CA242 ELISA 试剂盒

• Pre-coated, ready to use 96-well strip plate
• Plate sealer for 96 wells
• Standard
• Sample/Standard Dilution Buffer
• Biotin-labeled Antibody (Concentrated)
• Antibody Dilution Buffer
• HRP-Streptavidin Conjugate (SABC)
• SABC Dilution Buffer
• TMB Substrate
• Stop Solution
• Wash Buffer (25 x concentrate)
• Instruction manual

• Microplate reader (wavelength: 450nm)
• 37 °C incubator
• Automated plate washer
• Precision single and multi-channel pipette and disposable tips
• Clean tubes and Eppendorf tubes
• Deionized or distilled water

Sandwich ELISA, Double Antibody

Take the Elisa kit from the fridge around 20 minutes earlier and equilibrate to room temperature(18-25℃). For repeated assays, please just take the strips and standards required for the current assay, store the rest materials according to the relevant condition.

Wash Buffer

• Dilute 30ml (15ml for 48T) concentrated wash buffer to 750ml (375ml for 48T) wash buffer with deionized or distilled water and mix well. (The recommended resistivity of ultrapure water is 18MΩ.) Alternatively, take appropriate amount of concentrated wash buffer according to the assay requirement, then create a 25-fold dilution and mix well. Store the rest solution at 2-8℃. Crystals formed in the concentrated wash buffer can be heated by water bath at 40℃ till complete dissolution. (Heating temperature should be below 50℃.) Mix well for the next step. It's better to use up the prepared wash buffer in one day. Store the rest buffer at 2-8℃ within 48h.

• Centrifuge standards tube for 1min at 10000xg. Label it as Zero tube.
• Add 1ml sample dilution buffer into the standard tube. Tighten the tube cap and Let it stand for 2min at room temperature. Invert the tube several times to mix gently. (Or you can mix it using a low speed vortex mixer for 3-5 seconds.)
• Centrifuge the tubes for 1min at 1000xg, making the liquid towards the bottom of tube and removing possible bubbles.
• Standard dilution: Label 7 EP tubes with 1/2, 1/4, 1/8, 1/16, 1/32, 1/64 and blank respectively. Add 0.3ml of the sample dilution buffer into each tube. Add 0.3ml solution from zero tube into 1/2 tube and mix them thoroughly. Transfer 0.3ml from 1/2 tube into 1/4 tube and mix them thoroughly. Transfer 0.3ml from 1/4 tube into 1/8 tube and mix them thoroughly, so on till 1/64 tube. Now blank tube only contain 0.3ml sample dilution buffer. The standard concentration from zero tube to blank tube is 2000pg/ml, 1000pg/ml, 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml, 31.25pg/ml, 0pg/ml.
• Notes: Store the zero tube with dissolved standards at 2-8℃ and use it within 12h. Other diluted working solutions containing standards should be used in 2h.

• The working solution should be prepared within 30min before the assay and can't be stored for a long time.
• Calculate required total volume of the working solution: 100ul/well x quantity of wells. (It's better to prepare additional 100ul-200ul.)
• Centrifuge for 1min at 1000xg in low speed and bring down the concentrated biotin-labeled antibody to the bottom of tube.
• Dilute the biotinylated detection antibody with antibody dilution buffer at 1:99 and mix them thoroughly. (e.g. Add 10ul concentrated biotin-labeled antibody into 990ul antibody dilution buffer.)

• The working solution should be prepared within 30min before the assay and can't be stored for a long time.
• Calculate required total volume of the working solution: 100ul/well x quantity of wells. (It's better to prepare additional 100ul-200ul.)
• Centrifuge for 1min at 1000xg in low speed and bring down the concentrated SABC to the bottom of tube.
• Dilute the concentrated SABC with SABC dilution buffer at 1:99 and mix them thoroughly. (e.g. Add 10ul concentrated SABC into 990ul SABC dilution buffer.)

• It is recommended to use fresh samples without long storage, otherwise protein degradation and denaturation may occur in these samples, leading to false results. Samples should therefore be stored for a short period at 2 - 8 °C or aliquoted at -20 °C (≤1 month) or -80 °C (≤ 3 months). Repeated freeze-thaw cycles should be avoided. Prior to assay, the frozen samples should be slowly thawed and centrifuged to remove precipitates.
• If the sample type is not specified in the instructions, a preliminary test is necessary to determine compatibility with the kit.
• If a lysis buffer is used to prepare tissue homogenates or cell culture supernatant, there is a possibility of causing a deviation due to the introduced chemical substance. The recommended dilution factor is for reference only.
• Please estimate the concentration of the samples before performing the test. If the values are not in the range of the standard curve, the optimal sample dilution for the particular experiment has to be determined.

1. When diluting samples and reagents, they must be mixed completely. It's recommended to plot a standard curve for each test.
2. Set standard, pilot samples, control (blank) wells on the pre-coated plate respectively, and then, records their positions. It's recommended to measure each standard and sample in duplicate to decrease experimental errors.
3. Standards and samples loading: Aliquot 100ul of zero tube, 1st tube, 2nd tube, 3rd tube, 4th tube into each standard well. Also add 100ul sample dilution buffer into the control (blank) well. Then, add 100ul pilot samples into each sample well. Seal the plate and static incubate for 90 minutes at 37°C. (Add the solution to the bottom of each well. Mix gently and without touch the sidewall and foam the sample.)
4. Wash twice: Remove the cover, then absorb the liquid in the plate or tap the plate on a clean absorbent paper two or three times. Add 350ul wash buffer into each well without immersion. Discard the liquid in the well and tap on the absorbent paper again. Repeat the washing step twice.
5. Biotin-labeled Antibody: Add 100ul biotin-labeled antibody working solution into each well. Seal the plate and static incubate for 60 minutes at 37°C.
6. Wash three times: Remove the cover, then absorb the liquid in the plate or tap the plate on a clean absorbent paper two or three times. Add 350ul wash buffer into each well and immerse for 1min. Discard the liquid in the well and tap on the absorbent paper again. Repeat the washing step three times.
7. HRP-Streptavidin Conjugate (SABC): Add 100ul SABC working solution into each well. Seal the plate and static incubate for 30 minutes at 37°C. (Put the whole bottle of TMB into the 37°C incubator to equilibrate for 30min.)
8. Wash five times: Remove the cover, and then wash the plate with wash buffer five times. Read washing method in step 5.
9. TMB Substrate: Add 90ul TMB Substrate into each well, seal the plate and static incubate at 37°C in dark within 10-20 minutes. Run the microplate reader and preheat for 15min.
   (Notes: Please do not use the reagent reservoirs used by HRP couplings. The reaction time can be shortened or extended according to the actual color change, but not more than 30 minutes. You can terminate the reaction when apparent gradient appeared in standard wells. Weaker or stronger color intensity is unacceptable.)
10. Stop: Keep the liquid in the well after staining. Add 50ul stop solution into each well. The color will turn yellow immediately. The order for adding stop solution and TMB substrate solution is the same.
11. OD Measurement: Read the O.D. absorbance at 450nm in a microplate reader immediately. (If your microplate reader has a choice of correction wavelength, set it to 570nm or 630nm. Correct the read value to the OD450 value minus the OD570 or OD630 value. In this way, the OD value of non-chromogenic substances can be corrected and removed, thus obtaining more accurate results. If the microplate reader does not have a 570nm or 630nm wavelength, the original OD450 value can be used.)

1. Calculate the mean OD450 value (using the original OD450 value or the corrected OD450 value) of the duplicate readings for each standard, control, and sample. Then, obtain the value of calculation by subtracting the OD450 blank.
2. Create a four parameter logistic curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis. (Remove the OD450 blank during plotting.) Alternatively, you can use the curve fitting software offered by the microplate reader (e.g. Thermo SkanIt RE software, ).
3. Calculate the sample concentration by substituting OD450 value into the standard curve. Diluted samples should be multiplied by the relevant dilution ratio.