Instructions for using the Acid Glycoprotein Assay Kit

The usage method of acid glycoprotein (such as α 1-acid glycoprotein, α 1-AGP) assay kit varies depending on the detection principle and kit type. The following are the general operating steps and precautions based on enzyme-linked immunosorbent assay (ELISA) and immunoturbidimetry: Waiting for everything to be complete.

Sample processing:

Serum/plasma: Blood naturally coagulates at room temperature for 10-20 minutes, centrifuges for 20 minutes (2000-3000 rpm), and collects supernatant.

. Cell culture supernatant: Centrifuge for 20 minutes (2000-3000 rpm) to remove particles and polymers.

Other samples (such as urine, pleural and peritoneal fluid): Centrifuge according to the instructions.

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Storage: If not tested immediately, the sample should be packaged and stored at -20 ℃ or -70 ℃ to avoid repeated freezing and thawing.

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2. Standard dilution

According to the instructions, dilute the original standard with a standard dilution solution to obtain standard solutions of different concentrations (such as 400 ng/mL, 200 ng/mL, 100 ng/mL, etc.).

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3. Sample addition

Blank well: No sample or enzyme-linked immunosorbent assay is added, the rest of the steps are the same.

. Standard well: Add 50 μ L of diluted standard to each well.

Test sample well: First add 40 μ L of sample diluent, then add 10 μ L of test sample (with a final dilution of 5 times).

Sample addition technique: 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

After sealing the plate with a sealing film, incubate at 37 ℃ for 30-60 minutes (specific time according to the instructions).

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5. Washing

Carefully remove the sealing film, discard the liquid, and shake dry.

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Fill each well with detergent, let it stand for 30 seconds, discard it, repeat washing 3-5 times, and pat dry.

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6. Enzyme addition

Add 50 μ L of enzyme labeled reagent to each well (except for blank wells), gently shake and mix well.

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7. Incubate and wash again

Repeat steps 4 and 5.

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8. Color development

Add 50 μ L of color developer A to each well, then add 50 μ L of color developer B, gently shake and mix, and develop color at 37 ℃ in the dark for 10-15 minutes.

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9. Termination

Add 50 μ L of termination solution to each well to terminate the reaction (at this point, the blue color turns yellow).

10. Measurement

Zero the blank well and measure the absorbance (OD value) of each well in sequence at a wavelength of 450nm.

. The measurement should be completed within 15 minutes after adding the termination solution.

11. Result Calculation

Standard Curve: Plot the standard curve with the concentration of the standard substance as the horizontal axis and the OD value as the vertical axis.

. Sample concentration: Find the corresponding concentration on the standard curve based on the OD value of the sample, and multiply it by the dilution factor to obtain the actual concentration.

2. Operation steps of immunoturbidimetry

1. Experimental preparation

Reagents and instruments: Ensure that the reagent kit (including reagents R1, R2, standards, etc.), fully automatic biochemical analyzer (or spectrophotometer), etc. are complete.

. Sample processing: Same as ELISA method.

2. Reagent Preparation

Reagents R1 and R2 are liquid reagents that can be used directly.

. If dilution is required, follow the instructions in the manual.

3. Parameter settings

Main wavelength: 340nm.

Reaction method: endpoint method.

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Reaction temperature: 37 ℃. Reaction direction: upward.

4. Operation steps

Take 3 μ L of sample, add 225 μ L of R1, mix well, incubate at 37 ℃ for 3-5 minutes, and read the first point absorbance A1.

Add 75 μ L of R2, mix well, incubate at 37 ℃ for 5 minutes, and read the second point absorbance A2.

Calculate Δ A=A2-A1.

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5. Result Calculation

Calculate the concentration of alpha 1-acid glycoprotein in the sample based on the standard curve or formula provided in the instruction manual.

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III. Precautions

Reagent Balance: After removing the reagent kit from the refrigerator, it needs to be equilibrated at room temperature for 30 minutes before use.

. Accurate sample addition: Use a sampler to add samples and avoid experimental errors caused by inaccurate sample amounts. Incubation time: Strictly control the incubation time to ensure consistent incubation time for all reaction wells. Thoroughly washed: Insufficient washing can lead to a decrease in accuracy or an incorrect increase in OD value.

Avoid contamination: Avoid cross contamination during the experiment and use disposable suction tips.

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Result interpretation: If the OD value of the sample is higher than the OD value of the first well of the standard, it needs to be diluted with sample diluent before measurement, and the calculation should be multiplied by the total dilution factor.

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Waste disposal: All samples, detergents, and waste must be treated as infectious agents.

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