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Human von Willebrand Factor / Restomycin Cofactor (VWF) Enzyme Linked Immunoassay (ELISA)

Kit instruction manual

This reagent is for research use only

Purpose: This kit is used to determine the content of von Willebrand factor / Restomycin cofactor (VWF) in human serum, plasma and related liquid samples.

Experimental principle:

This kit uses the double antibody sandwich method to determine the level of human von Willebrand factor / Restomycin cofactor (VWF) in the specimen. Microporous plates were coated with purified human hemophilia factor / restomycin cofactor (VWF) antibody to prepare solid-phase antibodies, and von Willebrand factor was sequentially added to the monoclonal antibody-coated microwells / Restromycin cofactor (VWF), and then combined with HRP-labeled von Willebrand factor / Restromycin cofactor (VWF) antibody to form an antibody-antigen-enzyme labeled antibody complex, after thorough After washing, the substrate TMB is added for color development. TMB is converted into blue under the catalysis of HRP enzyme, and into the final yellow under the action of acid. The color depth is positively correlated with the von Willebrand factor / Restomycin cofactor (VWF) in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the concentration of human von Willebrand factor / Restromycin cofactor (VWF) in the sample was calculated by a standard curve.

Sample processing and requirements:

1. Serum: room temperature blood coagulates naturally for 10-20 minutes, centrifuged for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully and centrifuge again if a precipitate appears during storage.

2. Urine: collected in a sterile tube and centrifuged for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, centrifuge again. Pleural and ascites, cerebrospinal fluid reference implementation.

3. Plasma: EDTA or sodium citrate should be selected as the anticoagulant according to the requirements of the specimen. After mixing for 10-20 minutes, centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, it should be centrifuged again.

4. Cell culture supernatant: When detecting secreted components, collect with a sterile tube. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. When detecting the components inside the cells, dilute the cell suspension with PBS (PH7.2-7.4), and the cell concentration will reach about 1 million / ml. Through repeated freezing and thawing, the cells are destroyed and the intracellular components are released. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, it should be centrifuged again.

5. Organize the specimen: after cutting the specimen, weigh it. Add a certain amount of PBS, PH7.4. Quickly freeze and save with liquid nitrogen for later use. After the specimen melts, it still maintains a temperature of 2-8 ° C. Add a certain amount of PBS (PH7.4) and homogenize the specimen with a manual or homogenizer. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. After aliquoting, a portion is to be tested, and the rest is frozen for future use.

6. The specimen should be extracted as soon as possible after collection. The extraction should be carried out according to relevant literature. The experiment should be carried out as soon as possible after extraction. If the test cannot be performed immediately, the specimen can be stored at -20 ℃, but repeated freezing and thawing should be avoided.

7. The sample containing NaN3 cannot be detected because NaN3 inhibits the activity of horseradish peroxidase (HRP).

Precautions:

1. Crystals may be precipitated in the concentrated washing liquid, which can be heated and dissolved in a water bath during dilution, and the results will not be affected during washing.

2. The sealing film is limited to one-time use to avoid cross-contamination.

3. The sampler should be used at each step of sample addition, and the accuracy should be regularly checked to avoid test errors. It is best to control the sampling time within 5 minutes. If there are many specimens, it is recommended to use a volley gun to add samples.

4. The kit should be equilibrated at room temperature for 15-30 minutes before being taken out of the refrigerated environment. If the enzyme label coated plate is unopened, the strip should be stored in a sealed bag.

5. Please make a standard curve at the same time of each measurement, it is best to make a double hole. If the content of the test substance in the specimen is too high (the OD value of the sample is greater than the OD value of the first well of the standard well), please first dilute it with a certain multiple (n times) of the sample diluent and then determine it. When calculating, please multiply the total dilution Multiple (× n × 5).

6. Please keep the substrate away from light.

7. Strictly follow the instructions, and the test results must be determined by the microplate reader.

8. All samples, washing liquids and various wastes should be treated as infectious agents.

9. The components of different batches of this reagent shall not be mixed.

10. If there is any difference with the English manual, the English manual shall prevail.

Kit composition:

Kit composition

48 hole configuration

96-well configuration

save

Instructions

1 serving

1 serving

Sealing film

2 pieces (48)

2 pieces (96)

sealed bag

1

1

Enzyme coated plate

1 × 48

1 × 96

Store at 2-8 ℃

Standard product: 2700ng / L

0.5ml × 1 bottle

0.5ml × 1 bottle

Store at 2-8 ℃

Standard dilution

1.5ml × 1 bottle

1.5ml × 1 bottle

Store at 2-8 ℃

Enzyme reagent

3 ml × 1 bottle

6 ml × 1 bottle

Store at 2-8 ℃

Sample diluent

3 ml × 1 bottle

6 ml × 1 bottle

Store at 2-8 ℃

Developer A liquid

3 ml × 1 bottle

6 ml × 1 bottle

Store at 2-8 ℃

Developer B liquid

3 ml × 1 bottle

6 ml × 1 bottle

Store at 2-8 ℃

Stop solution

3ml × 1 bottle

6ml × 1 bottle

Store at 2-8 ℃

Concentrated washing liquid

(20ml × 20 times) × 1 bottle

(20ml × 30 times) × 1 bottle

Store at 2-8 ℃

Steps

1. Add sample: set up blank wells separately (the blank control wells do not add samples and enzyme-labeled reagents, the rest of the steps are the same) and the sample wells to be tested. Add 40μl of sample diluent to the test sample well of the enzyme-coated plate, and then add 10μl of the sample to be tested (the final dilution of the sample is 5 times). Add the sample and add the sample to the bottom of the well of the microplate, try not to touch the wall of the well, shake gently to mix.

2. Dilution and loading of standard products: set 10 standard wells on the enzyme-coated plate, add 100 μl of the standard products in the first and second wells, and then add the standard products in the first and second wells. 50μl of diluent, mix well; then take 100μl from the first well and the second well and add them to the third and fourth wells respectively, and then add 50μl of standard diluent to the third and fourth wells respectively, mix well; Then take 50μl each in the third and fourth wells and discard it, then add 50μl each to the fifth and sixth wells, and then add 50ul of the standard dilution solution to the fifth and sixth wells respectively, and mix well; After mixing, take 50μl from the fifth and sixth wells and add them to the seventh and eighth wells respectively. Then add 50μl of the standard dilution solution to the seventh and eighth wells respectively. Take 50μl from the eight wells and add them to the ninth and tenth wells. Then add 50μl of the standard dilution solution to the ninth and tenth wells. After mixing, take 50μl from the ninth and tenth wells and discard. (After dilution, the volume of each well is 50μl, and the concentration is 1800ng / L, 1200ng / L, 600ng / L, 300ng / L, 150ng / L)

3. Incubation: Seal the plate with a sealing plate and incubate at 37 ° C for 30 minutes.

4. Mixing solution: Dilute 30 times (20 times of 48T) concentrated washing liquid with distilled water 30 times (20 times of 48T) and then use.

5. Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with the washing liquid, let it stand for 30 seconds and then discard, repeat this 5 times, pat dry.

6. Incubation: The operation is the same as 3.

7. Washing: The operation is the same as 5.

8. Color development: Add 50μl of developer A to each well, and then add 50μl of developer B, mix gently, and develop at 37 ° C in the dark for 15 minutes.

9. Add enzyme: add 50μl of enzyme label reagent to each well, except blank well.

10. Termination: Add 50μl of stop solution to each well to stop the reaction (at this time the blue will turn to yellow).

11. Determination: Measure the absorbance (OD value) of each well in sequence with the blank air conditioner at zero and 450 nm wavelength. The measurement should be carried out within 15 minutes after adding the stop solution.

Storage conditions and validity period:

1. Store the kit: 2-8 ℃.

2. Validity: 6 months

Kit performance:

1. The correlation coefficient R between the linear regression of the sample and the expected concentration is above 0.95.

2. The batch and approval shall be less than 9% and 11% respectively

examination range:

100ng / L -2000ng / L

Calculation:

Taking the concentration of the standard as the abscissa and the OD value as the ordinate, draw a standard curve on the coordinate paper, and find the corresponding concentration from the standard curve according to the OD value of the sample; then multiply by the dilution factor; Calculate the linear regression equation of the standard curve with the OD value, substitute the OD value of the sample into the equation, calculate the sample concentration, and multiply it by the dilution factor to obtain the actual concentration of the sample.

Hypochlorous acid (HOCl or HClO) is a weak acid that forms when chlorine dissolves in water, and itself partially dissociates, forming hypochlorite, ClO−. HClO and ClO− are oxidizers, and the primary disinfection agents of chlorine solutions.[2] HClO cannot be isolated from these solutions due to rapid equilibration with its precursor. Sodium hypochlorite (NaClO) and calcium hypochlorite (Ca(ClO)2), are bleaches, deodorants, and disinfectants.

Hypochlorous acid was discovered in 1834 by the French chemist Antoine Jérôme Balard (1802–1876) by adding, to a flask of chlorine gas, a dilute suspension of mercury oxide in water.He also named the acid and its compounds.

In organic synthesis, HClO converts alkenes to chlorohydrins.
In biology, hypochlorous acid is generated in activated neutrophils by myeloperoxidase-mediated peroxidation of chloride ions, and contributes to the destruction of bacteria.
In the cosmetics industry it is used on the skin.[citation needed] It is also used in baby products.[citation needed]
In food service and water distribution, specialized equipment to generate weak solutions of HClO from water and salt is sometimes used to generate adequate quantities of safe (unstable) disinfectant to treat food preparation surfaces and water supplies.
In water treatment, hypochlorous acid is the active sanitizer in hypochlorite-based products (e.g. used in swimming pools).
Similarly, in ships and yachts, marine sanitation devices use electricity to convert seawater into hypochlorous acid to disinfect macerated faecal waste before discharge into the sea.

Virusuperclean produces hypochlorous acid that has an expiration date of up to one year, supports customization in a variety of package sizes, and has been Tested and approved for EU1276:2019 and REACH, please contact us for more information.

Disinfection Water (Non Alcohol)

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