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Instruction manual of fish citric acid (CS) ELISA Kit

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Brief introduction


? Fish citric acid CS Kit ELISA)

an instruction manual

?

l? The kit is used for quantitative detection of serum, plasma, tissue, cell supernatant and related liquid samples in vitro Fish citric acid CS )The content of.

l? term of validity: 6 months

l? Storage conditions: two -8℃

l? This kit is only used for in vitro study, not for clinical diagnosis

?

Experimental principle

One step sandwich enzyme-linked immunosorbent assay (ELISA) was used( ELISA)。 Go ahead and pack cover Fish citric acid CS )The antibody was captured in the coated micropore, and the sample, standard substance, and HRP labeled antibody was incubated and washed thoroughly. TMB was used as the substrate for color development. TMB was converted into blue under the catalysis of peroxidase and finally yellow under the action of acid. The depth of the color and the Fish citric acid CS )Present Positive correlation. The enzyme labeled instrument was used to determine the content of The absorbance (OD value) was measured at 450 nm and the concentration of the sample was calculated.

?

Sample processing and requirements

one ? serum The whole blood sample should be kept at room temperature two Hours or 4℃ After the night 1000g centrifugal twenty The supernatant can be detected, or the specimen can be placed in the -20℃ or -80℃ Storage, but avoid repeated freezing and thawing.
two ? plasma : available EDTA Or heparin was used as anticoagulant thirty Minutes on 2 - 8°C 1000g centrifugal twenty Minutes, or place the specimen in the -20℃ or -80℃ Storage, but avoid repeated freezing and thawing.

3. ? Tissue homogenate : with precooled PBS (0.01M, pH=7.4) The tissue was rinsed to remove residual blood (red blood cells that split in the homogenate would affect the measurement results), and the tissue was cut to pieces after weighing. Compare the shear structure with the corresponding volume PBS (generally press 1:9 Weight to volume ratio, e.g 1g Corresponding to the tissue samples 9mL Of PBS The specific volume can be adjusted according to the needs of the experiment and recorded. Recommended in PBS Add protease inhibitor to glass homogenizer and grind it on ice. In order to further lyse the tissue cells, the homogenate can be broken by ultrasound or freeze-thaw repeatedly. Finally, the homogenate was mixed with five thousand × g centrifugal 5~10 Minutes, take the supernatant for detection.

four . ? Cell culture supernatant or other biological specimens 1000g centrifugal twenty The supernatant can be detected, or the specimen can be placed in the -20℃ or -80℃ Storage, but avoid repeated freezing and thawing.
Note: hemolysis of the sample will affect the final test results, so it is not suitable to carry out this test on hemolytic samples.

Specimen treatment

1. ? We are not responsible for the consumption of the sample before using the kit.

2. ? Before the experiment, the sample content should be predicted. If the sample concentration is too high, the sample should be diluted to make the diluted sample meet the detection range of the kit, and then multiply the corresponding dilution multiple. Specimen use 0.01mol/L Of PBS dilution (PH=7.0-7.2)

3. ? If the samples are not included in the samples listed in the instruction manual, it is recommended to carry out pre experiment to verify its effectiveness, and pay attention to the retention of samples.

4. ? Tissue homogenates or cell extracts prepared with chemical lysates may be caused by the introduction of certain chemicals ELISA The deviation of experimental results.

5. ? If the sample is cell culture supernatant, it may not be detected due to many interference factors, such as cell state, cell number, sampling time, etc.

6. ? Some natural or recombinant proteins, including prokaryotic and eukaryotic recombinant proteins, may not be detected because they do not match the detection and capture antibodies used in this product.

7. ? It is suggested that fresh samples should be used. If the storage time is too long, protein degradation or denaturation may lead to deviation of experimental results.

?

Reagents and equipment not required

1.? Microplate reader( 450nm

2.? High precision sampler and gun head: 0.5-10uL 2-20uL 20-200uL 200-1000uL

3.? 37℃ Incubator

4.? Distilled water or deionized water

?

Kit composition

name

ninety-six Hole configuration

forty-eight Hole configuration

remarks

Microplate

eight hole × twelve strip

eight hole × six strip

nothing

Standard

zero three mL*6 Tube

zero three mL*6 Tube

nothing

Sample diluent

6mL

3mL

nothing

Detection of antibodies -HRP

10mL

5mL

nothing

20× Washing buffer

25mL

15mL

Dilute according to the instructions

substrate A

6mL

3mL

nothing

substrate B

6mL

3mL

nothing

Termination fluid

6mL

3mL

nothing

Sealing film

two Zhang

two Zhang

nothing

instructions

one share

one share

nothing

Self sealing bag

one individual

one individual

nothing

prepare notes

1. ? The concentration of standard substance was as follows: twenty-four twelve six three one point five zero U/L

2. ? After a large number of normal samples were tested, the normal concentration values of the samples were within the detection range provided by the kit fifty μL Sample loading is enough. When some sample values exceed the maximum standard concentration, the sample diluent can be used for appropriate dilution before the experiment.

?

matters needing attention

1.? In order to ensure the accurate results, the temperature and time should be strictly followed. All reagents must reach room temperature before use 20-25℃ Refrigerate the reagent immediately after use.

2.? Incorrect plate washing can lead to inaccurate results. Before adding the substrate, make sure that the liquid in the hole is dried as much as possible. Do not let the micropores dry out in the process of warm cultivation.

3.? Eliminate the residual liquid and finger print at the bottom of the plate, otherwise the effect will be affected OD Value.

4.? The substrate solution should be colorless or very light color, and the substrate solution that has turned blue cannot be used.

5.? Avoid cross contamination of reagents and samples to avoid wrong results.

6.? Avoid direct exposure to strong light during storage and incubation.

7.? After balancing to room temperature, open the sealing bag to prevent water droplets from condensing on the cold strip.

8.? Any reaction reagent should not contact with bleaching solvent or strong gas emitted by bleaching solvent. Any bleaching component will destroy the biological activity of the reagent in the kit.

9.? Expired products cannot be used.

10.? If there is a risk of disease transmission, all samples should be managed and treated according to the specified procedures

?

Reagent preparation

The kit should be taken out from the refrigerated environment and should be balanced at room temperature before use.

two Dilution of washing buffer solution: distilled water as per one twenty Dilution, i.e one share 20× Washing buffer solution adding nineteen Parts distilled water.

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Operation steps

one ?? Equilibrium from room temperature 20min After that, take out the required aluminum foil bag 4℃

two ?? The standard sample hole and sample hole are set, and the standard sample hole is added with different concentrations of standard substance 50μL

three ?? Sample hole in plus enter Sample to be tested five 0μL The blank hole is not added.

four ?? In addition to the blank hole, horseradish peroxidase was added into each well of the standard sample hole and the sample hole( HRP )Labeled detection antibody 100μL The reaction hole was sealed with a sealing plate film, 37℃ Water bath or incubator warming 60min

five ?? Discard liquid, pat dry on absorbent paper, and fill each hole with detergent three hundred and fifty μL , stand still 1min Shake off the washing solution, pat dry on the absorbent paper, and repeat the washing five Times (also can be washed by washing machine).

six ?? Substrate is added to each well A B various 50μL 37℃ Incubation in dark 15min

seven ?? Add the termination solution into each hole 50μL 15min Inside, in 450nm At the wavelength, the OD Value.

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Calculation of experimental results

with Of the standard OD value Is the abscissa, Concentration of standard substance Value is ordinate, which is on the coordinate paper Or draw with relevant software standard curve And get Linear regression equation The OD The value is substituted into the equation to calculate the sample Of concentration

?

Performance of kit

1.? ? Detection range 0.75 U/L ?24 U/L

2.? ? Sensitivity: the lowest detection concentration is less than zero point one ? U/L

3.? ? Specificity: no cross reaction with other soluble structural analogues.

4.? ? Repeatability: the coefficient of variation within the plate is less than ten % ? The coefficient of variation between plates is less than one five % ?

?

explain

1. ? Due to the existing conditions and the level of science and technology can not fully identify and analyze all raw materials provided by all suppliers, this product may have certain quality and technical risks.

2. ? Make sure that the sufficient preparation of the reagents and the experimental results are closely related to the laboratory environment.

3. ? Different batches of the same product may have some differences, such as detection limit, sensitivity and color development time. Please carry out the experiment according to the instructions in the kit. The electronic manual on the website is only for reference.

4. ? Only when all the matching reagents of this kit are used can the detection effect be guaranteed, and the products of other manufacturers cannot be mixed. The best detection results can be obtained only by strictly following the experimental instructions of this kit.

?

Question and answer

If the experimental effect is not good, please take photos of the color development results in time, keep the strips and unused reagents, and keep them properly, and then contact our technical support to solve the problem for you. You can also refer to the following materials:

problem

Possible causes

Solution

Standard curve deviation

Inadequate absorption and washing

Adequate absorption and washing

Inaccurate transfer

Check and correct the pipette

Low precision

Inadequate washing

Wash and soak thoroughly according to instructions

Insufficient mixing and insufficient reagent absorption

Mix well and absorb the reagent

Reusing suction head, container and film covering

When using the sampler, replace the syringe with a new suction head, use a new container and cover the film

Inaccurate sample addition

Check and correct the pipette

O.D Low value

The amount of reagent added per well is not accurate

Correct the pipette and add reagent accurately

Incorrect incubation time

Ensure sufficient warm breeding time

The temperature of incubation is not correct

The reagent should be balanced to room temperature and accurate incubation temperature should be ensured

Enzyme marker or substrate failure

By mixing the enzyme marker and substrate, the color should appear quickly to check

No termination solution was added

Add the termination solution according to the instruction

Over reading time reading

Read within the reading time recommended in the manual

Sample value

Incorrect sample storage

Store samples correctly and use fresh samples for experiments

Incorrect sample collection and handling methods

Adopt the correct method of sample collection and treatment

The content of the substance to be tested is low in the sample

Using fresh samples, repeat the experiment

?


The kit is used for in vitro quantitative detection of fish citric acid (CS) in serum, plasma, tissue, cell supernatant and related liquid samples.
Expiry date: 6 months
Storage conditions: 2-8 ℃
The kit is only used for in vitro research, not for clinical diagnosis

Experimental principle
One step sandwich ELISA was used in the kit. Samples, standard samples and HRP labeled antibodies were added to the coated micropores in advance, and then incubated and washed thoroughly. TMB was used as the substrate for color development. TMB was converted into blue under the catalysis of peroxidase and finally yellow under the action of acid. The color of citric acid is positively correlated with the color of the fish. The absorbance (OD value) was measured at 450 nm wavelength by enzyme labeled instrument, and the concentration of the sample was calculated.

Sample processing and requirements
1. Serum: the whole blood sample should be placed at room temperature for 2 hours or 4 ℃ overnight, and centrifuged at 1000g for 20 minutes. The supernatant can be detected, or the sample can be stored at - 20 ℃ or - 80 ℃, but repeated freezing and thawing should be avoided.
2. Plasma: EDTA or heparin can be used as anticoagulant. The samples should be centrifuged at 2-8 ° C 1000g for 20 minutes within 30 minutes after collection, or stored at - 20 ℃ or - 80 ℃, but repeated freezing and thawing should be avoided.
3. Tissue homogenate: rinse the tissue with pre cooled PBS (0.01M, pH = 7.4) to remove the residual blood (red blood cells in the homogenate will affect the measurement results), and cut the tissue into pieces after weighing. Cut the tissue and the corresponding volume of PBS (generally according to the weight volume ratio of 1:9, for example, 1g tissue sample corresponds to 9ml PBS, the specific volume can be adjusted according to the needs of the experiment, and the record should be made. It is recommended to add protease inhibitor into PBS) into glass homogenizer and grind it on ice. In order to further lyse the tissue cells, the homogenate can be broken by ultrasound or freeze-thaw repeatedly. Finally, the homogenate was centrifuged at 5000 × g for 5-10 minutes, and the supernatant was detected.
4. Cell culture supernatant or other biological samples: centrifuged at 1000g for 20 minutes, the supernatant can be detected, or the specimens can be stored at - 20 ℃ or - 80 ℃, but repeated freezing and thawing should be avoided.
Note: hemolysis of the sample will affect the final test results, so it is not suitable to carry out this test on hemolytic samples.

Specimen treatment
1. The company is only responsible for the kit itself, not for the sample consumption caused by the use of the kit. Please fully consider the possible use of samples and reserve sufficient samples before use.
2. The sample content should be predicted before the experiment. If the sample concentration is too high, the sample should be diluted to make the diluted sample meet the detection range of the kit, and then multiply the corresponding dilution multiple. The samples were diluted with 0.01 mol / L PBS (pH = 7.0-7.2).
3. If the tested samples are not included in the samples listed in the instruction manual, it is recommended to conduct pre experiment to verify its effectiveness, and pay attention to the retention of samples.
4. Tissue homogenate or cell extract prepared by chemical lysate may lead to the deviation of ELISA results due to the introduction of some chemicals.
5. If the sample is cell culture supernatant, it may not be detected because of many interference factors, such as cell state, cell number, sampling time, etc.
6. Some natural proteins or recombinant proteins, including prokaryotic and eukaryotic recombinant proteins, may not be detected because they do not match the detection and capture antibodies used in this product.
7. It is suggested that fresh samples should be used. If the storage time is too long, protein degradation or denaturation may lead to deviation of experimental results.

Reagents and equipment required but not provided
1. ELISA (450nm)
2. High precision sampler and gun head: 0.5-10ul, 2-20ul, 20-200ul, 200-1000ul
3.37 ℃ incubator
4. Distilled water or deionized water

Kit composition
Name 96 hole configuration 48 hole configuration remarks
The microplate was 8-well × 12, 8-well × 6, none
Standard 0.3ml * 6 tubes 0.3ml * 6 tubes none
Sample diluent 6ml 3ml none
Antibody HRP 10 ml 5ml no
20 × washing buffer 25ml, 15ml, diluted according to the instructions
Substrate a 6ml 3ml none
Substrate B 6ml 3ml none
Termination solution 6ml 3ml none
Two sheets of sealing film, two sheets of none
One copy of instruction manual and one copy of none
Self sealing bag 1 No
remarks:
1. The concentration of standard substance was 24, 12, 6, 3, 1.5, 0 U / L
2. After a large number of normal samples were tested, the normal concentration values of the samples were within the detection range provided by the kit. During the experiment, 50 μ l sample can be directly taken for sample loading. When some sample values exceed the maximum standard concentration, the sample diluent can be used for appropriate dilution before the experiment.

matters needing attention
1. Carry out temperature cultivation in strict accordance with the specified time and temperature to ensure accurate results. All reagents must reach room temperature 20-25 ℃ before use. Refrigerate the reagent immediately after use.
2. Incorrect plate washing can lead to inaccurate results. Before adding the substrate, make sure that the liquid in the hole is dried as much as possible. Do not let the micropores dry out in the process of warm cultivation.
3. Eliminate the residual liquid and finger print at the bottom of the plate, otherwise the OD value will be affected.
4. The substrate solution should be colorless or light color, and the substrate solution that has turned blue cannot be used.
5. Avoid cross contamination of reagents and samples to avoid wrong results.
6. Avoid direct exposure to strong light during storage and incubation.
7. After balancing to room temperature, open the sealing bag to prevent water droplets from condensing on the cold strip.
8. Any reaction reagent should not contact with bleaching solvent or strong gas emitted by bleaching solvent. Any bleaching component will destroy the biological activity of the reagent in the kit.
The product cannot be used out of date.
10. If the disease is likely to spread, all samples should be well managed and the samples and detection devices should be handled according to the specified procedures.

Reagent preparation
The kit should be taken out from the refrigerated environment and should be balanced at room temperature before use.
Dilution of 20 × washing buffer: dilute distilled water by 1:20, that is, 1 part of 20 × washing buffer solution plus 19 parts of distilled water.

Operation steps
1. Take out the required strips from the aluminum foil bags after 20 minutes of room temperature balance, and put the remaining strips back to 4 ℃ with self sealing bags.
The concentration of standard sample was 50 μ L, and the standard sample was set at 2 μ L;
3. Add 50 μ l sample into the sample hole; do not add the blank hole.
4. In addition to the blank hole, 100 μ l HRP labeled antibody was added into each well of standard sample hole and sample hole. The reaction hole was sealed with sealing plate membrane, and incubated in 37 ℃ water bath or incubator for 60 min.
5. Discard the liquid, pat dry on the absorbent paper, fill each hole with washing solution (350 μ L), let stand for 1 min, throw away the washing liquid, pat dry on the absorbent paper, and wash the plate for 5 times in this way (also use washing machine to wash the plate).
6. 50 μ l substrate a and 50 μ L B were added into each well and incubated for 15 min at 37 ℃.
7. Add 50 μ l termination solution into each well, and within 15 minutes, determine the OD value of each pore at 450 nm.

Calculation of experimental results
Taking the OD value of the standard substance as the abscissa and the concentration value of the standard as the ordinate, the standard curve is drawn on the coordinate paper or by using relevant software, and the linear regression equation is obtained. The OD value of the sample is substituted into the equation to calculate the concentration of the sample.

Performance of kit
1. Detection range: 0.75 U / L – 24 U / L.
2. Sensitivity: the minimum detection concentration is less than 0.1 U / L.
Specificity: no cross reaction with other soluble analogues.
4. Repeatability: the coefficient of variation within the plate is less than 10%, and the coefficient of variation between plates is less than 15%.

explain
1. Due to the existing conditions and the level of science and technology can not fully identify and analyze all raw materials provided by all suppliers, this product may have certain quality and technical risks.
2. The final experimental results are closely related to the effectiveness of the reagent, the relevant operation of the experimenter and the experimental environment at that time. Please be sure to prepare sufficient specimen backup.
3. Different batches of the same product may have some differences, such as detection limit, sensitivity and color development time. Please carry out the experiment according to the instructions in the kit. The electronic version of the manual on the website is only for reference.
4. Only when all the matching reagents of this kit are used can the detection effect be guaranteed, and the products of other manufacturers cannot be mixed. The best detection results can be obtained only by strictly following the experimental instructions of this kit.

Question and answer
If the experimental effect is not good, please take photos of the color development results in time, keep the strips and unused reagents, and keep them properly, and then contact our technical support to solve the problem for you. You can also refer to the following materials:
Possible causes and Solutions
Standard curve difference absorption and washing insufficient absorption and washing
Pipette inaccuracy check and correct pipette
The precision is low, the washing is not enough, and the washing and soaking are sufficient according to the requirements of the manual
Insufficient mixing and insufficient reagent absorption
To reuse the syringe, container and lamination, use a new pipette, use a new container and a new film
Inspection and calibration of pipette for inaccurate sampling
O. When the D value is low, the amount of reagent added per well is not accurate. Correct the pipette and add reagent accurately
Improper warm-up time to ensure sufficient warm-up time
If the incubation temperature is not correct, the reagent should be balanced to room temperature and the accurate incubation temperature should be ensured
The failure of enzyme marker or substrate should be checked by mixing enzyme marker and substrate, and the color should appear quickly
The termination solution was not added, and the termination solution was added according to the operation steps of the manual
The reading time beyond the reading time should be read within the reading time recommended in the manual
If the sample value is not correct, store the sample correctly and use fresh sample for experiment
Incorrect sample collection and processing methods take the correct sample collection and processing methods
The content of the substance to be tested is low in the sample, fresh sample is used, and the experiment is repeated
Related products: Instruction manual of fish citric acid (CS) ELISA Kit ??

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