Precautions for using the acid glycoprotein assay kit

When using acid glycoprotein (such as alpha 1 acid glycoprotein) assay kits, the following precautions should be paid attention to to ensure the accuracy of experimental results and operational safety:

1. Preparation before the experiment

Reagent balance and storage

After removing the kit from the refrigerated environment (2-8 ℃), it should be equilibrated at room temperature for 15-30 minutes to avoid sudden temperature changes affecting reagent performance.

After the enzyme label coated plate is opened, the unused Flat noodles should be immediately put into a sealed bag and stored with desiccant to prevent moisture. Different batches of reagents must not be mixed and used within their shelf life. Expired reagents may become invalid.

Sample processing

Serum/plasma: Use test tubes free of pyrogens and endotoxins to avoid cell irritation.

. After collection, centrifuge at 1000-3000 rpm for 10-20 minutes to separate the supernatant. If precipitation occurs, it needs to be centrifuged again. Cell supernatant: Centrifuge to remove particles and polymers (1000 × g, 10 minutes).

Tissue homogenate: Add an appropriate amount of physiological saline, crush and centrifuge (1000-3000 × g, 10-20 minutes), and take the supernatant.

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Storage: If the sample is not used immediately, it should be packaged and frozen at -70 ℃ to avoid repeated freeze-thaw cycles.

. Thawing should be evenly done at room temperature, and heating above 37 ℃ is prohibited.

Standard dilution

The standard should be diluted in a gradient according to the instructions (such as 400 μ g/mL → 200 μ g/mL → 100 μ g/mL, etc.). The diluted standard should be discarded and cannot be stored.

. If the sample concentration exceeds the detection range of the kit (such as 10-320 μ g/mL), it needs to be diluted with a special diluent and retested. The result should be multiplied by the total dilution factor.

2. Experimental operation specifications

Sample addition and incubation

All sample addition operations require the use of a sampler and regular calibration to avoid errors.

. It is best to control the sample addition time within 5 minutes, and it is recommended to use a sampling gun when the sample size is large.

The sample should be added to the bottom of the enzyme-linked immunosorbent assay (ELISA) plate well to avoid touching the well wall. Gently shake and mix well.

. During incubation, it is necessary to seal the plate with a sealing film to prevent water evaporation. The incubation conditions (such as 37 ℃) must be strictly followed according to the instructions.

Washing and color development

When washing, it is necessary to fully pat the liquid in the hole to avoid direct contact of absorbent paper with the bottom of the hole and prevent cross contamination.

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Color developing agents (such as TMB) should be stored in the dark, and the color development time (such as 15 minutes in the dark at 37 ℃) should be strictly controlled to avoid colors that are too dark or too light.

. Termination solution (such as sulfuric acid) is sensitive to light and should be immediately measured for OD value after addition.

Result determination

The experimental results must be based on the reading of the enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450nm, and the OD values of each well must be measured after zeroing the blank wells.

. When calculating the sample concentration based on the standard curve, it is necessary to multiply it by the dilution factor (if the sample has been diluted 5 times, the result needs to be multiplied by 5).

III. Safety and Protection

Personal Protection

During the experiment, it is necessary to wear laboratory clothes and latex gloves to avoid direct contact with reagents and samples.

. Eating, drinking, smoking, or using cosmetics are prohibited in the laboratory to prevent contamination.

Waste disposal

All samples, detergents, and waste must be treated as infectious agents to avoid environmental pollution.

. The termination solution and substrate solutions A and B are corrosive and should be immediately rinsed with water upon contact.

IV. Common Problems and Solutions

Light color development

Possible reasons: insufficient incubation time, ineffective color developer, or low sample concentration.

. Solution: Extend the color development time, replace the color developer, or re dilute the sample.

Abnormal OD value

Possible reasons: The unsealed sealing film causes water evaporation during incubation, or incomplete washing leads to cross contamination.

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Solution: Ensure that the sealing film is well sealed, and fully dry the liquid in the hole during washing.

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Sample hemolysis or hyperlipidemia

Possible reasons: Improper handling during sample collection or improper sample storage.

. Solution: Re collect the sample to avoid hemolysis or hyperlipidemia, centrifuge or filter before testing.