Human anti-SARS-CoV-2 nucleoprotein(NP)  IgM/IgG/IgA total antibody ELISA Kit

Please read this instruction manual carefully before beginning this assay.

About This Assay

It is for the qualitative determination of anti-SARS-CoV-2 N protein- IgM/IgG/IgA in human serum, plasma.

Test  Principle 

The ELISA is based on the the qualitative enzyme immunoassay technique. The microplate  provided  in  this  kit  has  been  pre-coated  with  human novel coronavirus nucleoprotein. Samples are pipetted into the wells with anti-human  IgM/IgG/IgA antibody conjugated Biotin. Next, Streptavidin-HRP is added to each microplate well and incubated. After TMB Substrate solution is added, Any antibodies specific for the antigen present will bind to the pre-coated antigen. Following a wash to remove any unbound reagent, a substrate solution is added to the wells and color develops in proportion to the amount of human novel coronavirus nucleoprotein (SARS-CoV-2 NP)  IgM/IgG/IgA antibody bound in the initial step. The color development is stopped and the intensity of the color is measured.

Materials Supplied

 Store  as follows for Unopened kit,  it is 12 months shelf  life. Store at 4℃ for opened kit, it is one month shelf  life.

Materials Needed But Not Supplied

1.  Microplate reader (wavelength: 450 nm)

2.  37℃  incubator

3.  Automated plate washer

4.  Precision single and multi-channel pipette and disposable tips

5.  Clean tubes and Eppendorf  tubes

6.  Deionized or distilled water

7.  0.01M Phosphate Buffered Saline(PBS), pH 7.2-7.4

Samples 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 25 minutes at approximately   1,000×g. Assay

 freshly prepared serum immediately or store samples in aliquot     

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 20 minutes at 

1,000×g at 2-8 ℃ within 30 minutes of collection. Remove plasma and assay immediately or store 

samples in aliquot at -20 ℃ or -80 ℃ for later use. Avoid repeated freeze-thaw cycles.

Note:

1. Samples to be used within 5 days may be stored at 4 ℃, otherwise samples must be

 stored at -20℃ (≤6 months) or -80℃ (≤12 months) to avoid loss of bioactivity and contamination.

2. Samples hemolysis will influence the result, so hemolytic specimen should not be used.

3. When performing the assay, bring samples to room temperature.

4. It is highly recommended to use serum instead of plasma for the detection based on quantity of our in-house data.

Samples Preparation

1. Dilute the serum or plasma samples with Universal Diluent(1:200) before test. The suggested 

200-fold dilution can be achieved by adding 1.5 μL sample to 298.5 μL of Universal Diluent.

2. We are only responsible for the kit itself, but not for the samples consumed during the assay.

 The user should calculate the possible amount of the  samples used in the whole test.  Please reserve

 sufficient  samples in advance.

3.Several trials may be necessary. The test sample must be well mixed with the

  Universal Diluent. And also Positive Control （Negative Control）and samples should be making in pre-experiment. 

 4. If the samples are not indicated in the manual, a preliminary experiment to determine

 the validity of the kit is necessary.

Reagent  Preparation

Bring all kit components and samples to room temperature (18-25 ℃) before use.

 If the kit will not be used up in one time, please only take out strips and reagents for 

present experiment, and leave the remaining strips and reagents in required condition.

1.  Positive Control and Negative Control - Dissolve them to the working concentration with

     400 μL Universal  Diluent.

2.  Biotin-antibody(1×) ａnd Streptavidin-HRP(1×) - Briefly spin or centrifuge the stock Biotin-antibody(100×) and

 Streptavidin-HRP(100×) before Dilution. Dilute them to the working concentration with Universal  Diluent. A suggested 100-fold dilution is 10 μl of  Biotin-antibody(100×) or 

Streptavidin- HRP(100×) + 990 μl of  Universal  Diluent.

3. Wash Buffer(1×) - Dilute 15 mL of Wash Buffer (30×) with 435 mL of deionized or  

distilled water to prepare 450 mL of  Wash Buffer (1×).

4. TMB Substrate - Aspirate the needed dosage of the solution with sterilized tips and do

      not dump the residual solution into the vial again.

Note:

1.  Making serial dilution in the wells directly is not permitted.

2.  Prepare Positive Control and Negative Control within 15 minutes before assay. Please

do not dissolve the reagents at 37 ℃ directly.

3. Please carefully reconstitute Positive Control and Negative Control according to the instruction

 ａnd avoid foaming and mix gently until the crystals are completely dissolved. To minimize 

imprecision caused by pipetting, use small volumes and ensure that pipettors are calibrated.

 It is recommended to suck more than 10 μL for one pipetting.

  4.  The reconstituted Positive Control and Negative Control can be used only once.

5.  If crystals have formed in the Wash Buffer (30×), warm to room temperature and mix gently until the crystals are completely dissolved.

6. Contaminated water or container for reagent preparation will influence the detection result.

Plate Set Up

The 96-wells plate included with this kit is supplied ready to use. It is not necessary to rinse the plate prior 

to adding the reagents.

A suggested plate format is shown in Figure 1 below. The user may vary the location and type of wells present 

as necessary for each particular experiment.The plate format provided below has been designed to allow 

for easy data analysis using a convenient spreadsheet offered by NewEastBio

PC=Postive Control;   NC=Negative Control;   BLK=Blank;  S=Sample

Assay Procedure

1.  Determine wells for Positive Control and Negative Control, blank and sample. Prepare Blank 

well without any solution.

2.  Add 100 μL of  Negative Control, Positive Control or diluted Sample per well. Cover 

with the Plate sealer. Incubate for 30 minutes at 37 ℃.

3.  Aspirate each well and wash with  300 μL  of   Wash Buffer(1×)  to  each  well  using

    a squirt  bottle, multi-channel pipette, manifold dispenser or autowasher, and let it sit for

   1~2 minutes. Remove the remaining liquid from all wells completely by snapping the

 plate onto absorbent paper. Repeat wash 3 times.After the last wash, remove any 

remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against 

absorbent paper.

   4.  Add 100 μL of  Biotin-antibody(1×) to each well(not to blank). Cover with the Plate 

    sealer.Incubate for  30 minutes at 37 ℃.

   5.Repeat the aspiration/wash process for total 3 times as conducted in step 3

   6. Add 100 μL of Streptavidin-HRP(1×) to each well(not to blank), Cover the wells with the 

plate sealer and incubate for 30 minutes at 37 ℃.

 7.  Repeat the aspiration/wash process for total 5 times as conducted in step 3.

8.  Add 90 μL of TMB Substrate  to each well. Incubate for 15-20 minutes at 37 ℃. Protect from  light. 

 The  liquid  will  turn  blue  by  the  addition of  TMB Substrate.

9. Add 50 μL of Stop Solution to each well. The liquid will turn yellow by the addition of Stop solution. Mix the

 liquid by tapping the side of the plate. If color change does not appear uniform, gently tap the plate to ensure

 thorough mixing.

10. Remove any drop of water and fingerprint on the bottom of the plate and confirm there is no bubble on the surface of the liquid. Then, run the microplate reader and conduct measurement at 450 nm immediately.

Assay Procedure Summary

1.  Prepare all reagents, samples and Negative Control, Positive Control.

2.  Add 100 μL Negative Control, Positive Control or sample to each well. Incubate for  30 minutes at 37℃ .

3.  Aspirate and wash 3 times.

4.  Add 100 μL  Biotin-antibody(1×) to each well(not to blank). Incubate for  30 minutes at 37℃ .

5.  Aspirate and wash 3 times.

6.  Add 100 μL Streptavidin-HRP(1×) to each well(not to blank). Incubate for 30 minutes at 37℃ .

7.  Aspirate and wash 5 times.

8.  Add 90 μL TMB Substrate. Incubate for 15-20 minutes at 37℃ .

9   Add 50 μL Stop Solution ａnd  read at 450 nm immediately.

Note:

     1.  Assay preparation: Keep appropriate numbers of wells for each experiment and 

     remove extra wells from microplate. Rest wells should be resealed and stored at -20 ℃.

2. Samples or reagents addition: Please use the freshly prepared Negative Control, Positive Control. Please carefully add samples to wells and mix gently to avoid foaming. Do not touch the well wall.

 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 Negative Control, Positive Control and specimens, although not required, is recommended. To  avoid  cross-contamination,  change  pipette  tips between additions of Negative Control, Positive Control, samples, and reagents. Also, use separated 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 are

 added to the well strips, do not let the strips dry at any time during the assay. Incubation time and temperature 

must be controlled.

4.  Washing:  The  wash  procedure  is  critical.  Complete  removal  of  liquid  at  each  step  is  essential  for 

 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 false elevated absorbance reading.

  5. Controlling of reaction time:  Observe the change of color after adding TMB             

Substrate(e.g. observation once every 7 minutes), if the color is too deep, add Stop Solution in advance

 to avoid excessively strong reaction which will result in inaccurate absorbance reading.

7.  TMB Substrate is easily contaminated. Please protect it from light.

Precision

Intra-assay Precision (Precision within an assay): CV%< 10%

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.

Calculation Of Results

For calculation the valence of human novel coronavirus nucleoprotein (SARS-CoV-2 NP)  IgM/IgG/IgA antibody, compare the sample well with control.

When OD(Positive Control)  ≥0.5 and OD(Negative Control) ≤0.2,  it represents the results are valid. If OD(sample) close to the Cut off value, we recommend repeating the experiment.

OD_（blank）＜0.1

Cut off  value= OD_{(Negative Control)} + 0.2

Negative Result : OD_(sample)＜ Cutoff  value

Positive Result : OD_(sample)≥ Cutoff  value

Declaration

1.  Limited by current conditions and scientific technology, we can't conduct comprehensive

   identification and analysis on all the raw material provided. So there might be some

qualitative and technical risks for users using the kit.

2.  Do not mix or substitute reagents from one kit to another. Use only the reagents  

supplied by us, please ask for our advice if  you use  any other reagents.

3.  This product is for research only, not for human or veterinary diangostic or   therapeutic use !