Instruction Manual for Mouse Leptospira IgG ELISA Kit

Instruction Manual for Mouse Leptospira IgG ELISA Kit

This kit is for research use only.

Drug Name:

Generic name: Mouse Leptospira IgG ELISA kit

purpose of usage:

This kit for qualitative determination of Leptospira IgG in mouse blood or other related tissues

Experimental principle:

This kit uses double-antibody sandwich enzyme-linked immunoassay (ELISA) to determine mouse Leptospira IgG in the specimen. Microporous plates are coated with purified mouse Leptospira IgG antibody to make solid phase antibody, which can be combined with mouse Leptospira IgG in the sample, washed to remove unbound antigen and other components and then labeled with HRP The goat anti-mouse antibody binds to form an antibody-antigen-enzyme-labeled antibody complex. 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 mouse Leptospira IgG in the specimen.

Kit composition:

1

20 times concentrated washing liquid

30ml × 1 bottle

7

Stop solution

6ml × 1 bottle

2

Enzyme reagent

6ml × 1 / bottle

8

Standard (positive control)

0.5ml × 1 bottle

3

Enzyme coated plate

12 holes × 8

9

Standard dilution (negative control)

0.5ml × 1 bottle

4

Sample diluent

6ml × 1 bottle

10

Instructions

1 serving

5

Developer A liquid

6ml × 1 bottle

11

Sealing film

2 sheets

6

Developer B liquid

6ml × 1 bottle

12

sealed bag

1

Specimen processing and requirements:

1. Specimen processing: (1) Serum or plasma: can be directly used for detection

(2) Cell culture: remove the cell culture fluid, collect the cells in the culture bottle and add them to the centrifuge tube.

Discard the supernatant at 2500 rpm for 5 minutes. Add 500 μl of physiological saline to the cell mass and incubate for 1 hour at room temperature (vortex mixing from time to time) to be tested.

2. Specimen requirements: samples containing NaN3 cannot be detected because NaN3 inhibits horseradish peroxidase (HRP) activity.

Steps:

1. Numbering: serially number the corresponding microwells of the sample, each plate should be set with 2 wells for negative control, 2 wells for positive control, and 1 well for blank control (the blank control well does not add sample and enzyme reagent, the rest of the steps are the same)

2. Add sample: add 50μl of negative control and positive control (standard) 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 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 plate and incubate at 37 ° C for 30 minutes.

4. Mixing solution: add 20 times concentrated washing liquid to distilled water to 1000ml

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 5 times and pat dry.

6. Add enzyme: add 50μl of enzyme label reagent (or one drop) to each well, except for blank wells.

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

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

9. Color development: add 50μl (or one drop) of developer A to each well, then add 50μl (or one drop) of developer B, mix gently, and develop color at 37 ℃ for 15 minutes in the dark

10. Termination: Add 50μl of stop solution (or one drop) to each well to stop the reaction (in this case, 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.

Result judgment:

Test effectiveness: the average of positive control wells ≥1.00; the average of negative control wells ≤0.15

Calculation of critical value (CUT OFF): critical value = average value of negative control wells + 0.20

Negative judgment: the OD value of the sample <CUT OFF is negative for mouse Leptospira IgG

Positive judgment: the sample OD value ≥ critical value (CUT OFF) is positive for mouse Leptospira IgG

Precautions

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

2. 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.

3. 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.

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

8. If there is any difference with the English manual, the English manual shall prevail ..

specification:

96 servings / box

Storage conditions and validity period

1. Kit storage :; 2-8 ℃.

2. Validity: 6 months

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