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.

3.If the sample OD value is higher than the upper limit of the standard curve, it should be re-measured after appropriate dilution.

4.Typical data

Due to different experimental operating conditions (such as operator, pipetting technology, plate washing technology, and stable conditions, etc.), the OD value of the standard curve may be different. The trial kit provides data and curves in the range of 0.25-16 ng / mL for reference. The experimenter can select points within this range to establish a standard curve.

1）Measurement range 0.25-16ng/mL Standard curve line

10.Precautions

1.Storage: Please store the reagents in the kit reasonably according to the instructions. Avoid exposing the reagents to strong light during storage and incubation. All reagent bottles need to be screwed tight to prevent evaporation and microbial contamination, otherwise false results may occur.

2.ELISA plate: There may be a little water-like substance in the well of the just-opened microtiter plate. This is normal and will not affect the experimental results. The slat should be removed and put into a spare ziplock bag and stored at the recommended temperature if won’t be used in near future.

3.Adding sample: When adding samples and adding the same reagent, an excessively long sampling interval between the first well and the last well will result in different "pre-incubation" times, which will obviously affect the accuracy and repeatability of the measured values. It is better to add samples within 10 minutes. Setting duplicate holes is recommended.

4.Incubation: In order to prevent the sample from evaporating, the microtiter plate must be coated during the experiment. The next step should be performed as soon as possible after washing the plate to prevent the microtiter plate from being dry. Strict adherence to the given incubation time and temperature.

5.Washing: The washing liquid remaining in the reaction wells during the washing process should be patted dry on absorbent paper. Do not put the filter paper directly into the reaction wells to absorb water. Before reading, remove the residual liquid and fingerprints on the bottom of the microplate reader to avoid affecting the microplate reader reading.

6.Reagent preparation: Liquid may adhere to the tube wall or bottle cap during transportation, so use 1000 rpm / separation center for one minute to deposit the liquid attached to the tube wall or bottle cap to the bottom of the tube. Before use, carefully pipette 4-5 times with a pipette to mix the solution. All reagents should be prepared exactly as indicated in the instructions.

7.Control of color rendering time: After adding the substrate, please observe the color change of the reaction hole regularly (for example, every five minutes). If the gradient is obvious, please add the termination solution in advance to stop the reaction, so as to avoid too deep color and affect the reading of the enzyme reader.

8.Substrate: Please keep the substrate away from light. Avoid direct exposure to strong light when storing and warming.

9.Mixing: Sufficient and slight mixing is particularly important for the reaction results. It is better to use a micro oscillator with the lowest frequency. If there is no micro oscillator, you can manually tap the enzyme label plate frame to mix before the reaction.

10.Different batches of kit components shall not be mixed (except detergent and termination solution)

11.About sample recovery rate

1) This kit uses the determination of the antigenicity of the recombinant bovine enterokinase instead of its activity to estimate the protein residue. Therefore, the standard in the kit uses the extinction coefficient of the recombinant bovine enterokinase (E1% 1cm = 20.01D, that is, when A280nm = 2.01 At that time, bovine enterokinase concentration was 1mg / ml) to calculate its protein content, and internal quality control was used to keep the standard content as accurate and constant as possible.

2) Different measurement methods and differences in product purity can lead to inconsistent protein content of bovine enterokinase from different sources and different batches of products, and also cause differences in recovery rates. In order to avoid the influence of the above factors, it is recommended to use the spectrophotometric method (A280nm) to estimate the protein content of the sample used for the recovery test, and then dilute it to a suitable concentration for the recovery test to minimize the recovery error.

11.Problem analysis

If there are problems with the experimental results, please take photos of the color development results in a timely manner, and properly store unused slats and reagents, and then contact technical support. Also refer to the information below to find out why.