Trypsin ELISA 试剂盒

Details for Product Trypsin ELISA Kit No. ABIN415936, 供应商: Log in to see
抗原
  • wu:fb57g08
  • zgc:109701
  • LOC397853
  • Trypsin
  • trp1
  • try1
  • trypsin
  • trypsin
  • trypsinogen
  • protease, serine, 1
  • try
  • LOC397853
  • prss1
  • Deipr_2253
适用
猪, 野猪
其他选择
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7
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2
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2
2
1
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1
实验类型
Sandwich ELISA
检测范围
1.56-100 ng/mL
最低检测浓度
1.56 ng/mL
应用范围
ELISA
选项
Supplier
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Supplier Product No.
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'独立验证'标志
Lot Number L141028604
Method validated ELISA
Positive Control Trypsin from porcine pancreas
Negative Control Chicken serum (non-reactive species)
Notes Target protein was detected in the positive control sample and not in the negative control sample as expected.
Controls
  • Positive control: Trypsin from porcine pancreas (Sigma-Aldrich. Catalog # T6567) dissolved in STANDARD diluent (provided in the kit) by rocking the mixture for 30 minutes at room temperature.
  • Negative control: Chicken serum (Jackson ImmunoResearch Laboratories Inc. Catalog #: 003-000-120, Lot #: 112503)
实验流程
  • 1. All reagents in the ELISA kit were brought up to room temperature (RT) before use.
  • 2. 100 μL of standard or sample were added to wells in ELISA plate pre-coated with capture antibody.
  • 3. All samples and standards were assayed in triplicate.
  • 4. The plate was covered with sealer (provided in kit) and incubated for 2 hours at 37°C. Unbound material was aspirated but the wells were NOT washed.
  • 5. 100 μL of Detection Reagent-A Working Solution was added to each well. Plate was covered with sealer (provided in kit) and incubated for 1 hour at 37°C. Unbound material was removed from each well and plate was washed three times with 350 μL of 1x Wash Solution (provided in the kit). After the last wash the plate was inverted and blotted against clean absorbent paper to remove any remaining liquid.
  • 6. 100 μL of Detection Reagent-B Working Solution was added to each well. Plate was covered with sealer (provided in kit) and incubated for 30 minutes at 37°C.
  • 7. Unbound material was removed by washing five times with 350 μL of 1x Wash Solution (provided in the kit). After the last wash the plate was inverted and blotted against clean absorbent paper to remove any remaining liquid.
  • 8. 90 μL of Substrate Solution was added to wells and the plate was covered with a new plate sealer. The plate was gently tapped to ensure mixing and incubated for 25 minutes at 37°C in the dark.
  • 9. After 25 minutes, when an apparent gradient appeared in the standard wells, the reaction was terminated by adding 50 μL of Stop Solution to each well.
  • 10. The optical density (OD value) of each well was read using a microplate reader set to 450 nm.
  • 11. The triplicate readings for each sample were averaged and the average zero standard optical density subtracted to yield ‘corrected absorbance at 450 nm’. A standard curve was generated by plotting the mean OD value for each standard on the X-axis against the concentration on the Y-axis using Excel. Standard curve was generated by regression analysis.
  • 12. An equation (y = 67.557x - 1.4478) was derived from the standard curve and used to calculate trypsin concentrations in samples based on their Average Absorbance values.
Experimental Notes The positive control was trypsin from porcine pancreas (Sigma-Aldrich Catalog # T6567). As per instruction from the manufacturer, the trypsin powder should be dissolved in 1 mM HCl. However, the acid solublization process renders the sample almost undetectable by the kit. Therefore trypsin powder was dissolved in STANDARD diluent (provided in the kit) by rocking the mixture for 30 minutes at room temperature.
Validation Images
ELISA validation image for Trypsin ELISA Kit (ABIN415936) Figure 1: Graph of ‘Corrected’ OD450 nm plotted for standard curve samples. Standard ...
ELISA validation image for Trypsin ELISA Kit (ABIN415936) Figure 2: Table of absorbance values for standard curve, spike control and samples. ...
原理 The kit is a sandwich enzyme immunoassay for in vitro quantitative measurement of TRY in Serum,Plasma,Tissue Homogenate,Cell Lysate,Cell Culture Supernatant,Biological Fluids
样品类型 Serum, Plasma, Tissue Homogenate, Cell Lysate, Cell Culture Supernatant, Biological Fluids
Analytical Method Quantitative
检测方法 Colorimetric
特异性

This assay has high sensitivity and excellent specificity for detection of Trypsin (TRY).
No significant cross-reactivity or interference between Trypsin (TRY) and analogues was observed.

交叉反应 (详细) No significant cross-reactivity or interference between Trypsin (TRY) and analogues was observed.
灵敏度 0.62 ng/mL
组件
  • Pre-coated, ready to use 96-well strip plate
  • Plate sealer for 96 wells
  • Standard Diluent
  • Assay Diluent A
  • Assay Diluent B
  • Stop Solution
  • Standard
  • Detection Reagent A
  • Detection Reagent B
  • TMB Substrate
  • Wash Buffer (30 × concentrate)
  • Instruction manual
试剂未包括
  • Microplate reader with 450 nm 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
应用备注
  • Limited by the current condition and scientific technology, we cannot 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.
  • 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.
  • Kits from different batches may be a little different in detection range, sensitivity and color developing time.
  • Do not mix or substitute reagents from one kit lot to another. Use only the reagents supplied by manufacturer.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Kits from different manufacturers for the same item might produce different results, since we have not compared our products with other manufacturers.
说明

Information on standard material:
The standard might be recombinant protein or natural protein, that will depend on the specific kit. Moreover, the expression system is E.coli or yeast or mammal cell. There is 0.05% proclin 300 in the standard as preservative.

Information on reagents:
The stop solution used in the kit is sulfuric acid with concentration of 1 mol/L. And the wash solution is TBS. The standard diluent contains 0.02 % sodium azide, assay diluent A and assay diluent B contain 0.01% sodium azide. Some kits can contain is BSA in them.

Information on antibodies:
The provided antibodies and their host vary in different kits.

样本量 100 μL
实验时间 3 h
板类型 Pre-coated
实验流程 The test principle applied in this kit is Sandwich enzyme immunoassay. The microtiter plate provided in this kit has been pre-coated with an antibody specific to Trypsin (TRY). Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to Trypsin (TRY). Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added, only those wells that contain Trypsin (TRY), biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of Trypsin (TRY) in the samples is then determined by comparing the O.D. of the samples to the standard curve.
试剂准备
  • Bring all kit components and samples to room temperature (18-25°C) before use.
  • 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 200ng/mL. Please firstly dilute the stock solution to 100ng/mL and the diluted standard serves as the highest standard (100ng/mL). Then prepare 7 tubes containing 0.5mL Standard Diluent and use the diluted standard to produce a double dilution series. Mix each tube thoroughly before the next transfer. Set up 7 points of diluted standard such as 100ng/mL, 50ng/mL, 25ng/mL, 12.5ng/mL, 6.25ng/mL, 3.12ng/mL, 1.56ng/mL, and the last EP tubes with Standard Diluent is the blank as 0ng/mL.
  • Assay Diluent A and Assay Diluent B - Dilute 6mL of Assay Diluent A or B Concentrate(2×) with 6mL of deionized or distilled water to prepare 12 mL of Assay Diluent A or B. (In fact, more than 6mL Assay Diluent A and Assay Diluent B are contained in the bottles. Therefore, in every test, please precisely pipette required amount of Diluent and make double dilution in a new container. The prepared working dilution cannot be frozen.)
  • Detection Reagent A and Detection Reagent B - Briefly spin or centrifuge the stock Detection A and Detection B before use. Dilute to the working concentration with working Assay Diluent A or B, respectively (1:100).
  • Wash Solution - Dilute 20mL of Wash Solution concentrate (30×) with 580mL of deionized or distilled water to prepare 600 mL of Wash Solution (1×).
  • TMB substrate - Aspirate the needed dosage of the solution with sterilized tips and do not dump the residual solution into the vial again.
Note:
  • 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.
  • 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 once 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 (30×), 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.
样品收集 Serum: Allow samples to clot for two hours at room temperature or overnight at 4°C before centrifugation for 20 minutes at approximately 1000 × g. Assay immediately or store samples in aliquot at -20°C or -80°C. Avoid repeated freeze/thaw cycles.

Plasma: Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 × g within 30 minutes of collection. Remove plasma and assay immediately or store samples in aliquot at -20°C or -80°C. Avoid repeated freeze/thaw cycles.

Tissue Homogenates: The preparation of tissue homogenates will vary depending upon tissue type. For this assay, rinse tissues in ice-cold PBS (0.02mol/L,pH 7.0-7.2) to remove excess blood thoroughly and weigh before homogenization. Mince the tissues to small pieces and homogenize them in 5-10 mL of PBS with a glass homogenizer on ice (Micro Tissue Grinders work, too). Sonicate the resulting suspension with an ultrasonic cell disrupter or subject it to two freeze-thaw cycles to further break the cell membranes. Centrifugate the homogenates for 5 minutes at 5000 × g. Remove the supernate and assay immediately or aliquot and store at -20°C

Cell Lysate: Cells must be lysed before assaying according to the following directions. Adherent cells should be detached with trypsin and then collected by centrifugation (suspension cells can be collected by centrifugation directly). Wash cells three times in cold PBS. Resuspend cells in PBS (1×) and subject them to ultrasonication for 4 times (or Freeze cells at -20 °C. Thaw cells with gentle mixing. Repeat the freeze/thaw cycle for 3 times.) Centrifuge at 1500 × g for 10 minutes at 2 - 8°C to remove cellular debris.

Cell Culture Supernatant: Centrifuge samples for 20 minutes at 1000 × g. Remove particulates and assay immediately or store samples in aliquot at -20 °C or -80 °C for later use. Avoid repeated freeze/thaw cycles.

Biological Fluids: Centrifuge samples for 20 minutes at 1000 × g. Remove particulates and assay immediately or store samples in aliquot at -20 °C or -80 °C for later use. Avoid repeated freeze/thaw cycles.
样品制备
  • 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.
实验流程
  1. Prepare all reagents, samples and standards,
  2. Add 100μL standard or sample to each well. Incubate 2 hours at 37 °C,
  3. Aspirate and add 100μL prepared Detection Reagent A. Incubate 1 hour at 37 °C,
  4. Aspirate and wash 3 times,
  5. Add 100μL prepared Detection Reagent B. Incubate 30 minutes at 37 °C,
  6. Aspirate and wash 5 times,
  7. Add 90μL Substrate Solution. Incubate 10-20 minutes at 37 °C,
  8. Add 50μL Stop Solution. Read at 450nm immediately.
结果分析

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 TRY 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.

实验精密度

Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level Trypsin (TRY) were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level Trypsin (TRY) were tested on 3 different plates, 8 replicates in each plate.
CV(%) = SD/meanX100
Intra-Assay: CV<10%
Inter-Assay: CV<12%

限制 仅限研究用
注意事项 The Stop Solution suggested for use with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material.
注意事项

The stability of kit is determined by the loss rate of activity. The loss rate of this kit is less than 5 % within the expiration date under appropriate storage condition.
To minimize extra influence on the performance, operation procedures and lab conditions, especially room temperature, air humidity, incubator temperature should be strictly controlled. It is also strongly suggested that the whole assay is performed by the same operator from the beginning to the end.

储存条件 4 °C
储存方法
  • For unopened 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 opened kit: When the kit is opened, the remaining reagents still 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 reseal along entire edge of zip-seal.
    Note: It is highly recommended to use the remaining reagents within 1 month provided this is within the expiration date of the kit.
  • For ELISA kit, 1 day storage at 37°C can be considered as 2 months at 4°C, which means 3 days at 37°C equaling 6 months at 4°C.
有效期 6 months
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