Procollagen, Type III (0) ELISA Kit

Antigen: Procollagen, Type III (0)

Epitope: N-Term

Reactivity: Rat (Rattus)

Application: ELISA

Certificates: ISO 9001:2008, ISO 13485:2003

Catalog no.: ABIN416400

Additional Information

Characteristics: The kit is a sandwich enzyme immunoassay for the in vitro quantitative measurement of PIIINP in rat serum, plasma and other biological fluids.

Alternative name: Procollagen III N-Terminal Propeptide (PIIINP)

Sample Type: Serum, Plasma, Biological Fluids

Description: Important Note: 1. 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. 2. The final experimental results will be closely related to validity of the products, operation skills of the end users and the experimental environments. Please make sure that sufficient samples are available. 3. Kits from different batches may be a little different in detection range, sensitivity and color developing time. 4. Do not mix or substitute reagents from one kit lot to another. Use only the reagents supplied by manufacturer. 5. Protect all reagents from strong light during storage and incubation. All the bottle caps of reagents should be covered tightly to prevent the evaporation and contamination of microorganism. 6. There may be some foggy substance in the wells when the plate is opened at the first time. It will not have any effect on the final assay results. Do not remove microtiter plate from the storage bag until needed. 7. Wrong operations during the reagents preparation and loading, as well as incorrect parameter setting for the plate reader may lead to incorrect results. A microplate plate reader with a bandwidth of 10nm or less and an optical density range of 0-3 O.D. or greater at 450 ± 10nm wavelength is acceptable for use in absorbance measurement. Please read the instruction carefully and adjust the instrument prior to the experiment. 8. Even the same operator might get different results in two separate experiments. In order to get better reproducible results, the operation of every step in the assay should be controlled. Furthermore, a preliminary experiment before assay for each batch is recommended. 9. Each kit has been strictly passed Q.C test. However, results from end users might be inconsistent with our in-house data due to some unexpected transportation conditions or different lab equipments. Intra-assay variance among kits from different batches might arise from above factors, too. 10. Kits from different manufacturers for the same item might produce different results, since we haven't compared our products with other manufacturers. 11. Valid period: six months. 12. The instruction manual also suit for the kit of 48T, but all reagents of 48T kit is reduced by half. Precaution: The Stop Solution suggested for use with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material.

Specificity: This assay has high sensitivity and excellent specificity for detection of rat PIIINP. No significant cross-reactivity or interference between rat PIIINP and analogues was observed. Note: Limited by current skills and knowledge, it is impossible for us to complete the cross- reactivity detection between rat PIIINP and all the analogues, therefore, cross reaction may still exist.

Sensitivity: 0.47-30ng/mL. The standard curve concentrations used for the ELISA's were 30ng/mL, 15ng/mL, 7.5ng/mL, 3.75ng/mL, 1.88ng/mL, 0.94ng/mL, 0.47ng/mL. The minimum detectable dose of rat PIIINP is typically less than 0.18ng/mL. The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero. It was determined the mean O.D. Value of 20 replicates of the zero standard added by their three standard deviations.

Application Details

Principle: The microtiter plate provided in this kit has been pre-coated with a monoclonal antibody specific to PIIINP. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for PIIINP. 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. Only those wells that contain PIIINP, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. 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 450nm ± 10nm. The concentration of PIIINP in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Protocol: 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 indeed the independent variable while O.D. value is the dependent variable. Further, in this part, in order to help the customer perform the assay more visual, we provide the customer with the raw data (not the log of data). However, plotting log of the data to construct the curve will be recommended. 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). This curve is provided for demonstration only. The customers should establish their own standard curve for each test conducted. Typical Standard Curve for Rat PIIINP ELISA.

Reagent Preparation: 1. Bring all kit components and samples to room temperature (18-25°C) before use. 1. 2. Standard - Reconstitute the Standard with 1.0mL 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 30ng/mL. Please prepare 7 tubes containing 0.5mL Standard Diluent and 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 30ng/mL, 15ng/mL, 7.5ng/mL, 3.75ng/mL, 1.88ng/mL, 0.94ng/mL, 0.47ng/mL, and the last EP tubes with Standard Diluent is the blank as 0ng/mL. 3. Assay Diluent A and Assay Diluent B - Dilute 6mL of Assay Diluent A or B Concentrate(2x) with 6mL of deionized or distilled water to prepare 12 mL of Assay Diluent A or B. The prepared working dilution can't be frozen. Detection Reagent A and Detection Reagent B - Briefly spin or centrifuge the stock Detection A and 4. Detection B before use. Dilute to the working concentration with working Assay Diluent A or B, respectively (1:100). 5. Wash Solution - Dilute 20mL of Wash Solution concentrate (30x) with 580mL of deionized or distilled water to prepare 600 mL of Wash Solution (1x). 6. TMB substrate - Aspirate the needed dosage of the solution with sterilized tips and do not dump the residual solution into the vial again. Note: 1. Making serial dilution in the wells directly is not permitted. Prepare standard within 15 minutes before assay. Please do not dissolve the reagents at 37 °C directly. 2. 3. 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. To minimize imprecision caused by pipetting, use small volumes and ensure that pipettors are calibrated. It is recommended to suck more than 10L for once pipetting. 4. The reconstituted Standards, Detection Reagent A and Detection Reagent B can be used only once once. 5. If crystals have formed in the Wash Solution concentrate (30x), warm to room temperature and mix gently until the crystals have completely dissolved. 6. Distilled water is recommended to be used to make the dilution for reagents or samples. Contaminated water or container for reagent preparation will influence the detection result.

Sample Collection: Serum - Use a serum separator tube and allow Samples to clot for two hours at room temperature or overnight at 4 C before centrifugation for 20 minutes at approximately 1000xg. Assay freshly prepared serum immediately or store Samples in aliquot at -20°C or -80 C for later use. Avoid repeated freeze/thaw cycles. Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge Samples for 15 minutes at 1000xg at 2 - 8 C within 30 minutes of collection. Remove plasma and assay immediately or store Samples in aliquot at -20°C or -80 C for later use. Avoid repeated freeze/thaw cycles. Other biological fluids: Centrifuge samples for 20 minutes at 1000 g. Remove particulates and assay immediately or store samples in aliquot at -20 or -80°C for later use. Avoid repeated freeze/thaw cycles.

Sample Preparation: The user should calculate the possible amount of the samples used in the whole test. Please reserve sufficient samples in advance. 2. 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. 3. Serum/plasma samples require about a 2-10 fold dilution. A suggested 10-fold dilution is 20L Sample + 180L PBS. Sample should be diluted by 0.02mol/L PBS(PH=7.0-7.2). 4. If the samples are not indicated in the manual, a preliminary experiment to determine the validity of the kit is necessary. 5. Tissue or cell extraction samples prepared by chemical lysis buffer may cause unexpected ELISA results due to the impacts of certain chemicals. 6. Owing to the possibility of mismatching between antigen from other resource 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. 7. Influenced by the factors including cell viability, cell number and also sampling time, samples from cell culture supernatant may not be detected by the kit. 8. 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: 1. Determine wells for diluted standard, blank and sample. Prepare 7 wells for standard, 1 well for blank. Add 100L each of dilutions of standard (read Reagent Preparation), blank and samples into the appropriate wells. Cover with the Plate sealer. Incubate for 2 hours at 37 °C. 2. Remove the liquid of each well, don't wash. Add 100L of Detection Reagent A working solution to each well. Incubate for 1 hour at 37 °C after covering 3. it with the Plate sealer. 4. Aspirate the solution and wash with 350L 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. Repeat 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 100L of Detection Reagent B working solution to each well. Incubate for 30 minutes at 37 °C after 5. covering it with the Plate sealer. 6. Repeat the aspiration/wash process for five times as conducted in step 4. 7. Add 90L of Substrate Solution to each well. Cover with a new Plate sealer. Incubate for 15 - 25 minutes at 37 °C (Don't exceed 30 minutes). Protect from light. The liquid will turn blue by the addition of Substrate Solution. 8. Add 50L 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. 9. 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. Note: 1. Assay preparation: Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20 °C until the kits expiry date. Samples or reagents addition Please use the freshly prepared Standard. Please carefully add samples 2. additionPlease to wells and mix gently to avoid foaming. Do not touch the well wall as possible. 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 each standard level, between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent. 3. 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 have been added to the well strips, DO NOT let the strips DRY at any time during the assay. Incubation time and temperature must be observed. 4. Washing: The wash procedure is critical. Complete removal of liquid at each step is essential to 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 falsely elevated absorbance reading. 5. 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. 6. TMB Substrate 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 on log-log graph paper, with PIIINP concentration on the y-axis and absorbance on the x-axis. Draw the best fit straight line through the standard points and it can be determined by regression analysis. Using some plot software, such as 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.

Application Notes: 1. Prepare all reagents, samples and standards, 2. Add 100L standard or sample to each well. Incubate 2 hours at 37 C, 3. Add 100L prepared Detection Reagent A. Incubate 1 hour at 37 C, 4. Aspirate and wash 3 times, 5. Add 100L prepared Detection Reagent B. Incubate 30 minutes at 37 C, 6. Aspirate and wash 5 times, 7. Add 90L Substrate Solution. Incubate 15-25 minutes at 37 C, 8. Add 50L Stop Solution. Read at 450nm immediately.

Components: Reagents (Quantity): Pre-coated, ready to use 96-well strip plate (1), Plate sealer for 96 wells (4), Standard (freeze dried) (2), Standard Diluent (1x20ml), Detection Reagent A (green) (1x120µl), Assay Diluent A (2 x concentrate) (1x6ml), Detection Reagent B (red) (1x120µl), Assay Diluent B (2 x concentrate) (1x6ml), TMB Substrate (1x9ml), Stop Solution (1x6ml), Wash Buffer (30 x concentrate) (1x20ml)

Material not included: 1. Microplate reader with 450 ± 10 nm filter. 2. Precision single and multi-channel pipettes and pipette tips with disposable tips. 3. Eppendorf Tubes for diluting samples. 4. Deionized or distilled water. 5. Absorbent paper for blotting the microtiter plate. 6. Container for Wash Solution

Storage: All the reagents should be kept according to the labels on vials. The Standard Detection Reagent A, Detection Standard, Reagent B and the 96-well strip plate should be stored at -20°C upon being received. The unused strips should be kept in a sealed bag with the desiccant provided to minimize exposure to damp air. The test kit may be used throughout the expiration date of the kit (six months from the date of manufacture). Opened test kits will remain stable until the expiring date shown, provided it is stored as prescribed above.

Restrictions: For Research Use only