AGE ELISA Kit

Catalog No. ABIN454153

Product Details AGE ELISA Kit

Target: AGE (Advanced Glycation End Products (AGE))

Reactivity: Mouse

Detection Method: Colorimetric

Method Type: Competition ELISA

Detection Range: 0.78-50 ng/mL

Minimum Detection Limit: 0.78 ng/mL

Application: ELISA

Purpose: This immunoassay kit allows for the in vitro quantitative determination of Mouse AGEs concentrations in serum, plasma and other biological fluids.

Sample Type: Plasma, Serum

Analytical Method: Quantitative

Specificity: This assay recognizes recombinant and natural Mouse AGEs.

Cross-Reactivity (Details): No significant cross-reactivity or interference was observed.

Sensitivity: < 1.17 ng/mL
The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest detectable concentration that could be differentiated from zero.

Characteristics: Advanced glycation end-product,AGE,

Components: Reagent (Quantity): Assay plate (1), Standard (2), Sample Diluent (1×20ml), Assay Diluent A (1×10ml), Assay Diluent B (1×10ml), Detection Reagent A 1 × 60 μ l Detection Reagent B (1×120 μl), Wash Buffer(25 x concentrate) (1×30ml), Substrate (1×10ml), Stop Solution (1×10ml), Plate sealer for 96 wells (5), Instruction (1)

Material not included: 2 Microplate reader. Pipettes and pipette tips. EP tube Deionized or distilled water.

Application Details

Sample Volume: 100 μL

Plate: Pre-coated

Protocol: The ELISA is based on the competitive binding enzyme immunoassay technique. The microtiter plate provided in this kit has been pre-coated with an antibody specific to AGEs, During the reaction, AGEs in the patient sample or standard competes with a fixed amount of bio-labeled AGEs for sites on a pre-coated rabbit polyclonal antibody specific to AGEs. Excess conjugate and unbound sample or standard are washed from the plate. Next , Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB substrate solution is added to each well. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of AGEs in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Reagent Preparation:

Bring all reagents to room temperature before use. Wash Buffer - If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have completely dissolved. Dilute 30 mL of Wash Buffer Concentrate into deionized or distilled water to prepare 750 mL of Wash Buffer. Standard - Reconstitute the Standard with 1.0 ml of Sample Diluent. This reconstitution produces a stock solution of 300 ng/mL. Allow the standard to sit for about 10 minutes with 3 gentle agitation prior to making serial dilutions (Making serial dilution in the wells directly is not permitted). The undiluted standard serves as the highest standard (300 ng/mL). The Sample Diluent serves as the zero standard (0 ng/mL). ng/mL 300 150 75 37.5 18.75 9.38 4.69 0 Detection Reagent A and B - Dilute to the working concentration using Assay Diluent A or B (1:100), respectively.

Sample Collection: Serum - Use a serum separator tube and allow samples to clot for 30 minutes before centrifugation for 20 minutes at approximately 1000 g. Remove serum and assay immediately or aliquot and store samples at -20 or -80 . Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 g at 2 - 8 within 30 minutes of collection. Store samples at -20 or -80 . Avoid repeated freeze-thaw cycles. Other biological fluids - Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20 or -80 . Avoid repeated freeze-thaw cycles. Sample preparation - Serum/plasma samples require a 100 fold dilution. A suggested 100-fold dilution is 10uL Sample + 990uL Sample Diluent. Sample should be diluted by 0.02 M PBS(PH=7.0-7.2). Note: Serum and plasma to be used within 7 days may be stored at 2-8 , otherwise samples must stored at -20 ( 1 month) or -80 ( 2 months) to avoid loss of bioactivity and contamination. Avoid freeze-thaw cycles. When performing the assay slowly bring samples to room temperature.

Assay Procedure:

Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37 directly.). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at 4 until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. 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.
1. Add 50 of Standard, Blank, or Sample per well.
2. Immediately add 50 μ l of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hours at 37C. Detection Reagent A working solution may appear cloudy. Warm to room temperature and mix gently until solution appears uniform.
3. Aspirate each well and wash, repeating the process three times for a total of three washes. Wash by filling each well with Wash Buffer (approximately 400 μ l) using a squirt bottle, multi-channel pipette, 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 μ l of Detection Reagent B working solution to each well. Cover with a new Plate sealer. Incubate for45minutes at 37 .
6. Repeat the aspiration/wash process for five times as conducted in step
4. 7. Add 90 μ l of Substrate Solution to each well. Cover with a new Plate sealer. Incubate for 15 - 30 minutes at 37 . Protect from light.
8. Add 50 μ l of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
9. Determine the optical density of each well at once, using a microplate reader set to 450 nm. 4
Important Note:
1. Absorbance is a function of the incubation time. Therefore, prior to starting the assay it is recommended that all reagents should be freshly prepared prior to use and all required strip-wells are secured in the microtiter frame. This will ensure equal elapsed time for each pipetting step, without interruption.
2. Please carefully reconstitute Standards or working Detection Reagent A and B according to the instruction, and avoid foaming and mix gently until the crystals have completely dissolved. The reconstituted Standards can be used only once. This assay requires pipetting of small volumes. To minimize imprecision caused by pipetting, ensure that pipettors are calibrated. It is recommended to suck more than 10 μ l for once pipetting.
3. 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 have been added to the well strips, DO NOT let the strips DRY at any time during the assay.
4. For each step in the procedure, total dispensing time for addition of reagents to the assay plate should not exceed 10 minutes.
5. To avoid cross-contamination, change pipette tips between additions of each standard level, between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent.
6. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings.
7. Duplication of all standards and specimens, although not required, is recommended.
8. Substrate Solution is easily contaminated. Please protect it from light.

Calculation of Results:

Average the duplicate readings for each standard, control, and samples and subtract the average zero standard optical density. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard 5 on the x-axis against the concentration on the y-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the AGEs concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. It is recommended to use some related software to do this calculation, such as curve expert 1.3 . This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.

Restrictions: For Research Use only

Handling

Handling Advice: 1. The kit should not be used beyond the expiration date on the kit label.
2. Do not mix or substitute reagents with those from other lots or sources.
3. If samples generate values lower than the highest standard, further dilute the samples and repeat the assay. Any variation in standard diluent, operator, pipetting technique, washing technique,incubation time or temperature, and kit age can cause variation in binding.
4. This assay is designed to eliminate interference by soluble receptors, ligands, binding proteins, and other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded.
5. Limited by the current condition and scientific technology, we can't completely conduct the comprehensive identification and analysis on the raw material provided by suppliers. So there might be some qualitative and technical risks to use the kit.

Storage: 4 °C/-20 °C

Storage Comment: The Standard, Detection Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20 °C upon being received. The other reagents can be stored at 4 °C.

Target Details for AGE

Target: AGE (Advanced Glycation End Products (AGE))

Alternative Name: AGE (AGE Products)

Synonyms: RBP ELISA Kit, RNBP ELISA Kit, renin binding protein ELISA Kit, RENBP ELISA Kit

Target Type: Chemical