Human IKZF3 ELISA Kit

SAMPLE COLLECTION AND STORAGE 

• Serum Use a serum separator tube (SST) and allow samples to clot for two hours at room temperature or overnight at 4°C before centrifugation for 15 minutes at 1000 ×g.Remove serum and assay immediately or aliquot and store samples at -20°C or -80°C.Avoid repeated freeze-thaw cycles.
• Plasma Collect plasma using EDTA, or heparin as an anticoagulant.Centrifuge for 15 minutes at 1000 xg, 2 - 8°C within 30 minutes of collection.Assay immediately or aliquot and store samples at -20°C or-80°C.Avoid repeated freeze-thaw cycles.Centrifuge the sample again after thawing before the assay.
• Tissue Homogenates 100mg tissue was rinsed with 1X PBS, homogenized in 1 ml of 1X PBS and stored overnight at -20°C.After two freeze-thaw cycles were performed to break the cell membranes, the homogenates were centrifuged for 5 minutes at 5000 xg, 2 - 8°C.The supernate was removed and assayed immediately.Alternatively, aliquot and store samples at -20°C or -80°C.Centrifuge the sample again after thawing before the assay.Avoid repeated freeze-thaw
• Cell Lysates (1) Adherent Cell: Remove media and rinse cells  once with ice-cold PBS (pH7.2-7.4).Scrape cells off the plate and transfer to an appropriate tube.Dilute cell suspension with 1xPBS (pH7.2-7.4), until cell concentration reached 100 million/ml.Then store overnight at -20°C.After two freeze-thaw cycles to break up the cell membranes, the cell lysates were centrifuged for 5 minutes at 5000 xg, 2 - 8°C.Collect the supernatant.Cell lysates should be assayed immediately or aliquotted and stored at -20°C.Centrifuge the sample again after thawing before the Avoid repeated freeze-thaw cycles. (2) Suspension Cell: Collect cells with appropriate tube, centrifuge for 5 minutes at 1000x g, 2 - 8°C.Remove the supernatant and resuspend cells with 1xPBS (pH7.2-7.4).Centrifuge for 5 minutes at 1000 xg, 2 - 8°C.Remove the supernatant.Dilute cell with 1xPBS (pH7.2-7.4), until cell concentration reached 100 million/ml.Then store overnight at -20°C.After two freeze-thaw cycles to break up the cell membranes, the cell lysates were centrifuged for 5 minutes at 5000 xg, 2 - 8°C.Collect the supernatant.Cell lysates should be assayed immediately or aliquoted and stored at -20°C.Centrifuge the sample again after thawing before the assay.Avoid repeated freeze-thaw cycles.

   

  REAGENT PREPARATION 

  Note:

  l Kindly use graduated containers to prepare the reagent. the kit.

  • Bring all reagents to room temperature (18-25°C) before use for
  • Prepare fresh standard for each assay.Use within 4 hours and discard after
  • Making serial dilution in the wells directly is not
  • Please carefully reconstitute Standards 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. before opening.

    Biotin-antibody requires a 100-fold dilution. (1x) -Centrifuge the vial before opening.

    HRP-avidin requires a 100-fold dilution. If crystals have formed in the concentrate, warm up to room temperature and mix gently until the crystals have completely dissolved. Dilute 20 ml of Wash Buffer Concentrate (25 x) into deionized or distilled water to prepare 500 ml of Wash Buffer (1x). 6000-10000rpm for 30s.

    Reconstitute the Standard with 1.0 ml of Sample Diluent.Do not substitute other diluents. 250 μl of Sample Diluent into each tube (S0-S6).Use the stock solution to produce a 2-fold dilution series (below).Mix each tube thoroughly before the next transfer.The undiluted Standard serves as the high standard (1600 pg/ml).Sample Diluent serves as the zero standard (0 pg/ml).

    

    Tube	S7	S6	S5	S4	S3	S2	S1	S0
    pg/ml	1600	800	400	200	100	50	25	0

     

    ASSAY PROCEDURE  

    Bring all reagents and samples  thawing before the assay.< /strong> It is recommended that all samples and standards be assayed in duplicate.

    1.Prepareall reagents, working standards, and samples as directed in the previous sections.

    2.Referto the Assay Layout Sheet to determine the number of wells to be used and put any remaining wells and the desiccant back into the pouch and seal the ziploc, store unused wells at 4°C.

    3.Add100μl of standard and sample per well.Cover with the adhesive strip provided.  Incubate for 2 hours at 37°C. each well, don’t wash.

    5. Add 100μl of Biotin-antibody (1x) to each well. cloudy.Warm up to room temperature and mix gently until solution appears uniform.)

    6.Aspirate each well and wash, repeating the process two times for a total of three washes.Wash by filling each well with Wash Buffer (200μl) using a squirt bottle, multi-channel pipette, manifold dispenser, or autowasher, and let it stand for 2 minutes, 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.

    7.Add100μl of HRP-avidin (1x) to each well.Cover the microtiter plate with a new adhesive strip.Incubate for 1 hour at 37°C.

    8.Repeat the aspiration/wash process for five times as in step 6.

    9.Add 90μl of TMB Substrate to each well.Incubate for 15-30 minutes at 37°C. Protect from light.

    10.Add50μl of Stop Solution to each well, gently tap the plate to ensure thorough mixing.

    11.Determine the optical density of each well within 5 minutes, using a microplate reader set to 450 nm. If correction is available, set to 540 nm or 570 nm. Subtract readings at 540 nm or 570 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate. Readings made directly at 450 nm without correction may be higher wavelength and less accurate.

    Note:

    1.Thefinal experimental results will be closely related to validity of the products, operation skills of the end users and the experimental environments.

    2.Samplesor reagents addition: Please use the freshly prepared Standard.Please carefully add samples 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 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. When using an automated plate washer, adding a 30 second soak assay period following the addition of wash buffer, and/or rotating the plate 180 degrees between wash steps may improve assay precision.

    5.Controlling of reaction time: Observe the change of color after adding TMB Substrate (eg observation once every 10 minutes), TMB Substrate should change from colorless or light blue to gradations of blue. If the color is too deep, add Stop Solution in advance to avoid excessively strong reaction which will result in inaccurate absorbance reading.

    6.TMBSubstrate is easily contaminated.TMB Substrate should remain colorless or light blue until added to the plate.Please protect it from light.

    7.StopSolution should be added to the plate in the same order as the TMB Substrate.The color developed in the wells will turn from blue to yellow upon addition of the Stop Solution. Wells that are green in color indicate that the Stop Solution has not mixed thoroughly with the TMB Substrate.
    
    

    ASSAY PROCEDURE SUMMARY
    
    
    
    

    CALCULATION OF RESULTS 

    Using the professional soft "Curve Expert" to make a standard curve is recommended.

    Average the duplicate readings for each standard and sample 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 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 IKZF3 concentrations versus the log of the OD and the best fit line can be determined by regression analysis.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.

    PRECISION  

    Intra-assay Precision (Precision within an assay): CV%<8%

    Three samples of known concentration were tested twenty times on one plate to assess.

    Inter-assay Precision (Precision between assays): CV%<10%

    Three samples of known concentration were tested in twenty assays to assess.

     

1.The Stop Solution provided with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material.

2.Samples to be used within 5 days may be stored at 2-8°C, otherwise samples must be stored at -20°C (≤1month) or -80°C (≤2month) to avoid loss of bioactivity and contamination.

3.Grossly hemolyzed samples are not suitable for use in this assay.

4.If the samples are not indicated in the manual, a preliminary experiment to determine the validity of the kit is necessary.

5.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.

6.Tissue or cell extraction samples prepared by chemical lysis buffer may cause unexpected ELISA results due to the impacts of certain chemicals.

7.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.

8.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.

9.Fresh samples without long time storage are recommended for the test. Otherwise, protein degradation and denaturalization may occur in those samples and finally lead to wrong results.