Human Fc quantification kit - Human Fc quantitative kit

This kit employs homogeneous time-resolved fluorescence technology and can be used to detect Human IgG antibodies in cell culture supernatants. The kit contains an antibody-labeled donor Eu (anti-Human IgG-Eu) and a Human IgG-labeled acceptor (Human IgG-Ac). When the anti-Human IgG-Eu binds to Human IgG-Ac, excitation of the donor at 320 nm transfers energy to the acceptor, causing it to emit light at a specific wavelength (665 nm).

Human IgG antibodies in the sample can competitively bind to anti-Human IgG-Eu, resulting in a signal that is inversely proportional to the concentration of Human IgG in the standards. Therefore, the amount of Human IgG in the sample can be quantified based on the standard curve. This homogeneous assay allows for accurate quantification of total Human IgG without the need for washing.

Note: After the first thaw, aliquot immediately and store at the recommended temperature; avoid storing diluted solutions or subjecting them to repeated freeze-thaw cycles.

1,Reagent preparation

Before use, allow all reagents to thaw at room temperature (equilibrate at room temperature for at least 30 minutes). The reaction volume in a 384-well plate is 20 μL (the reagent volumes for the reaction system are shown in Table 2), and when preparing… Previously, the required volume for the experiment was calculated and prepared as needed; the following preparation instructions are for reference only, using 500 reactions as an example.

Table 1.Reagent preparation

Table 2. Preparation of Standard Working Solutions

2,Sample addition and controls

Order of sample addition: 10muL standard or sample; 5muL Anti-Human IgG-Eu; 5muL Human IgG-Ac is sequentially added to a 384-well plate. (Note: Human IgG-Ac is added last.)

NC: Use 5muL'sDetection BufferInstead of 5muL Human IgG-Ac, 10muL Diluent BufferSubstitute sample.

Maximum value comparisonComparison: Use 10muL's Diluent BufferSubstitute sample.

3.Detection

Seal the plate wells with a sealing film, centrifuge at 1000 rpm for 1 minute, and incubate at room temperature for 1 hour. Detect using a microplate reader compatible with TR-FRET (excitation wavelength of 320 nm, emission wavelengths of 620 nm and 665 nm).

[Result Calculation]

1. TR-FRET Ratio Calculation

TR-FRET Ratio = 665 nm / 620 nm * 10000

2. Calculate CV (%)

CV (%) = Standard Deviation / Mean Ratio × 100%

[Data Example]

Please note that the following data cannot replace the data obtained in experiments; it is provided only as an example, and results may vary depending on the TR-FRET-compatible instrument.

Note: RecommendedMicroplate (384-well plate, white, shallow wells)