Working principle of B-factor assay kit

The B-factor assay kit is mainly based on the principle of enzyme-linked immunosorbent assay (ELISA), which detects the content of B-factor in the sample through antigen antibody specific binding reaction. Its core working principle and process are as follows:.

Enzyme labeling technology amplifies signals

Enzyme labeled secondary antibodies (such as horseradish peroxidase, HRP) are added to form a "solid-phase antibody B factor enzyme labeled secondary antibody" complex with already bound factor B.

. Enzyme markers can catalyze substrate coloration, and the concentration of factor B can be quantitatively analyzed by color intensity.

Color reaction and quantitative analysis

Adding a substrate (such as TMB), the enzyme catalyzes the substrate to produce a colored product (such as blue), which turns yellow under acidic conditions.

. The color depth is directly proportional to the concentration of factor B. The absorbance (OD value) is measured by an enzyme-linked immunosorbent assay (ELISA) reader, and a standard curve is drawn to calculate the content of factor B in the sample.

2. Operating Procedure (Taking Double Antibody Sandwich Method as an Example)

Sample Preparation

Serum/Plasma: Centrifuge the supernatant to avoid repeated freezing and thawing.

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Tissue homogenate: Rinse the tissue with pre cooled PBS, cut and homogenize, centrifuge to obtain the supernatant.

. Cell culture supernatant: Centrifuge to remove cell debris and take the supernatant for detection.

Sample addition and incubation

Add standard samples (gradient dilution) and test samples to the coated plate, and incubate to allow the antigen to bind to the solid-phase antibody.

. Wash to remove unbound components and reduce non-specific interference.

Add enzyme labeled secondary antibody

Add HRP labeled secondary antibody and incubate to form a complex.

. Wash again to remove unbound enzyme-linked antibodies.

Color development and termination

Add substrates A and B, and incubate in the dark for color development.

. Add termination solution (such as sulfuric acid), and the color changes from blue to yellow.

Result determination

OD value was measured at 450nm wavelength using an enzyme-linked immunosorbent assay (ELISA) reader.

. Calculate the concentration of factor B in the sample based on the standard curve.

III. Key Technical Features

High Sensitivity

Enzyme catalyzed signal amplification, capable of detecting factor B as low as ng/mL, suitable for early disease diagnosis or micro sample analysis.

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High specificity

Antigen antibody specific binding, avoiding cross reactivity and ensuring accuracy of results.

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Easy to operate

Standardized process, matching kit containing all necessary components, suitable for rapid detection in clinical or scientific research.

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Quantitative analysis

Absolute quantification is achieved through standard curves, providing objective data support for disease diagnosis.

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IV. Application Scenarios

Complement system function evaluation: detecting the level of factor B reflects the activity of the bypass pathway, assisting in the diagnosis of hereditary angioedema (HAE), paroxysmal nocturnal hemoglobinuria (PNH) and other diseases.

. Autoimmune disease monitoring: Overactivation of factor B in patients with systemic lupus erythematosus (SLE) may exacerbate inflammation, and detecting its levels can help evaluate disease activity. Drug development: Verify the efficacy of complement system inhibitors (such as Eculizumab) and monitor changes in factor B before and after treatment.