Materials required but not supplied in kit
  • Microplate reader with 450± 10nm filter.
  • Precision single or multi-channel pipettes and disposable tips.
  • Microcentrifuge tubes for dilutions.
  • Deionized or distilled water.
  • Absorbent paper for blotting the microtiter plate.
  • Container for the diluted wash buffer.
Sample Collection and Storage
Serum

Use a serum separator tube and allow samples to clot for two hours at room temperature or overnight at 4℃ before centrifugation for 20 minutes at approximately 1000×g. Assay freshly prepared serum immediately or store samples in aliquots at -20℃ or -80℃ 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℃ within 30 minutes of collection. Remove plasma and assay immediately or store samples in aliquots at -20℃ or -80℃ for later use. Avoid repeated freeze/thaw cycles.

Tissue homogenates

The preparation of tissue homogenates will vary depending on tissue type. In general, tissues should be rinsed thoroughly in ice-cold PBS (0.02mol/L,pH 7.0-7.2) to remove excess blood 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 work as well). The resulting suspension should be sonicated with an ultrasonic cell disrupter or subjected to two freeze-thaw cycles to further break the cell membranes. After that, the homogenates should be centrifugated for 5 minutes at 5000×g. Remove the supernatant and assay immediately or aliquot and store at ≤-20 ℃.

Cell Lysates

Cells must be lysed before assaying according to the following directions:

  1. Adherent cells should be detached with trypsin and then collected by centrifugation (suspension cells can be collected by centrifugation directly).
  2. Wash cells three times in cold PBS.
  3. Resuspend cells in 1X PBS  and subject to ultrasonication 4 times (or Freeze cells at≤-20℃. Thaw cells with gentle mixing. Repeat the freeze/thaw cycle 3 times.)
  4. Centrifuge at 1500×g for 10 minutes at 2 - 8℃ to remove cellular debris. Cell culture supernates and 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℃. Avoid repeated freeze/thaw cycles.
Sample Preparation
  • Reddot Biotech is only responsible for the kit itself, not for the samples consumed during the assay. The experimenter should always calculate the total amount of samples needed prior to beginning the experiment.
  • The experimenter should also predict the approximate concentration before beginning. If the expected values are not within the range of the standard curve, it is recommended to determine the optimal dilution for your sample.
  • If the sample type you are using is not indicated in the manual, you should conduct a preliminary experiment to ensure the effectiveness of the kit before beginning the actual experiment.
  • Using a chemical lysis buffer to prepare tissue or cell extraction samples is not recommended as it may cause unexpected reactions and results due to the contents of the lysis buffer.
  • Due to the possibility of mismatching between antigens of other origins and the antibodies used in our kits, it is NOT recommended to use native or recombinant proteins from other manufacturers as they may not be recognized by our kit.
  • Samples from cell culture supernatant may not be detected by our kit as they can easily be influenced by factors such as cell viability, cell number and/or sampling time.
  • Fresh samples that have not been stored for long periods are recommended for use with our kits. If they are stored for a long time, protein degradation and denaturation can occur, resulting in contradictory or incorrect results.
Precision

The general method to determine precision for all our kits is described below. Specifications for individuals lots of kits are available by request.

  • Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level the index were tested 20 times on one plate, respectively.
  • Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level the index were tested on 3 different plates, 8 replicates in each plate.
  • CV(%) = SD/mean(X)100
    • Intra-Assay: CV<10%
    • Inter-Assay: CV<12%
Stability

The stability of our ELISA kits are determined by the rate of the loss of activity. The loss rate of our kits is generally less than 5% within the expiration date under appropriate storage conditions.

To minimize any outside influences on the performance of our kits, operation procedures and lab conditions should be controlled at all times. Factors to pay specific attention to include:  room temperature, air humidity, and incubator temperature. It is also strongly suggested that the whole assay is performed by the same experimenter from the beginning to the end.

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