Bovine Aprotinin (AP) ELISA Kit
For reference only. Please follow the manual included in your kit for instructions.
Catalog Number
RDR-AP-b
The Bovine Aprotinin (AP) ELISA Kit (RDR-AP-b) is an enzyme-linked immunosorbent assay for quantitative detection of AP in Bovine/Cow/Cattle samples. This kit uses the Ready to Use ELISA format for colorimetric detection of natural AP in samples including serum, plasma, tissue homogenates, cell lysates, cell culture supernatants and other biological fluids. The kit is easy to use and comes with a pre-coated microplate and all necessary reagents and buffers. The standard curve generated in the experiment is used to calculate the concentration of AP in samples. For research use only.
Product Specifications
Product Name
Bovine Aprotinin (AP) ELISA Kit
Catalog Number
RDR-AP-b
Detection Range
1.56-100ng/mL
Sensitivity
0.43ng/mL
Species Reactivity
Bovine/Cow/Cattle
Species
Bos taurus; Bovine Cattle
Assay Length
3 hours
Experimental Method
Competitive Inhibition
Detection Method
Colorimetric
Sample Type
serum, plasma, tissue homogenates, cell lysates, cell culture supernatants and other biological fluids
Gene ID
404172
UniProtKB
P00974
Shelf Life
16 months
Synonyms
BPTITrasylolPancreatic Trypsin InhibitorBasic protease inhibitor
Product Details
Intended Use
The kit is a competitive inhibition enzyme immunoassay technique for the in vitro quantitative measurement of AP in bovine serum, plasma, tissue homogenates, cell lysates, cell culture supernatants and other biological fluids.
Test Principle
This assay employs the competitive inhibition enzyme immunoassay technique. A monoclonal antibody specific to AP has been pre-coated onto a microplate. A competitive inhibition reaction is launched between biotin labeled AP and unlabeled AP (Standards or samples) with the pre-coated antibody specific to AP. After incubation, the unbound conjugate is washed off. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. The amount of bound HRP conjugate is reverse proportional to the concentration of AP in the sample. After addition of the substrate solution, the intensity of color developed is reverse proportional to the concentration of AP in the sample.
Application Data
Reagents and Materials Provided
| Reagents | Quantity | Reagents | Quantity |
|---|---|---|---|
| Pre-coated, ready to use 96-well strip plate | 1 | Plate sealer for 96 wells | 2 |
| Standard | 2 | Standard Diluent | 1 × 20 mL |
| Detection Solution A | 1 × 6 mL | Detection Solution B | 1 × 12 mL |
| TMB Substrate | 1 × 9 mL | Stop Solution | 1 × 6 mL |
| Wash Buffer (30× concentrate) | 1 × 20 mL | Instruction manual | 1 |
Materials Required but not Supplied
- Microplate reader with 450 ± 10 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.
Storage of the kits
- For unopened kits: All the reagents should be stored at -20°C upon receipt.
- For opened kits: Once the kit is opened, the remaining reagents still need to be stored according to the above storage conditions. In addition, return the unused wells to the foil pouch containing the desiccant pack, and reseal along entire edge of zip-seal.
- For the expiration date of the kit, please refer to the label on the kit box. All components are stable until this date.
- It is highly recommended to use the remaining reagents within 1 month of opening.
Sample Collection and Storage
- Serum - Use a serum separator tube and allow samples to clot for 2 hours at room temperature or overnight at 4°C before centrifugation for 20 minutes at approximately 1000 × g. Assay freshly prepared serum immediately or store samples in aliquots 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 1000 × g at 2-8°C within 30 minutes of collection. Remove plasma and assay immediately or store samples in aliquots at -20°C or -80°C for later use. Avoid repeated freeze/thaw cycles.
- Tissue homogenates - The preparation of tissue homogenates will vary depending upon tissue type. For this assay, tissues should be rinsed in ice-cold PBS (0.01 mol/L, pH 7.0-7.2) to remove excess blood thoroughly and weighed 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 also work). The resulting suspension should be sonicated with an ultrasonic cell disrupter or subjected to 2 freeze/thaw cycles to further break the cell membranes. Then, centrifuge the homogenates for 5 minutes at 5000 × g. Remove the supernatant and assay immediately or aliquot and store at ≤-20°C.
- Cell Lysates - 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 3 times in cold PBS.
- Resuspend cells in PBS (1×) and subject the cells to ultrasonication 4 times (or Freeze cells at ≤-20°C. Thaw cells with gentle mixing. Repeat the freeze/thaw cycle 3 times.).
- Centrifuge at 1500 × g for 10 minutes at 2-8°C to remove cellular debris.
- Cell culture supernatants and other biological fluids - Centrifuge samples for 20 minutes at 1000 × g. Remove particulates and assay immediately or store samples in aliquots at -20°C or -80°C. Avoid repeated freeze/thaw cycles.
- Samples to be used within 5 days may be stored at 4°C, otherwise samples must be stored at -20°C (≤1 month) or -80°C (≤2 months) to avoid loss of bioactivity and contamination.
- Noticeable hemolysis will affect antibody-antigen reactions. Samples with any sign of hemolysis are not acceptable for this assay.
- When performing the assay, bring samples to room temperature.
Reagent Preparation
- 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, keep for 10 minutes at room temperature, shake gently (not to foam). The concentration of the standard in the stock solution is 100 ng/mL. Prepare 7 tubes containing 0.2 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. Prepare a dilution series with 7 points; for example: 100 ng/mL, 50 ng/mL, 25 ng/mL, 12.5 ng/mL, 6.25 ng/mL, 3.125 ng/mL, 1.562 ng/mL, and the last EP tube with Standard Diluent is the blank at 0 ng/mL.
- Detection Solution A and Detection Solution B - Detection Solutions A and B are already at the correct concentrations and do not need to be diluted further.
- Wash Solution - Dilute 20 mL of Wash Solution concentrate (30×) with 580 mL 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. Do not dump the residual solution back into the vial.
- Do not perform a serial dilution directly in the wells.
- Prepare standard within 15 minutes before assay.
- Carefully reconstitute Standards according to the instruction, avoid foaming and mix gently until the crystals are completely dissolved.
- The reconstituted Standards 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.
- Any contaminated water or container used during reagent preparation will influence the detection result.
Assay Procedure Summary
- Prepare all reagents, samples and standards.
- Add 50 µL standard or sample to each well. And then add 50 µL Detection Solution A immediately. Shake and mix. Incubate 1 hour at 37°C.
- Aspirate and wash 3 times.
- Add 100 μL Detection Solution B. Incubate 1 hour at 37°C.
- Aspirate and wash 5 times.
- Add 90 μL Substrate Solution. Incubate 15-25 minutes at 37°C.
- Add 50 μL Stop Solution. Read at 450 nm immediately.
Calculation of Results
This assay employs the competitive inhibition enzyme immunoassay technique, so there is an inverse correlation between AP concentration in the sample and the assay signal intensity. Average the duplicate readings for each standard, control, and samples. Create a standard curve on log-log or semi-log graph paper, with the log of AP concentration on the y-axis and absorbance on the x-axis. Draw the best fit straight line through the standard points, or it can be determined by regression analysis. Using plotting 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.
Product Data
Sensitivity
- The minimum detectable dose of AP is typically less than 0.43 ng/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 by adding two standard deviations to the mean optical density value of twenty zero standard replicates and calculating the corresponding concentration.
Specificity
- This assay has high sensitivity and excellent specificity for detection of AP.
- No significant cross-reactivity or interference between AP and analogues was observed.
- Limited by current skills and knowledge, it is impossible to perform all possible cross-reactivity detection tests between AP and all analogues, therefore, cross reactivity may still exist.
Precision
- Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level AP were tested 20 times on one plate, respectively.
- Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level AP were tested on 3 different plates, 8 replicates in each plate.
- CV (%) = SD/mean × 100
- Intra-Assay: CV<10%
- Inter-Assay: CV<12%
Stability
- The stability of ELISA 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 unnecessary influences on the performance, operation procedures and lab conditions, especially room temperature, air humidity, and incubator temperatures should be strictly regulated. It is also strongly suggested that the whole assay is performed by the same experimenter from the beginning to the end.
Research Areas