ELISA analysis of rat superoxide dismutase (SOD)

ELISA analysis of rat superoxide dismutase (SOD)

Kit instruction manual

This reagent is for research purposes only: This kit is used to determine the content of superoxide dismutase (SOD) in rat serum, plasma and related liquid samples.

Experimental principle:

This kit uses the double antibody sandwich method to determine the level of rat superoxide dismutase (SOD) in the specimen. Microporous plates were coated with purified rat SOD antibody to make solid phase antibodies. Superoxide dismutase (SOD) was added to the microwells coated with mAb in sequence, and then combined with HRP-labeled goat anti-mouse antibody The antibody-antigen-enzyme-labeled antibody complex is thoroughly washed and added with substrate TMB 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 superoxide dismutase in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the concentration of rat superoxide dismutase (SOD) in the sample was calculated by a standard curve.

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: 225U / 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 ℃

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

3. 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.

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).

Steps:

1. Dilution and sample loading of standard products: set 10 standard wells on the enzyme-coated plate, add 100 μl of standard products in the first and second wells, then add 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 150U / L, 100U / L, 50U / L, 25U / L, 12.5U / L).

2. Add samples: set up blank wells (the blank control wells do not add samples and enzyme 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.

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

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

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

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

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.

Precautions:

1. 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.

2. 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.

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. 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).

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

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.

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 it by the dilution factor; or use the concentration of the standard 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. (This picture is for reference only)

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:

10U / L -200U / L

Storage conditions and validity period:

1. Store the kit: 2-8 ℃.

2. Validity: 6 months

FOR RESEARCH USE ONLY

Drug Names

Generic Name: Rat Super Oxidase Dimutase (SOD) ELISA Kit.

Purpose

This kit allows for the determination of SODconcentrations in Rat serum, blood plasma, and other biological fluids.

Principle of the assay

The kit assay Rat SOD level in the sample, use Purified RatSOD antibody to coat microtiter plate wells, make solid-phase antibody, then add SOD to wells, Combined antibody which With HRP labeled goat anti-Rat become antibody-antigen-enzyme-antibody complex, after washing Completely, Add TMB substrate solution, TMB substrate becomes blue color At HRP enzyme-catalyzed, reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm. The concentration of SOD in the samples is then determined by comparing the OD of the samples to the standard curve.

Materials provided with the kit

Materials provided with the kit

48determinations

96determinations

Storage

User manual

1

1

Closure plate membrane

2

2

Sealed bags

1

1

Microelisa stripplate

1

1

2-8 ℃

Standard: 225U / L

0.5ml × 1 bottle

0.5ml × 1 bottle

2-8 ℃

Standard diluent

1.5ml × 1 bottle

1.5ml × 1 bottle

2-8 ℃

HRP-Conjugate reagent

3ml × 1 bottle

6ml × 1 bottle

2-8 ℃

Sample diluent

3ml × 1 bottle

6ml × 1 bottle

2-8 ℃

Chromogen Solution A

3ml × 1 bottle

6ml × 1 bottle

2-8 ℃

Chromogen Solution B

3ml × 1 bottle

6ml × 1 bottle

2-8 ℃

Stop Solution

3ml × 1 bottle

6ml × 1 bottle

2-8 ℃

wash solution

(20ml × 20 fold)

× 1bottle

(20ml × 30 fold)

× 1bottle

2-8 ℃

Specimen requirements

1. serum- coagulation at room temperature 10-20 mins, centrifugation 20-min at the speed of 2000-3000 rpm remove supernatant, If precipitation appeared, Centrifugal again.

2. plasma-use suited EDTA or citrate plasmaas an anticoagulant, mix 10-20 mins, centrifugation 20-min at the speed of 2000-3000 rpm remove supernatant, If precipitation appeared, Centrifugal again.

3. Urine-collect sue a sterile container, centrifugation 20-min at the speed of 2000-3000 rpm remove supernatant, If precipitation appeared, Centrifugal again. The Operation of Hydrothorax and cerebrospinal fluid Reference to it.

4. cell culture supernatant-detect secretory components, collect sue a sterile container, centrifugation 20-min at the speed of 2000-3000 rpm remove supernatant, detect the composition of cells, Dilut cell suspension with PBS (PH7.2-7.4), Cell concentration reached 1 million / ml, repeated freeze-thaw cycles, damage cells and release of intracellular components, centrifugation 20-min at the speed of 2000-3000 rpm remove supernatant, If precipitation appeared, Centrifugal again.

5. Tissue samples- After cutting samples, check the weight, add PBS (PH7.2-7.4), Rapidly frozen with liquid nitrogen, maintain samples at 2-8 ℃ after melting, add PBS (PH7.4), Homogenized by hand or Grinders, centrifugation 20-min at the speed of 2000-3000 rpm remove supernatant.

6. extractas soon as possible after Specimen collection, and according to the relevant literature, and should be experiment as soon as possible after the extraction. If it can't, specimen can be kept in -20 ℃ to preserve, Avoid repeated freeze- thaw cycles.

7. Can't detect the sample which contain NaN3, because NaN3 inhibits HRP active.

Assay procedure

1.Dilute and add sample to Standard: set 10 Standard wells on the ELISA plates coated, add Standard 100μl to the first and the second well, then add Standard dilution50μl to the first and the second well, mix; take out 100μl form the first and the second well then add it to the third and the forth well separately. then add Standard dilution50μl to the third and the forth well, mix; then take out 50μl from the third and the forth well discard, add 50μl to the fifth and the sixth well, then add Standard dilution50μl to the fifth and the sixth well, mix; take out 50μl from the fifth and the sixth well and add to the seventh and the eighth well, then add Standard dilution50μl to the seventh and the eighth well, mix ; take out 50μl from the seventh and the eighth well and add to the ninth and the tenth well, add Standard dilution 50μl to the ninth and the tenth well, mix, take out 50μl from the ninth and the tenth well discard (add Sample 50μl to each well after Diluting, (den sity: 150U / L, 100U / L, 50U / L, 25U / L, 12.5U / L)

2.add sample: Set blank wells separately (blank comparison wells don't add sample and HRP-Conjugate reagent, other each step operation is same). Testing sample well. Add Sample dilution 40μl to testing sample well, then add testing sample 10μl (sample final dilution is 5-fold), add sample to wells, don't touch the well wall as far as possible, and Gently mix.

3.Incubate: After closing plate with Closure plate membrane, incubate for 30 min at 37 ℃.

4.Configurate liquid: 30-fold (or 20-fold) wash solution diluted 30-fold (or 20-fold) with distilled water and reserve.

5.washing: Uncover Closure plate membrane, discardLiquid, dry by swing, add washing buffer to every well, still for 30s then drain, repeat 5 times, dry by pat.

6.add enzyme: Add HRP-Conjugate reagent 50μl to each well, except blank well.

7.incubate: Operation with 3.

8.washing: Operation with 5.

9.color: Add Chromogen Solution A 50ul and Chromogen Solution B to each well, evade the light preservation for 15 min at 37 ℃

10.Stop the reaction: Add Stop Solution50μl to each well, Stop the reaction (the blue color change to yellow color).

11.assay: take blank well as zero, Read absorbance at 450nm after Adding Stop Solution and within 15min.

Important notes

1. The kit takes out from the refrigeration environment should be balanced 15-30 minutes in the room temperature, ELISA plates coated if has not use up after opened, the plate should be stored in Sealed bag.

2. washing buffer will Crystallization separation, it can be heated the water helps dissolve when dilute. Washing does not affect the result.

3. add Sample with sampler Each step, And proofread its accuracy frequently, avoids the experimental error. Add sample within 5 mins, if the number of sample is much, recommend to use Volley.

4. if the testing material content is excessively higher (The sample OD is bigger than the first standard well), please dilute Sample (n-fold), Please diluenteand multiplied by the dilution factor. (× n × 5).

5. Closure plate membrane only limits the disposable use, to avoid cross-contamination.

6. The substrate evade the light preservation.

7. Please according to use instruction strictly, The test result determination must take the microtiter plate reader as a standard.

8. All samples, washing buffer and each kind of reject should according to infective material process.

9. Do not mix reagents with those from other lots.

Assay range

10U / L -200U / L

Storage and validity

1. Storage: 2-8 ℃.

2. validity: six months.