Human Fc quantification kit - human Fc定量试剂盒

This kit employs homogeneous time-resolved fluorescence technology and can be used to detect Human IgG antibodies in cell culture supernatants. The kit includes an antibody-labeled donor Eu (anti-Human IgG-Eu) and a Human IgG-labeled acceptor (Human IgG-Ac). The anti-Human IgG-Eu binds to Human IgG-Ac, and when excited by 320 nm light, the donor transfers energy to the acceptor, which then emits light at a specific wavelength (665 nm).

Human IgG antibodies in the sample competitively bind to anti-Human IgG-Eu, resulting in a signal inversely proportional to the concentration of Human IgG in the standard. This allows quantification of Human IgG in the sample based on a standard curve. This homogeneous assay requires no washing and enables accurate quantification of total Human IgG.

Note: Aliquot immediately after first thaw and store at recommended temperatures. Avoid storage after dilution or repeated freeze-thaw cycles.

1. Reagent Preparation

Thaw all reagents at room temperature before use (equilibrate at room temperature for at least 30 min). The reaction volume for the 384-well shallow plate is 20 μL (reagent volumes for the reaction system are shown in Table 2). Calculate the required volume for the experiment before preparation and prepare as needed; the following preparation is for reference only, using 500 assays as an example.

Table 1. Reagent Preparation

Table 2. Preparation of Standard Working Solutions

2. Sample Addition and Controls

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

NC: Replace 5μL Human IgG-Ac with 5μL Detection Buffer, and replace sample with 10μL standard diluent.

Maximum Control: Replace sample with 10μL standard diluent.

3.Detection

Seal the plate wells with a plate sealer, centrifuge at 1000 rpm for 1 min, and incubate at room temperature for 1 h. Detect on a TR-FRET compatible microplate reader (excitation wavelength 320 nm, emission wavelengths 620 nm and 665 nm).

[Result Calculation]

1. TR-FRET Ratio Calculation

TR-FRET Ratio = 665nm / 620nm *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 and is provided only as an example. Results may vary depending on the TR-FRET compatible instrument.

Note: Recommended microplate (384-well plate, white, shallow well)