What Is a Serum ELISA?

There are many kinds of ELISA kits in China, such as: ELISA detection kit, ELISA Kit, enzyme-linked immunoassay kit, enzyme-linked immunosorbent assay kit, enzyme-linked immunoassay kit, enzyme-free kit, etc. Its name is ELISA detection kit, enzyme-linked immunosorbent assay kit and so on.

elisa kit

First, high-efficiency, sensitive and specific antibodies;
Second, stable repeatability and reliability;
Solid phase carrier with good adsorption performance, low blank value, and high pore bottom transparency;
Fourth, suitable for serum, plasma, tissue homogenate, cell culture supernatant,

It includes the following steps:

1) Computer screening of epitopes;

2) preparation of antigens;

3) Collection of serum;

4) Establishment of indirect ELISA method;

5) Verification of the sensitivity and specificity of the indirect ELISA method;

6) Stability verification of the kit;

7) Perform clinical tests on the slaughterhouse. The method is simple, fast, high sensitivity, strong specificity, qualified stability and good repeatability. The kit prepared by the invention can effectively detect the hepatitis E virus infected pigs, is suitable for the detection of a large number of diseased samples, can be used for the rapid diagnosis of pig hepatitis E, and can prevent and control the epidemic and resistance of pig hepatitis E Hepatitis E virus is transmitted from pig to human.

1. Serum: Avoid any cell stimulation during operation. Use pyrogen- and endotoxin-free test tubes. After the blood was collected, the red blood cells were quickly and carefully separated by centrifugation at 1000 × g for 10 minutes.

2. Plasma: EDTA, citrate, heparin plasma can be used for detection. Centrifuge at 1000 × g for 30 minutes to remove particles.

3. Cell supernatant: Centrifuge at 1000 × g for 10 minutes to remove particles and polymers.

4. Tissue homogenate: Tissue is mashed by adding an appropriate amount of physiological saline. Centrifuge at 1000 × g for 10 minutes and take the supernatant.

5. Storage: If the sample is not used immediately, it should be divided into small parts -70 to avoid repeated freezing. Do not use hemolysis or hyperlipidemia whenever possible. If there are a lot of particles in the serum, centrifuge or filter before testing. Do not thaw at 37 ° C or higher. Thaw at room temperature and ensure that samples are thawed uniformly and adequately.

This IBL kit can be used for the quantitative detection of interleukin-6 in mouse serum, EDTA plasma, and cell supernatant. Component 1 Pre-coated plate: anti-mouse interleukin-6 rabbit IgG, affinity purification 96T
2 Enzyme-labeled antibody: (30-fold concentrated) HRP-labeled anti-mouse interleukin-6 rabbit IgG, affinity purified 0.4mL x 1
3 Standards: Recombinant Mouse Interleukin-6 0.5mL x 2
4 EIA buffer: containing 1% BSA, 0.05% Tween 20 BPS 30mL x 1
5 Labeled antibody dilution: 1% BSA, 0.05% Tween 20 BPS 12mL x 1
6 Developer: TMB substrate solution 15mL x 1
7 Stop solution: 1N sulfuric acid 12mL x 1
8 Concentrated washing solution: (40x concentrated) Contains 1% BSA, 0.05% Tween 20 BPS 50mL x 1 Operating Instructions 1 Equipment required for the experiment (but not provided with the kit) Microplate reader (450nm) Micropipette and Nozzle graduated cylinder and beaker Deionized water refrigerator (4 ° C) Graph paper (log / log) Water-absorbing paper test tube (for standard dilution) Incubator (37 ° C ± 1 ° C) Wash bottle (for washing plate) once Reagent tubes (for concentrating enzyme-labeled antibodies and developers)

Double antibody sandwich

1. Coating: Dilute the antibody with 0.05MPH9. carbonate coating buffer to a protein content of 1-10 g / ml. Add 0.1 ml to the reaction wells of each polystyrene plate and incubate at 4 ° C overnight. The next day, the solution in the well was discarded and washed 3 times with washing buffer for 3 minutes each time. (Referred to as washing, the same below).

2. Adding a sample: Add 0.1ml of a certain dilution of the sample to be tested to the coated wells, and incubate at 37 ° C for 1 hour. Then wash. (Make blank holes at the same time,

The development of clinical measurement technology mainly lies in the development of methodology, and the development of methodology depends on the continuous advancement of reagent production technology and the application of type markers. Molecular biology is and will surely give a comprehensive and thorough understanding of the entire life science. Its impact on the development of immunoassay technology is also direct and effective.

Reagent

(1) Coating buffer (pH 9.60.05M carbonate buffer):

Na2CO3 1.59 g

NaHCO3 2.93 g

Add distilled water to 1000ml

(2) Washing buffer (PH7.4PBS): 0.15M

KH2PO4 0.2 g

Na2HPO4 · 12H2O 2.9 g

NaCl 8.0 g

KCl 0.2 g

Tween-20 0.05% 0.5ml

Add distilled water to 1000ml

(3) Diluent:

Make a comparison

In the formal test, the test conditions should be controlled with positive control and negative control respectively, and the samples to be tested should be made in duplicate to ensure the accuracy of the experimental results. Sometimes the background is high, indicating that there is a non-specific reaction, and can be blocked with sheep serum, rabbit serum or BSA.

Selection of experimental conditions

In ELISA, it is important to choose the conditions for each experiment, including:

(1) Choice of solid phase support: Many materials can be used as solid phase support, such as polyvinyl chloride, polystyrene, polyacrylamide, and cellulose. Its form can be a recessed flat plate, a test tube, beads, and the like. At present, a 40-well polystyrene concave well plate is commonly used. No matter what kind of vector, you can screen before use: coated with the same amount of antigen, reacted under the same experimental conditions, and observed

ELISA is a new technology in immunodiagnosis, and has been successfully applied to immunodiagnosis of infectious diseases, parasitic diseases and non-infectious diseases caused by a variety of pathogenic microorganisms. It has also been applied to the quantitative determination of large-molecule antigens and small-molecule antigens. According to the results that have been used, ELISA methods are considered to be sensitive, specific, simple, fast, stable, and easy to automate. Not only is it suitable for the examination of clinical specimens, but because hundreds or even thousands of specimens can be inspected in one day, it is also suitable for seroepidemiological investigations. This method can be used not only to measure antibodies, but also to measure circulating antigens in body fluids, so it is also a good method for early diagnosis. Therefore, the application of ELISA method in various fields of biomedicine is expanding.

Four aspects can be summarized:

1. Localization of various intracellular components by immunoenzyme staining.

2. Study the synthesis of anti-enzyme antibodies.

3. A slight amount of immunoprecipitation is shown.

4. Quantitative detection of antigen or antibody components in body fluids.

Precautions during ELISA test

I. General rules of ELISA experiment 1. To ensure the accuracy of the pipette, the error cannot exceed 2%. This can be determined with water and electronic balances. But it is best to have a professional to correct it. 2. Each of 20ul, 50ul, 100ul, 1000ul, and row gun should be equipped. After aspiration of different liquids, the tip must be changed. Even when pipetting standards. 3. Remove the kit from the refrigerator 1 hour before the experiment, and return all the reagents to room temperature to make the results more stable. 4. Keep substrates protected from light during experiments. 5. When using a gun to suck up liquid, the speed should not be too fast, so as not to generate bubbles and make the pick-up amount inaccurate. 6. When sucking liquid, use a gun with a range close to the required amount to suck it to reduce the error. 7. When adding liquid to the enzyme standard wells, avoid contact between the pipette tip and the liquid in the well, and make the droplets on the pipette tip contact the wall of the well, and the droplets will flow down naturally. 8. After all the liquid has been added, shake the microplate in parallel on the table for 30 seconds to mix the liquid. The shaking function of the microplate reader can also be used. 9. When the bath is warmed, seal the enzyme plate with stickers or tape to prevent water evaporation. 10. When washing the plate, it should be left for 1 minute after each washing solution is added to make the washing more thorough. When there is no plate washer, after the liquid is drained, pat the plate firmly on a newspaper or wool paper. 11. When the washing solution is not enough, you can use distilled water to prepare a phosphate buffer solution with pH 7.4 and 0.02M, and add 0.1% Tween 20 as the washing solution. Add 1/1000 sodium azide for long-term storage. 12. The substrate is light-sensitive and should be prepared immediately before use. 13. Before detection, turn on the microplate reader and allow it to stabilize for more than 10 minutes. 14. The substrate is toxic, and the stop solution is corrosive to the skin. Avoid contact as much as possible. 15. The samples to be tested must be clarified, otherwise the results will be affected. 16. Warm-up time should comply with the provisions of the kit. 17. Double-well experiments should be done as far as possible to ensure the accuracy of the data. 18. Samples with questionable results should be confirmed by other methods. Second, the problems mentioned below are related to our company's ELISA products and solutions 1. After adding the reaction stop solution, there is no absorbance value or the absorbance value is low. a. Reason: No enzyme label was added. Solution: Please follow the operation steps. b. Reason: The contents of the kit were not fully returned. Solution: Make sure the contents of the kit are warmed before proceeding. c. Reason: The room temperature is too low. Solution: If the room temperature is too low (<19 ), please proceed to step 4 to extend the incubation time. d. Reason: The cleaning solution was not used for cleaning. Solution: Please use the cleaning solution provided in the kit. e. Reason: The expiration date of the kit has expired. Solution: Please replace the kit with the one that is still valid. 2. The standard curve has poor linearity or poor parallelism. a. Reason: The incubation position is not well protected from light or interfered by air flow. Solution: Make sure that the incubation location is neither light nor air (such as inside a drawer). b. Reason: The operation time is too long. Solution: Please operate after the method is proficient. c. Reason: mutual contamination between micropores. Solution: When adding samples, be careful not to splash the microwells to avoid contamination of other microwells. d. Reason: Inconsistent amounts of standards or enzyme markers were added. Solution: Use a calibrated pipette and make sure it has a high degree of adhesion to the tip. e. Reason: The method of adding samples or reagents is incorrect. Solution: When adding samples or reagents, do not touch the tip with the microwell. f. Reason: There is a problem in the washing process (such as the pipeline is blocked, and the next step is not performed immediately after washing the plate). Solution: Please monitor the plate washing process of the plate washer at any time, and proceed to the next step immediately after washing. 3. The absorbance is high (or the linearity is not steep enough). a. Reason: The room temperature is too high (> 25 ° C). Solution: If the indoor temperature cannot be reduced, shorten the incubation time of step 4 appropriately. b. Cause: The substrate solution is contaminated. Solution: If the substrate solution is found to be light blue, please do not use it again. c. Reason: The reaction time is too long. Solution: Please control the response time. d. Reason: The enzyme label has contaminated other reagents in the box. Solution: The used tips should be discarded immediately to avoid mutual contamination between the reagents, and all containers contacted by the reagents (especially the enzyme label and substrate solution) should be cleaned immediately. (Soak in bleach first, then rinse with water to ensure no residue) 4. The absorbance of the negative standard is lower than that of the positive standard. a. Reason: The reagents in the microwells are not sufficiently mixed. Solution: After the reagents are added, tap the sides of the disk to make them fully mixed. b. Reason: A problem occurred during the washing process (same as 2-f.). Solution: Please refer to 2-f. c. Reason: The amount of the added sample or enzyme marker is not consistent. Solution: Please refer to 2-d.