Lipocalin 2 ELISA Kit (LCN2) ELISA Kit
- Lipocalin 2 (LCN2)
- Detection Method
- Method Type
- Sandwich ELISA
- Detection Range
- 0.16 ng/mL - 10 ng/mL
- Minimum Detection Limit
- 0.16 ng/mL
- The kit is a sandwich enzyme immunoassay technique for the in vitro quantitative measurement in various sample types.
- Sample Type
- Plasma, Serum
- Analytical Method
- This kit recognizes natural and recombinantHumanNGAL. No significant cross-reactivity or interference between HumanNGAL and analogues was observed. Note: Limited by existing techniques, cross reaction may still exist, as it is impossible for us to complete the cross-reactivity detection between HumanNGAL and all the analogues.
- Cross-Reactivity (Details)
- No significant cross-reactivity or interference between human Neutrophil Gelatinase Associated Lipocalin and analogues was observed. Note: Limited by current skills and knowledge, it is impossible for us to complete the cross-reactivity detection between Human bFGF and all the analogues, therefore, cross reaction may still exist.
- 0.09 ng/mL
- Pre-coated, ready to use 96-well strip plate, flat buttom
- Plate sealer for 96 wells
- Reference Standard
- Reference Standard & Sample Diluent
- Biotinylated Detection Antibody (100 x concentrate)
- HRP Conjugate (100 x concentrate)
- Biotinylated Detection Antibody Diluent
- HRP Conjugate Diluent
- Substrate Reagent
- Stop Solution
- Wash Buffer (25 x concentrate)
- Instruction manual
- Material not included
Microplate reader with 450nm wavelength filter
High-precision transferpettor, EP tubes and disposable pipette tips
Deionized or distilled water
Loading slot for Wash Buffer
- Discover our best selling LCN2 ELISA Kit
- Application Notes
ELISA Plate: The just opened ELISA Plate may appear water-like substance, which is normal and will not have any impact on the experiment results.
Add Sample: The interval of sample adding between the first well and the last well should not be too long, otherwise will cause different pre-incubation time, which will significantly affect the experiment’s accuracy and repeatability. For each step in the procedure, total dispensing time for addition of reagents or samples to the assay plate should not exceed 10 minutes. Parallel measure ment is recommended.
Incubation: To prevent evaporation and 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. Do not let the strips dry at any time during the assay. Strict compliance with the given incubation time and temperature.
Washing: The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings. Residual liquid in the reaction wells should be pat dry against absorbent paper in the washing process. But don’t put absorbent paper into reaction wells directly. Note that clear the residual liquid and fingerprint in the bottom before measurement, so as not to affect the micro-titer plate reader.
Reagent Preparation: As the volume of Detection Ab and HRP Conjugate is very small, liquid may adhere to the tube wall or tube cap when being transported. You better hand-throw it or centrifugal it for 1 minute at 1000rpm. Please pipette the solution for 4-5 times before pippeting. Please carefully reconstitute Standards, working solutions of Detection Ab and HRP Conjugate according to the instructions. To minimize imprecision caused by pipetting, ensure that pipettors are calibrated. It is recommended to suck more than 10μL for once pipetting. Do not reuse standard solution, working solution of Detection Ab and HRP Conjugate, which have been diluted. If you need to use standard repeatedly, you can divide the standard into small pack according to the amount of each assay, keep them at -20°C to -80°C and avoid repeated freezing and thawing.
Reaction Time Control: Please control reaction time strictly following this product description!
Substrate: Substrate Solution is easily contaminated. Please protect it from light.Stop Solution: As it is an acid solution, please pay attention to the protection of your eyes, hands, face and clothes when using this solution.
Mixing: You’d better use micro-oscillator at the lowest frequency, as sufficient and gentle mixing is particularly important to reaction result. If there is no micro-oscillator available, you can knock the ELISA plate frame gently with your finger before reaction.
Security: Please wear lab coats and latex gloves for protection. Especially detecting samples of blood or other body fluid, please perform following the national security columns of biological laboratories.
Do not use component from different batches of kit(washing buffer and stop solution can be an exception)
To avoid cross-contamination, change pipette tips between adding of each standard level, between sample adding, and between reagent adding. Also, use separate reservoirs for each reagent. Otherwise, the results will be inaccurate!
Information on standard material:
The formulation of the standard is 0.01 M PBS. The standard contains additives (1 % BSA).
Information on reagents:
Reagents include 1 M SO2. Azide, thimerosal, 2-mercaptoethanol (2-ME) or any other poisonous materials are not used.
Information on antibodies:
The provided antibodies and their host vary in different kits. All antibodies are affinity purified
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.
- Sample Volume
- 100 µL
- Add 100 µL standard or sample to each well. Incubate for 90 min at 37 °C.
- Remove the liquid. Add 100 µL Biotinylated Detection Antibody. Incubate for 1 hour at 37 °C.
- Aspirate and wash 3 times.
- Add 100 µL HRP Conjugate. Incubate for 30 min at 37 °C.
- Aspirate and wash 5 times.
- Add 90 µL Substrate Reagent. Incubate for 15 min at 37 °C.
- Add 50 µL Stop Solution. Read at 450 nm immediately.
- Calculation of results.
- Reagent Preparation
- Bring all reagents to room temperature (18~25 °C) before use. Follow the Microplate reader manual for set-up and preheat it for 15 min before OD measurement.
- Wash Buffer: Dilute 30 mL of Concentrated Wash Buffer with 720 mL of deionized or distilled water to prepare 750 mL of Wash Buffer.Note: if crystals have formed in the concentrate, warm it in a 40 °C water bath and mix it gently until the crystals have completely dissolved
- Standard working solution: Centrifuge the standard at 10,000xg for 1 min. Add 1.0 mL of Reference Standard &Sample Diluent, let it stand for 10 min and invert it gently several times. After it dissolves fully, mix it thoroughly with a pipette. This reconstitution produces a working solution of 10 ng/mL. Then make serial dilutions as needed. The recommended dilution gradient is as follows: 10, 5, 2.5, 1.25, 0.63, 0.32, 0.16, 0 ng/mL. Dilution method: Take 7 EP tubes, add 500 μLof Reference Standard & Sample Diluent to each tube. Pipette 500 μLof the 10 ng/mL working solution to the first tube and mix up to produce a 5 ng/mL working solution. Pipette 500 μLof the solution from the former tube into the latter one according to these steps. The illustration below is for reference. Note: the last tube is regarded as a blank. Don't pipette solution into it from the former tube.
- Biotinylated Detection Antibody working solution: Calculate the required amount before the experiment (100 μL/well). In preparation, slightly more than calculated should be prepared. Centrifuge the stock tube before use, dilute the 100x Concentrated Biotinylated Detection Antibody to 1xworking solution with Biotinylated Detection Antibody Diluent.
- Concentrated HRP Conjugate working solution: Calculate the required amount before the experiment (100 μL/well). In preparation, slightly more than calculated should be prepared. Dilute the 100x Concentrated HRP Conjugate to 1x working solution with Concentrated HRP Conjugate Diluent.
- Sample Collection
Samples should be clear and transparent and be centrifuged to remove suspended solids.
Serum: Allow samples to clot for 2 hours at room temperature or overnight at 4°C before centrifugation for 15 minutes at 1000×g. Collect the supernatant and carry out the assay immediately. Blood collection tubes should be disposable, non-pyrogenic, and non-endotoxin.
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. Collect the supernatant and carry out the assay immediately. Hemolysis samples are not suitable for ELISA assay!
Other biological fluids: Centrifuge samples for 20 minutes at 1000×g at 2 - 8°C. Collect the supernatant and carry out the assay immediately.
Tissue homogenates: You’d better get detailed references from other literatures before assay aiming at different tissue types. For general information, hemolysis blood may affect the result, so you should mince the tissues to small pieces and rinse them in ice-cold PBS (0.02M, pH=7.4) to remove excess blood thoroughly. Tissue pieces should be weighed and then homogenized in PBS (the volume depends on the weight of the tissue) with a glass homogenizer on ice. To further break the cells, you can sonicate the suspension with an ultrasonic cell disrupter or subject it to freeze-thaw cycles. The homogenates are then centrifugated for 5minutes at 5000×g to get the supernate.
Samples should be used within 7 days when stored at 2-8°C, otherwise samples must be divided and stored at -20°C (≤ 1month) or -80°C (≤ 6months) to avoid the loss of bioactivity and contamination. Avoid repeated freeze-thaw cycles.
Please take the samples to room temperature (18-25°C) without extra heating before performing the assay.
Please predict the concentration before assaying. If the sample concentration is not within the range of the standard curve, users must determine the optimal sample dilutions for their particular experiments.
- Calculation of Results
Average the duplicate readings for each standard and samples, then subtract the average zero standard optical density. Create a standard curve by plotting the mean OD value for each standard on the y-axisagainst the concentration on the x-axis and draw a best fit curve through the points on the graph. It is recommended to use some professional software to do this calculation, such as curve expert 1.3 or 1.4. In the software interface, a best fitting equation of standard curve will be calculated using OD values and concentrations of standard sample. The software will calculate the concentration of samples after entering the OD value of samples. If samples have been diluted, the concentration calculated from the standard curve must be multiplied by the dilution factor. If the OD of the sample surpasses the upper limit of the standard curve, you should re-test it after appropriate dilution. The actual concentration is the calculated concentration multiplied dilution factor.
- For Research Use only
- Handling Advice
- All the reagents in the kit should be stored according to the labels on vials. Unused wells should be returned to the foil pouch with the desiccant pack and resealed along entire edge of zip-seal. Substrate Reagent shouldn't be kept at -20 °C (Check!). Exposure of reagents to strong light should be avoided in the process of incubation and storage. All the taps of reagents should be tightened to prevent evaporation and microbial contamination. If not to store reagents according to above suggestions, erroneous results may occur.
- 4 °C/-20 °C
- Storage Comment
- The unopened kit can be stored at 4℃ for 1 month. If the kit is not used within 1 month, store the items separately according to the conditions since the kit is received.
Bongers, Alsady, Nijenhuis, Tulp, Eijsvogels, Deen, Hopman: "Impact of acute versus prolonged exercise and dehydration on kidney function and injury." in: Physiological reports, Vol. 6, Issue 11, pp. e13734, 2018 (PubMed).
- Bongers, Alsady, Nijenhuis, Tulp, Eijsvogels, Deen, Hopman: "Impact of acute versus prolonged exercise and dehydration on kidney function and injury." in: Physiological reports, Vol. 6, Issue 11, pp. e13734, 2018 (PubMed).