RBP4 ELISA Kit (Retinol Binding Protein 4, Plasma) ELISA Kit
- Detection Method
- Method Type
- Sandwich ELISA
- Detection Range
- 0.312 ng/mL - 20 ng/mL
- Minimum Detection Limit
- 0.312 ng/mL
- The kit is a sandwich enzyme immunoassay for in vitro quantitative measurement of RBP4 in rat serum, plasma.
- Sample Type
- Plasma, Serum
- Analytical Method
- This assay has high sensitivity and excellent specificity for detection of Retinol Binding Protein 4 (RBP4)
- < 0.151 ng/mL
- Pre-coated, ready to use 96-well strip plate
- Plate sealer for 96 wells
- Standard Diluent
- Detection Reagent A
- Detection Reagent B
- Assay Diluent A
- Assay Diluent B
- TMB Substrate
- Stop Solution
- Wash Buffer (30 x concentrate)
- Instruction manual
- Material not included
- Microplate reader with 450 ± 10nm filter.
- Precision single or multi-channel pipettes and disposable tips.
- Eppendorf Tubes for diluting samples.
- Deionized or distilled water.
- Absorbent paper for blotting the microtiter plate.
- Container for Wash Solution
- 0.01Mol/L (or 1x) Phosphate Buffered Saline(PBS), pH 7.0-7.2.
Retinol Binding Protein 4, Plasma (RBP4) ELISA Kit ELISA Kit
RBP4 Reactivity: Mouse Colorimetric Sandwich ELISA 0.312 ng/mL - 20 ng/mL Cell Culture Supernatant, Cell Lysate, Plasma, Serum, Tissue HomogenateRetinol Binding Protein 4, Plasma (RBP4) ELISA Kit ELISA Kit
RBP4 Reactivity: Chicken Colorimetric Sandwich ELISA 0.156 ng/mL - 10 ng/mL Cell Culture Supernatant, Cell Lysate, Plasma, Serum, Tissue HomogenateRetinol Binding Protein 4, Plasma (RBP4) ELISA Kit ELISA Kit
RBP4 Reactivity: Cow Colorimetric Sandwich ELISA 0.312 ng/mL - 20 ng/mL Cell Culture Supernatant, Cell Lysate, Plasma, Serum, Tissue Homogenate
- Sample Volume
- 100 μL
- Assay Time
- 3 h
- Prepare all reagents, samples and standards,
- Add 100μL standard or sample to each well. Incubate 1 hours at 37 °C,
- Aspirate and add 100μL prepared Detection Reagent A. Incubate 1 hour at 37 °C,
- Aspirate and wash 3 times,
- Add 100μL prepared Detection Reagent B. Incubate 30 minutes at 37 °C,
- Aspirate and wash 5 times,
- Add 90μL Substrate Solution. Incubate 10-20 minutes at 37 °C,
- Add 50μL Stop Solution. Read at 450nm immediately.
- Reagent Preparation
- Bring all kit components and samples to room temperature (18-25 °C) before use. If the kit will not be used up in one time, please only take out strips and reagents for present experiment, and leave the remaining strips and reagents in required condition.
- Standard - Reconstitute the Standard with 1.0 mL of Standard Diluent, kept for 10 minutes at room temperature, shake gently(not to foam). The concentration of the standard in the stock solution is 80 ng/mL. Please firstly dilute the stock solution to 20 ng/mL and the diluted standard serves as the highest standard (20 ng/mL). Then prepare 7 tubes containing 0.5 mL Standard Diluent and use the diluted standard to produce a double dilution series according to the picture shown below. Mix each tube thoroughly before the next transfer. Set up 7 points of diluted standard such as 20 ng/mL, 10 ng/mL, 5 ng/mL, 2.5 ng/mL, 1.25 ng/mL, 0.625 ng/mL, 0.312 ng/mL, and the last EP tubes with Standard Diluent is the blank as 0 ng/mL.
- Detection Reagent A and Detection Reagent B - Briefly spin or centrifuge the stock Detection A and Detection B before use. Dilute them to the working concentration 100-fold with Assay Diluent A and B, respectively.
- Wash Solution - Dilute 20 mL of Wash Solution concentrate (30x) with 580 mL of deionized or distilled water to prepare 600 mL of Wash Solution (1x).
- TMB substrate - Aspirate the needed dosage of the solution with sterilized tips and do not dump the residual solution into the vial again.
- Making serial dilution in the wells directly is not permitted.
- Prepare standards within 15 minutes before assay. Please do not dissolve the reagents at 37 °C directly.
- Please carefully reconstitute Standards or working Detection Reagent A and B according to the instruction, and avoid foaming and mix gently until the crystals are completely dissolved. To minimize imprecision caused by pipetting, use small volumes and ensure that pipettors are calibrated. It is recommended to suck more than 10μL for one pipetting.
- The reconstituted Standards, Detection Reagent A and Detection Reagent B can be used only once.
- If crystals have formed in the Wash Solution concentrate (30x), warm to room temperature and mix gently until the crystals are completely dissolved.
- Contaminated water or container for reagent preparation will influence the detection result.
- Sample Preparation
- We are only responsible for the kit itself, but not for the samples consumed during the assay. The user should calculate the possible amount of the samples used in the whole test. Please reserve sufficient samples in advance.
- Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their particular experiments. Sample should be diluted by 0.01Mol/L PBS ( pH =7.0-7.2).
- If the samples are not indicated in the manual, a preliminary experiment to determine the validity of the kit is necessary.
- Tissue or cell extraction samples prepared by chemical lysis buffer may cause unexpected ELISA results due to the impacts from certain chemicals.
- Due to the possibility of mismatching between antigen from other origin and antibody used in our kits (e.g., antibody targets conformational epitope rather than linear epitope), some native or recombinant proteins from other manufacturers may not be recognized by our products.
- Influenced by the factors including cell viability, cell number or sampling time, samples from cell culture supernatant may not be detected by the kit.
- Fresh samples without long time storage is recommended for the test. Otherwise, protein degradation and denaturalization may occur in those samples and finally lead to wrong results.
- Assay Procedure
- Determine wells for diluted standard, blank and sample. Prepare wells for standard points and blank. Add 100μL each of dilutions of standard (read Reagent Preparation), blank and samples into the appropriate wells, respectively. Cover with the Plate sealer. Incubate for 1 hour at 37 °C.
- Remove the liquid of each well, don't wash.
- Add 100μL of Detection Reagent A working solution to each well, cover the wells with the plate sealer and incubate for 1 hour at 37 °C.
- Aspirate the solution and wash with 350μL of 1x Wash Solution to each well using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher, and let it sit for 1-2 minutes. Remove the remaining liquid from all wells completely by snapping the plate onto absorbent paper. Totally wash 3 times. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against absorbent paper.
- Add 100μL of Detection Reagent B working solution to each well, cover the wells with the plate sealer and incubate for 30 minutes at 37 °C.
- Repeat the aspiration/wash process for total 5 times as conducted in step 4.
- Add 90μL of Substrate Solution to each well. Cover with a new Plate sealer. Incubate for 10 - 20 minutes at 37 °C (Don't exceed 30 minutes). Protect from light. The liquid will turn blue by the addition of Substrate Solution.
- Add 50μL of Stop Solution to each well. The liquid will turn yellow by the addition of Stop solution. Mix the liquid by tapping the side of the plate. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
- Remove any drop of water and fingerprint on the bottom of the plate and confirm there is no bubble on the surface of the liquid. Then, run the microplate reader and conduct measurement at 450nm immediately.
- Assay preparation: Keep appropriate numbers of wells for each experiment and remove extra wells from microplate. Rest wells should be resealed and stored at -20 °C.
- Samples or reagents addition:Please use the freshly prepared Standard. Please carefully add samples to wells and mix gently to avoid foaming. Do not touch the well wall. For each step in the procedure, total dispensing time for addition of reagents or samples to the assay plate should not exceed 10 minutes. This will ensure equal elapsed time for each pipetting step, without interruption. Duplication of all standards and specimens, although not required, is recommended. To avoid cross-contamination, change pipette tips between additions of standards, samples, and reagents. Also, use separated reservoirs for each reagent.
- Incubation: To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary. Do not allow wells to sit uncovered for extended periods between incubation steps. Once reagents are added to the well strips, DO NOT let the strips DRY at any time during the assay. Incubation time and temperature must be controlled.
- Washing: The wash procedure is critical. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining Wash Solution by aspirating or decanting and remove any drop of water and fingerprint on the bottom of the plate. Insufficient washing will result in poor precision and false elevated absorbance reading.
- Controlling of reaction time: Observe the change of color after adding TMB Substrate (e.g. observation once every 10 minutes), if the color is too deep, add Stop Solution in advance to avoid excessively strong reaction which will result in inaccurate absorbance reading.
- TMB Substrate is easily contaminated. Please protect it from light.
- The environment humidity which is less than 60 % might have some effects on the final performance, therefore, a humidifier is recommended to be used at that condition.
- Calculation of Results
Average the duplicate readings for each standard, control, and samples and subtract the average zero standard optical density. Construct a standard curve by plotting the mean O.D. and concentration for each standard and draw a best fit curve through the points on the graph or create a standard curve on log-log graph paper with target concentration on the y-axis and absorbance on the x-axis. Using some plot software, for instance, curve expert 1.30, is also recommended. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.
In order to make the calculation easier, we plot the O.D. value of the standard (X-axis) against the known concentration of the standard (Y-axis), although concentration is the independent variable and O.D. value is the dependent variable. However, the O.D. values of the standard curve may vary according to the conditions of assay performance (e.g. operator, pipetting technique, washing technique or temperature effects), plotting log of the data to establish standard curve for each test is recommended. Typical standard curve below is provided for reference only.
- Assay Precision
- Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level of target were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level of target were tested on 3 different plates, 8 replicates in each plate.
CV(%) = SD/meanX100
Intra-Assay: CV < 10%
Inter-Assay: CV < 12%
- For Research Use only
- 4 °C,-20 °C
- Storage Comment
- For unused kit: All the reagents should be kept according to the labels on vials. The Standard, Detection Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20 °C upon receipt while the others should be at 4 °C.
- For used kit: When the kit is used, the remaining reagents need to be stored according to the above storage condition. Besides, please return the unused wells to the foil pouch containing the desiccant pack, and zip-seal the foil pouch.
It is highly recommended to use the remaining reagents within 1 month provided this is prior to the expiration date of the kit. For the expiration date of the kit, please refer to the label on the kit box. All components are stable up to the expiration date.
- Expiry Date
- 6 months
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- Alternative Name
- Retinol Binding Protein 4 (RBP4) (RBP4 ELISA Kit Abstract)
- RDCCAS, rbp4, MGC54038, srbp, RBP, PRBP, RBPA, Rbp-4, fb23c12, rbp, wu:fb23c12, wu:fb58d04, wu:fb72b04, zgc:86911, retinol binding protein 4, retinol binding protein 4 S homeolog, retinol binding protein 4 A, plasma, retinol binding protein 4, plasma, retinol binding protein 4 L homeolog, RBP4, rbp4.S, rbp4, Rbp4, RBP4A, rbp4.L
- Regulatory RNA Pathways, Positive Regulation of Peptide Hormone Secretion, Carbohydrate Homeostasis, Production of Molecular Mediator of Immune Response