1.Intended use

This  ELISA kit is intended for use in quantitating Enterokinase, recombinant  from bovine pancreas. The kit is for research use only and is not  intended for diagnostic use in human or animals.

2.Basic parameters of ELISA Kit

Sensitivity ≤ 1 ng/ml

Detection Range: 0.25 -16 ng/ml

Specificity:  Recombinant bovine enterokinase can be detected without significant  cross-reactivity with other recombinant coliform expression system  related proteins.

Repeatability: Coefficients of variation between plates and in a single plate are both less than 10%

Operating time: Skilled experimenters can complete a determination in 2.5 hours.

Shelf life: 6 months

3.Kit composition and storage

Unopened  kits can be stored at 2-8 °C for one week. If the kit will be used  after one week, disassemble the kit and store each component separately  according to the conditions in the table below.

Explanation:

1. All reagent bottle caps must be screwed tight to prevent evaporation and microbial contamination.

2. The microtiter plate can be stored at -20 °C for 6 months, and not stored at 2-8 °C for more than one week.

4.Self provided experimental equipment

1. Microplate reader (filter wavelength 450 nm and 650 nm)

2. High precision pipette, EP tube and disposable tips

3. 37 °C incubator, double distilled water or deionized water

4. absorbent paper

5.Note for samples to be tested

1.  If the sample is tested within 1 week after collection, it can be  stored at 2-8 °C. If it cannot be detected in time, please distribute it  according to the amount used once and store it frozen at -20 °C (test  within 1 month) Or -80 °C (tested within 3 months), avoid repeated  freeze-thaw cycles.

2.  The detection range of the kit is not the same as the concentration  range of the sample. If the concentration of the test substance in your  sample is higher than the highest value of the standard, please make an  appropriate multiple dilution before measuring.

3.  If a sample or cell extract is prepared using a chemical lysate, the  introduction of certain chemicals may cause deviations in ELISA  measurements.

6.Preparation before testing

1.  Remove the kit from the refrigerator 20 minutes in advance and  equilibrate to room temperature. Turn on the microplate reader 15  minutes in advance to warm up.

2. Diluent preparation

Dilute  the concentrated standard & sample dilution or the concentrated  HRP-antibody dilution with double distilled water (1:25).

3. Preparation of washing liquid

The concentrated washing solution was diluted with double distilled water (1:25).

Tip:  The concentrated washing solution taken out of the refrigerator may  have crystals, which is a normal phenomenon. It can be heated in a 40 °C  water bath to completely dissolve the crystals and then prepare the  washing solution (the heating temperature should not exceed 50 °C. The  temperature of the washing solution should be room temperature when  using ). Please use it the same day.

4. Standard preparation

Place  the standard at 1000 rpm for 1 minute, add 1.0 mL of the standard &  sample diluent to the lyophilized standard, tighten the tube cap, leave  it for 10 minutes, and turn it upside down several times. After it is  fully dissolved, use Mix gently with a pipette (concentration of 16ng /  ml), and then perform a multiple dilution (Note: Do not directly perform  a multiple dilution in the reaction well). It is recommended to  formulate the following concentrations: 16, 8, 4, 2, 1, 0.5, 0.25ng /  ml. The sample dilution was directly used as a blank well at 0 ng / ml.

Double dilution method

Take  7 EP tubes, add 500 μL of standard & sample dilution solution to  each tube, pipet 500 μL from 16 ng / ml of standard working solution,  add to 500 μL of sample dilution EP tube and mix to make 8 ng / ml of  the standard working solution, follow this step and then suck and mix in  sequence. As shown below.

Standard dilution legend (500 μL as sample)

Note: The last tube is directly used as a blank hole.

5.HRP Labeled antibody working solution preparation

Centrifuge the antibody tube at low speed before the experiment to avoid residual antibody on the tube wall and tube cap. Calculate  the amount required for the experiment (calculated at 100 μL /well)  before the experiment. In actual preparation, 100-200 μL should be  prepared. 15 minutes before use, dilute the HRP-labeled antibody to the  working concentration with the antibody diluent (antibody: diluent = 1:  250) and use it the same day.

6.Preparation of coloring working solution (TMB)

Calculate  the amount required for the experiment (calculated at 100 μL /well)  before the experiment. In actual preparation, 100-200 μL should be  prepared. 15 minutes before use, dilute the TMB chromogenic substrate to  the working concentration (substrate: diluent = 1: 20) with the  substrate diluent and use it the same day.

7.Cleaning method

1.  Automatic plate washer: 350 μL of washing solution is added to each  well, and the injection and suction intervals are 60 seconds.

2.  Wash the plate by hand: Shake off the liquid in the hole, pat it dry on  a clean thick absorbent paper (or dry cloth), add 350 μL of washing  solution to each hole (you can also use a clean wash bottle to rinse  here), soak 2-3 Minutes, suck (do not touch the wall of the board) or  shake off the liquid in the board and pat dry on a clean thick absorbent  paper.

8.Operating procedure

1.Add  the working solution of the standard to the first two rows of wells in  sequence, and add two wells of each concentration of the working fluid  side by side, each well 100 μL. The sample to be tested is added to  other wells, 100 μL per well. If the concentration of the sample is  higher than the detection range, it needs to be diluted with the  standard & sample dilution solution and added. Cover the microplate  and incubate at 37 °C for 60 minutes.

Note: When  adding the sample, add the sample to the bottom of the microtiter plate  without touching the wall of the well, and shake gently to avoid air  bubbles. Add the sample within 10 minutes.

2.Washing:  Discard the liquid, spin dry, and pat dry on a clean thick absorbent  paper. Add 350 μL of washing solution to each well and soak for 3  minutes. Aspirate (do not touch the wall of the plate) or shake off the  liquid in the plate and pat dry on a clean thick absorbent paper. Repeat  this washing step 3 times.

3.Add  100 μL of HRP-antibody working solution to each well, mix well, cover  the microtiter plate and incubate at 37 °C for 30 minutes.

4.Washing: Wash the plate 4 times with washing solution, the method steps are the same as 2.

5.Add  100 μL of substrate coloring working solution (TMB) to each well, cover  the microtiter plate and incubate at 37 °C for 10 minutes.

Note: It  can be shortened or extended as appropriate according to the actual  color development, and the color development time is within the range of  5-30 minutes. When the standard well shows a significant gradient, it  can be terminated.

6.Add 50 μL of stop solution to each well to stop the reaction, and leave it at room temperature for 1 minute.

Note: The  order of addition of the stop solution should be the same as that of  the substrate solution. When adding liquid, do not touch the pipette tip  to avoid contamination, which will affect the experimental results.

7.Use  a microplate reader to measure the optical density (OD450 / 650 nm  value) of each well at 450 nm (reference wavelength 650nm).

Note: Please power on the microplate reader in advance, warm up the instrument, and set up the detection program.

9.Result judgment

1.Calculate  the average OD value of each group of wells. The average OD value of  each standard minus the OD value of the blank well was used as the  correction value. Take the concentration of the standard as the abscissa  (X) and the OD value as the ordinate (Y), draw a standard curve (the  value of the blank group can also be removed when plotting).

2.It  is recommended to use professional curve making software (such as curve  expert 1.3 or ELISA Calc, etc.) to draw a standard curve according to  the specific requirements of the experiment.