Instructions for human bile acid (CA) enzyme-linked immunoassay (ELISA)

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Human bile acid (CA) enzyme-linked immunoassay (ELISA)
Kit Instructions for Use This reagent is for research purposes only: This kit is used to determine the content of cholic acid (CA) in human serum, plasma and related liquid samples.
Experimental principle:
This kit uses the double antibody sandwich method to determine the level of human cholic acid (CA) in the specimen. Coated microtiter plates with purified human cholic acid (CA) antibody to prepare solid-phase antibodies. Add cholic acid (CA) to the monoclonal antibody-coated microwells in turn, and then bind to HRP-labeled cholic acid (CA) antibody To form an antibody-antigen-enzyme-labeled antibody complex. After thorough washing, the substrate TMB is added for color development. TMB
It 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 cholic acid (CA) in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the content of human cholic acid (CA) in the sample was calculated by a standard curve.
Kit composition:
Kit composition 48 well configuration 96 well configuration storage instructions 1 part 1 part sealing plate film 2 pieces (48) 2 pieces (96)
One sealed bag, one enzyme-labeled coating plate 1 × 48 1 × 96 2-8 ° C Store the standard: 90 pmol / L 0.5ml × 1 bottle 0.5ml × 1 2-8 ° C Store the standard dilution 1.5ml × 1 vial 1.5ml × 1 vial at 2-8 ° C, 3 ml × 1 vial, 6 ml × 1 vial at 2-8 ℃, sample diluent 3 ml × 1 vial, 6 ml × 1 vial, 2-8 Toner A solution 3 ml × 1 bottle 6 ml × 1 bottle 2-8 ℃ stored developer B solution 3 ml × 1 bottle 6 ml × 1 bottle 2-8 ° C storage stop solution 3ml × 1 bottle 6ml × 1 bottle 2 Store the concentrated washing solution at -8 ℃ (20ml × 20 times) × 1 bottle (20ml × 30 times) × 1 bottle at 2-8 ℃
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 anticoagulant according to the requirements of the specimen, mixed for 10-20 minutes, and centrifuged
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 sterile tubes 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 /
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Minute). Collect the supernatant carefully. When detecting the components in 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. If the test cannot be performed immediately, the specimen can be stored at -20 ℃, but repeated freezing and thawing should be avoided.
7. Samples containing NaN3 cannot be detected because NaN3 inhibits horseradish peroxidase (HRP) activity.
Steps
1. 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, then add 50μl of the standard diluent to the third and fourth wells,
Mix well; then take 50μl each in the third and fourth wells and discard them, then add 50μl each to the fifth and sixth wells, and then add 50ul of standard dilution solution to the fifth and sixth wells, Mix; 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. 7. Take 50μl from the eighth and eighth wells respectively and add it to the ninth and tenth wells. Then add 50μl of the standard dilution solution to the ninth and tenth wells respectively. After mixing, take 50μl from the ninth and tenth wells and discard. (After dilution, the volume of each well is 50μl,
The concentrations were 60 pmol / L, 40 pmol / L, 20 pmol / L, 10 pmol / L, and 5 pmol / L respectively.
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 test sample (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 the sealing film and incubate at 37 ° C for 30 minutes.
4. Mixing solution: Dilute 30 times (20 times of 48T) concentrated washing solution with distilled water 30 times (20 times of 48T) and then reserve.
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, then discard, repeat 5 times and 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, and then add 50μl of developer B, mix gently, and develop color at 37 ℃ in the dark
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 450nm wavelength. The measurement should be carried out within 15 minutes after adding the stop solution.
Precautions:
1. The kit should be taken out of the refrigerated environment and equilibrated at room temperature for 15-30 minutes before use. If the enzyme-coated plate is unsealed after opening, 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 substance to be tested in the sample is too high (the OD value of the sample is greater than the OD value of the first well of the standard well), please dilute it with a certain multiple (n times) of the sample diluent before measuring.
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When calculating, please multiply the total dilution factor (× 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, the OD value is the ordinate,
Draw a standard curve on coordinate paper, according to the OD of the sample
The corresponding concentration is found by the standard curve; multiplied by the dilution factor; or the linear regression equation of the standard curve is calculated from the concentration of the standard and the OD value, and the OD value of the sample is substituted into the equation to calculate the sample concentration, and then multiplied by The dilution factor is 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. Within and between batches should be less than 9% and 11%, respectively
examination range:
3 pmol/L -70 pmol / L
Storage conditions and validity period:
1. Store the kit: 2-8 ℃.
2. Validity: 6 months
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Human Cholic acid (CA)
FOR RESEARCH USE ONLY
Drug Names
Generic Name: Human Cholic acid (CA) ELISA Kit.
Purpose
This kit allows for the determination of CA concentrations in Human serum, blood plasma,
and other biological fluids.
Principle of the assay
The kit assay Human CA level in the sample, use Purified Human CA antibody to coat
microtiter plate wells, make solid-phase antibody, then add CA to wells, Combined CA antibody
which With HRP labeled, 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 CA in the samples is then determined by comparing the OD of the samples to
the standard curve.
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Materials provided with the kit
Materials provided with
the kit
48determinations 96 determinations Storage
User manual 1 1
Closure plate membrane 2 2
Sealed bags 1 1
Microelisa stripplate 1 1 2-8 ℃
Standard: 90 pmol / 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 plasma as 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.
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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. extract as 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 dilution 50μl 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 dilution 50μl 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 dilution 50μl 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
dilution 50μl 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, (density: 60 pmol / L, 40 pmol / L, 20 pmol / L, 10 pmol / L, 5 pmol / 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, discard Liquid, dry by swing, add washing buffer
to every well, still for 30s then drain, repeat 5 times, dry by pat.
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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 diluente and 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.
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Calculate
Assay range
3 pmol/L -70 pmol / L
Storage and validity
1. Storage: 2-8 ℃.
2. validity: six months.
Take the standard density as the horizontal, the OD
value for the vertical, draw the standard curve on graph
paper, Find out the corresponding density according to the
sample OD value by the Sample curve, multiplied by the
dilution multiple, or calculate the straight line regression
equation of the standard curve with the standard density and
the OD value, with the sample OD value in the equation,
calculate the sample density, multiplied by the dilution factor,
the result is the sample actual density.
This chartis for reference only

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