicate. ELISA Protocol may need further optimization. 1. Prepare all samples, reagents and working standards as directed in the previous sections. 2. Add 100 μL of Dilution Buffer to Blank wells (B). 3. Add 100 μL of standard dilutions in reverse order of serial dilution from #7 to #S, samples, or positive control (P) per well. Cover with the plate sealer. Incubate for 2 hours on microplate shaker (350-400 rpm) at room temperature. 4. Aspirate each well and wash, repeating the process three times for a total of four washes. Wash by filling each well with 1x Wash Buffer (300 μL) using a squirt bottle, manifold dispenser, or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels. 5. Add 100 μL of Detection Antibody working solution to each well. Cover with the plate sealer. Incubate for 2 hours on microplate shaker at room temperature. 6. Repeat the aspiration/wash as in step 4. 7. Add 100 μL of Streptavidin-HRP Conjugate working solution to each well. Incubate for 60 minutes on microplate shaker at room temperature. Protect from light . 8. Repeat the aspiration/wash as in step 4. 9. Add 100 μL of Substrate Solution to each well. Incubate for 14-18 minutes on microplate shaker at room temperature. Protect from light. 10. Add 100 μL of Stop Solution to each well. The color in the wells should change from blue to yellow. If the color in the wells is green, or if the color change does not appear uniform, gently tap the plate to ensure thorough mixing. 11. Determine the optical density of each well using a microplate reader set to 450 nm within 3 minutes.

Create a standard curve by plotting the log of the known concentrations of the standard dilutions (xaxis) versus the log of its corresponding O.D. (y-axis) and draw the best fit line through the points. It is recommended to use computer software capable of generating a log-log curve or 4-Parameter fit to more accurately quantify the standard dilutions. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor (pretreated as suggested will have a dilution factor of 4). Sample O.D. readings with a concentration exceeding that of standard 12.5 ng/mL may result in inaccurate, low human ACVR2B levels. Such samples require further external predilution according to expected human ACVR2B values with Dilution Buffer and then pretreated in order to precisely quantify the actual human ACVR2B level.

This standard curve is provided for demonstration only. A new standard curve should be generated for each set of samples assayed. Lot No.: 20114704 Positive Control: 1-5 ng/mL

Intra-assay Precision: 4-7% Inter-assay Precision: 8-12%

0.195-12.5 ng/mL

50 pg/mL

This kit should be handled by those persons who have been trained in and can follow the principles of good laboratory practice. Wear protective clothing such as laboratory overalls, safety glasses and gloves. Care should be taken while handling solutions in this kit to avoid contact with skin or eyes, especially with the stop solution because it contains diluted hydrochloric acid. Wash immediately with water in case of contact on skin or eyes. Antibody & HRP Diluent Solution need shake well prior to use.

1. FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES. 2. This ELISA kit should not be used beyond the expiration date on the kit label. 3. Do not mix reagents with those from other lots or sources. 4. It is important that the Dilution Buffer selected for the standard curve be consistent with the samples being assayed. 5. Each laboratory must determine the optimal dilution factors for the samples being assayed with a pretest. 6. Any modifications in buffers, pipetting technique, washing technique, incubation time or temperature, as well as kit age can cause a change in signal. 7. Not all interfering factors have been tested in the immunoassay, therefore the possibility of interference cannot be excluded.

For the quantitative determination of human Soluble ACVR2B or ACVR2B concentrations in cell cultures or circulating samples.

1. ACVR2B Microplate , 96 well polystyrene microplate (12 strips of 8 wells) coated with a monoclonal antibody against human soluble ACTRIIB. 1 plate. 2. ACVR2B-Fc Standard , 25 ng/vial of recombinant human soluble ACVR2B (HEK293) in a buffered protein base with preservative; lyophilized. 1 vial. 3. Detection Antibody Concentrate , 1.2 mL/vial, 10× concentrate of a purified biotinylated antibody against human soluble ACVR2B with preservative; lyophilized. 1 vial. 4. Positive Control , one vial of recombinant human soluble ActR-IIB; lyophilized. 1 vial. 5. Streptavidin-HRP Conjugate , 100 μL/vial, 100× concentrated solution of Streptavidin conjugate to HRP. 1 vial. 6. Dilution Buffer , 40 mL of buffered protein based solution with preservative. 1 bottle. 7. Antibody & HRP Diluent Solution , 25 mL of buffered protein based solution with preservative. 1 bottle. 8. Wash Buffer , 25 mL of 20× concentrated buffered surfactant, with preservative. 1 bottle. 9. TMB Substrate Solution , 11 mL of TMB substrate solution. 1 bottle. 10. Stop Solution , 11 mL of 0.25M HCl solution. 1 bottle. 11. Plate Sealer , 1 12. Plastic Pouch , 1

See the individual product datasheet

See the individual product datasheet

See the individual product datasheet

See the individual product datasheet