SARS-CoV-2 Nucleocapsid ELISA 试剂盒

• ELISA Microplate(Dismountable)
• Lyophilized 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(25X)
• Plate Sealer
• Product Description

Washing
Manual: Discard the solution in the plate without touching the side walls. Clap the plate on absorbent filter papers or other absorbent material. Fill each well completely with 350ul wash buffer and soak for 1 to 2 minutes, then aspirate contents from the plate, and clap the plate on absorbent filter papers or other absorbent material.
Automatic: Aspirate all wells, and then wash plate with 350ul wash buffer. After the final wash, invert plate, and clap the plate on absorbent filter papers or other absorbent material. It is recommended that the washer shall be set for soaking 1 minute. (Note: set the height of the needles; be sure the fluid can be sipped up completely)

Bring all reagents and samples to room temperature for 20 minutes before use.

1. Wash Buffer: If crystals have formed in the concentrate, you can warm it with 40°C water bath (Heating temperature should not exceed 50°C) and mix it gently until the crystals have completely been dissolved. The solution should be cooled to room temperature before use.
   Dilute 30ml Concentrated Wash Buffer into 750ml Wash Buffer with deionized or distilled water. Put unused solution back at 2-8°C.
2. Standards:
   • Add 0.3ml Sample Dilution Buffer into one tube(labeled as zero tube),transfer 0.3ml fromstandard tube (100ng/ml) to zero tube and mix them thoroughly.
   Note: If the standard tube concentration higher than the range of the kit，please dilute it and labeled as zero tube.
   • 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 int o each tube. Add 0.3ml of the above Standard solution (from zero tube) into 1st tube and mix them thoroughly. Transfer 0.3ml from 1st tube to 2nd tube and mix them thoroughly. Transfer 0.3ml from 2nd tube to 3rd tube and mix them thoroughly, and so on. Sample Dilution Buffer was used for the blank control.
3. Preparation of HRP-labeled Antibody Working Solution: Prepare it within 30min before experiment.
• Calculate required total volume of the working solution: 50ul / well × quantity of wells. (Allow 55-60ul more than the total volume.)
• Dilute the HRP-detection antibody with AntibodyDilution Buffer at 1:100 and mix them thoroughly. (i.e. Add 1μl HRPlabeled antibody into 99μl Antibody Dilution Buffer.)

• Plasma: Collect plasma using (EDTA-Na2 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. Avoid hemolysis, high cholesterol samples.
• Tissue Homogenates: As hemolysis blood has relation to assay result, it is necessary to remove residual blood by washing tissue with pre-cooling PBS buffer (0.01M, pH=7.4). Mince tissue after weighing it and get it homogenized in PBS (the volume depends on the weight of the tissue. Normal, 9mL PBS would be appropriate to 1 gram tissue pieces. Some protease inhibitors are recommended to add into the PBS) 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 centrifuged for 5minutes at 5000×g to get the supernatant. The total protein concentration was determined by BCA kit and the total protein concentration of each pore sample should not exceed 0.3mg.
• Cell Culture Supernatant: Centrifuge supernatant for 20 minutes at 1000×g at 2 - 8°C to remove insoluble impurity and cell debris. Collect the clear supernatant and carry out the assay immediately.
• Cell Culture Lysate: Commercial RIPA kits are recommended to follow the instructions provided. Generally, 0.5ml RIPA lysis buffer would be appropriate to 2x106 cells, DNA must to be removed. The total protein concentration was determined by BCA kit and the total protein concentration of each pore sample should not exceed 0.3mg.
• Other Biological Fluids :(throat swab ,nose swab)Centrifuge samples for 20 minutes at 1000×g at 2-8°C. Collect supernatant and carry out the assay immediately.

• It is recommended to use fresh samples without long storage, otherwise protein degradation and denaturationmay occur in these samples, leading to false results. Samples should therefore be stored for a short periodat 2 - 8 °C or aliquoted at -20 °C (≤1 month) or -80 °C (≤ 3 months). Repeated freeze-thawcycles should be avoided. Prior to assay, the frozen samples should be slowly thawed and centrifuged toremove precipitates.
• If the sample type is not specified in the instructions, a preliminary test is necessary to determinecompatibility with the kit.
• If a lysis buffer is used to prepare tissue homogenates or cell culture supernatant, there is a possibilityof 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 therange of the standard curve, the optimal sample dilution for the particular experiment has to be determined.Samples should then be diluted with PBS (pH =7.0-7.2).

When diluting samples and reagents, they must be mixed completely and evenly. Before adding TMB into wells, equilibrate TMB Substrate for 30 min at 37 °C. It is recommended to plot a standard curve for each test.

1. Set standard, test samples (diluted at least 1/2 with Sample Dilution Buffer), control (blank) wells on the pre-coated plate respectively, and then, records their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (blank) wells!
2. Prepare Standards: Aliquot 100μl of zero tube, 1sttube, 2ndtube, 3rdtube, 4thtube, 5thtube, 6thtube and Sample Dilution Buffer (blank) into the standard wells.
3. Add Samples: Add 100μl of properly diluted sample into test sample wells.
4. Incubate: Seal the plate with a cover and incubate at 37°C for 90 minutes.
5. Wash: Remove the cover and discard the plate content, and wash plate 2 times with Wash Buffer. Do NOT let the wells dry completely at any time.
6. Biotin-labeled Antibody: Add 100μl Biotin-labeled antibody working solution into above wells (standard, test sample and blank wells). Add the solution at the bottom of each well without touching the sidewall, cover the plate and incubate at 37°C for 60 minutes.
7. Wash: Remove the cover, and wash plate 3 times with Wash Buffer, and let the Wash Buffer stay in the wells for 1-2 minute each time.
8. HRP-Streptavidin Conjugate (SABC): Add 100μl of SABC Working Solution into each well, cover the plate and incubate at 37°C for 30 minutes.
9. Wash: Remove the cover and wash plate 5 times with Wash Buffer, and let the wash buffer stay in the wells for 1-2 minute each time.
10. TMB Substrate: Add 90μl TMB Substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 minutes. (Note: The reaction time can be shortened or extended according to the actual color change, but not more than 30minutes. You can terminate the reaction when apparent gradient appeared in standard wells.)
11. Stop: Add 50μl Stop Solution into each well. The color will turn yellow immediately. The adding order of Stop Solution should be as the same as the TMB Substrate Solution.
12. OD Measurement: Read the O.D. absorbance at 450 nm in Microplate Reader immediately after adding the stop solution.

Regarding calculation, (the relative O.D.450) = (the O.D.450 of each well) – (the O.D.450 of blank well). The standard curve can be plotted as the relative O.D.450 of each standard solution (Y) vs. the respective concentration of the standard solution (X). The target concentration of the samples can be interpolated from the standard curve. It is recommended to use some professional software to do this calculation, such as Curve Expert 1.3 or 1.4.
Note: If the samples measured were diluted, multiply the dilution factor to the concentrations from interpolation to obtain the concentration before dilution.