Human Salmonella (Salmonella) Elisa Kit

Instructions for Salmonella Elisa Kit

The Salmonella Elisa kit is for research use only.

Detection range: 96T

Purpose: This kit is used to determine the expression of Salmonella in human serum, plasma and related liquid samples.

Experimental principle: This kit uses the double antibody sandwich method to determine the expression of Salmonella in specimens. Microporous plates are coated with purified human Salmonella antibody to make solid-phase antibodies, which can be combined with Salmonella in the sample. After washing to remove unbound antigen and other components, they are then labeled with HRP (Salmonella Salmonella) antibody binds to form an antibody-antigen-enzyme-labeled antibody complex, and after thorough 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 absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and compared with the CUTOFF value to determine the presence or absence of Salmonella in the specimen.

The Salmonella Elisa kit consists of:

1 30 times concentrated washing solution 20ml × 1 bottle 7 stop solution 6ml × 1 bottle

2 Enzyme label reagent 6ml × 1 bottle 8 Positive control 0.5ml × 1 bottle

3 Enzyme label coated plate 12 well × 8 strips 9 negative control 0.5ml × 1 bottle

4 Sample diluent 6ml × 1 bottle 10 instructions 1 copy

5 Developer A solution 6ml × 1 bottle 11 2 sealing film

6 Developer B liquid 6ml × 1 / bottle 12 1 sealed bag

Specimen requirements:

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

2. The specimens 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

Steps:

1. Numbering: Number the samples corresponding to the microwells in sequence, each plate should be set with 2 wells for the negative control, 2 wells for the positive control, and 1 well for the blank control (the blank control wells do not add samples and enzyme reagents, the remaining steps are the same)

2. Add samples: add negative control and positive control 50μl to the negative and positive control wells respectively. Then add 40μl of sample diluent to the sample well, and then add 10μl of the sample to be tested. 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, gently shake to mix.

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

4. Mixing solution: Dilute 30 times concentrated washing liquid with distilled water 30 times and reserve

5. Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with the washing solution, 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, and then add 50μl of developer B, mix gently, and develop at 37 ° C in the dark for 15 minutes.

10. Termination: Add 50 μl of stop solution to each well to stop the reaction (at this time, the blue color turns 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.

Calculation and result judgment:

Calculation of cut-off value (CUT OFF): cut-off value = average value of negative control well + 0.15

Test effectiveness: the average of positive control wells ≥1.00; the average of negative control wells ≤0.10

Positive judgment: the sample with OD value ≥ critical value (CUT OFF) is positive for Salmonella

Negative judgment: The sample with OD value <cut-off value (CUT OFF) is negative for Salmonella.

Precautions

1. The operation is carried out in strict accordance with the instructions. The components of different batches of this reagent must not be mixed.

2. 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 slats should be stored in a sealed bag.

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

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

5. Please keep the substrate away from light.

6. The test result must be determined based on the reading of the microplate reader. When using dual wavelength detection, the reference wavelength is 630nm

7. All samples, washing liquid and various wastes should be treated as infectious agents. The stop solution is 2M sulfuric acid, you must pay attention to safety when using it.

Storage conditions and validity period:

1. Kit storage: 2-8 ℃.

2. Validity: 6 months