Instructions for using the fibrinogen assay kit

The usage method of a fibrinogen assay kit (such as an ELISA kit) usually includes steps such as reagent preparation, sample collection and processing, sample addition, incubation, washing, color development, reaction termination, and result determination. The following is a detailed introduction: Termination liquid, etc.

Reagent recovery: Before use, slowly equilibrate all reagents to room temperature (18-25 ℃), and do not dissolve reagents directly at 37 ℃.

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Standard dilution: According to the instructions, use standard dilution solution to perform gradient dilution on the standard to prepare standard solutions of different concentrations.

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Dilution of detection solution: Detection solution A and detection solution B need to be diluted before use, usually diluted with detection diluent A or B in a certain ratio (such as 1:100). Dilution of washing solution: Dilute the concentrated washing solution to the working concentration with distilled water or deionized water, such as 30 fold dilution.

II. Sample Collection and Processing

Serum samples: Collect blood in test tubes that do not contain pyrogens and endotoxins, and avoid any cellular irritation during the operation.

. After collecting blood, quickly and carefully separate the red blood cells according to the centrifugation speed and time requirements (such as centrifugation at 1000 × g for 17 minutes or centrifugation at 3000 rpm for 10 minutes), and take the supernatant for testing.

Plasma samples: Select EDTA, citrate, or heparin as anticoagulants according to the sample requirements. After collecting the sample, centrifuge it at 2-8 ℃ for a specified time (such as within 30 minutes) and collect the supernatant for testing.

. The centrifugation speed and time may vary depending on the requirements of the reagent kit, such as centrifuging at 1000 × g for 60 minutes or centrifuging at 3000 rpm for 30 minutes.

Cell supernatant samples: Centrifuge to remove particles and polymers. The centrifugation speed and time may vary depending on the requirements of the kit, such as centrifugation at 1000 × g for 32 minutes or centrifugation at 3000 rpm for 10 minutes.

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Tissue homogenate sample: Crush the tissue by adding an appropriate amount of physiological saline, or wash the tissue with pre cooled PBS (0.01M, pH=7.4), weigh and cut it, and add the corresponding volume of PBS for homogenization.

. The homogenate may require further centrifugation to remove sediment, and the centrifugation speed and time may vary depending on the requirements of the kit, such as centrifugation at 5000 × g for 5-10 minutes. Sample preservation: If the sample is not used immediately, it should be divided into small portions and frozen at -20 ℃ or -80 ℃ to avoid repeated freeze-thaw cycles. Thaw at room temperature and ensure that the sample is evenly thawed.

III. Sample addition

Set standard and sample wells: add different concentrations of standard to each standard well, and add the sample to be tested to the sample well.

. Blank wells do not contain samples or enzyme-linked immunosorbent assay reagents, and all other steps are the same.

Sample amount: According to the requirements of the kit, add a certain amount of standard, sample, or enzyme-linked immunosorbent assay (ELISA) reagent to each well, such as 50 μ L or 100 μ L.

Sample addition operation: Add the sample to the bottom of the enzyme-linked immunosorbent assay plate well, try not to touch the well wall, gently shake and mix well.

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4. Incubation

Plate sealing: Seal the reaction pores with a sealing film to prevent evaporation and contamination.

. Incubation conditions: Place the sealed enzyme-linked immunosorbent assay (ELISA) plate in a 37 ℃ constant temperature incubator or water bath for a certain period of time, such as 30 minutes, 60 minutes, or 2 hours.

Fifth, washing

Hand washing the plate: suction or shake off the liquid inside the enzyme-linked immunosorbent assay plate;

; Lay several layers of absorbent paper on the experimental platform and pat the enzyme-linked immunosorbent assay (ELISA) plate downwards several times with force; Inject the recommended washing buffer into the well, soak for a certain period of time (such as 1-2 minutes), and repeat this process several times as needed.

Automatic board washing: If there is an automatic board washing machine, it should be used proficiently before being used in the formal experimental process.

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Sixth, color development

Add substrate: Add color developer A to each well first, then add color developer B, gently shake and mix well.

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Color development conditions: Place the enzyme-linked immunosorbent assay (ELISA) plate at 37 ℃ and incubate it in the dark for a certain period of time, such as 10 minutes, 15 minutes, or 30 minutes, to allow the substrate to develop color.

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7. Termination Reaction

Add termination solution to each well to terminate the enzymatic reaction.

. After adding the termination solution, the blue color will immediately turn yellow.

8. Result determination

Measure absorbance: Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance (OD value) of each well at a wavelength of 450nm.

. The measurement should be conducted within a certain period of time after adding the termination solution, such as within 15 minutes. Draw a standard curve: Using the OD value of the measured standard as the horizontal axis and the concentration value of the standard as the vertical axis, draw the standard curve on a coordinate paper or with relevant software, and obtain a linear regression equation. Calculate sample concentration: Substitute the OD value of the sample into the linear regression equation to calculate the concentration of the sample. If the sample is diluted before adding, it needs to be multiplied by the corresponding dilution factor when calculating.