Human Fc quantification kit - human Fc定量试剂盒

Product Specification Host Human Stability & Storage -80℃ Background 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. Components Component Concentration 100T 500T 2500T 10000T Storage Temperature Anti-Human IgG-Eu* 50× 10μL 50μL 250μL 1000μL -80℃ Human IgG-Ac* 50× 10μL 50μL 250μL 1000μL -80℃ IgG Standard 1mg/ml 5μL 10μL 50μL 200μL -80℃ Assay Buffer 1× 1.5mL 6ml 30mL 120mL -80℃ Standard Diluent 1× 1.5mL 6ml 30mL 120mL -80℃ Note: Aliquot immediately after first thaw and store at recommended temperatures. Avoid storage after dilution or repeated freeze-thaw cycles. Protocol 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 Reagent Name Preparation Volume per Assay Well (μL) Anti-Human IgG-Eu Add 2450 μL Detection Buffer to 50 μL Anti-Human IgG-Eu, mix well, and keep on standby. 5 Human IgG-Ac Add 2450 μL Detection Buffer to 50 μL Human IgG-Ac, mix well, and keep on standby. 5 IgG Standard Take 10 μL of the standard stock solution, add 115 μL of standard diluent, mix thoroughly. Standard curve dilutions are as follows. 10 Table 2. Preparation of Standard Working Solutions Standard Working Solution Concentration (ng/mL) Preparation Method ① 80000.0 10μL standard original stock solution + 115μL standard diluent ② 20000.0 20μL ①+60μL standard diluent ③ 5000.0 20μL ②+60μL standard diluent ④ 1250.0 20μL ③+60μL standard diluent ⑤ 312.5 20μL ④+60μL standard diluent ⑥ 78.125 20μL ⑤+60μL standard diluent ⑦ 19.531 20μL ⑥+60μL standard diluent ⑧ 4.88 20μL ⑦+60μL standard diluent ⑨ 1.220 20μL ⑧+60μL standard diluent ⑩ 0.305 20μL ⑨+60μL standard diluent Blank 0.0 60μL standard diluent 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. Std Concentration (ng/mL) Standard Ratio CV% ① 80000 163.65 7.65% ② 20000 255.25 0.86% ③ 5000 467.2 0.29% ④ 1250 1581 6.71% ⑤ 312.5 3420.5 4.53% ⑥ 78.125 3980.1 3.49% ⑦ 19.531 3704.5 0.02% ⑧ 4.883 3785.5 3.90% ⑨ 1.221 3673.5 1.56% ⑩ 0.305 3940.5 8.06% Blank 0 3965 3.92% NC NC 121.75 5.52% Note: Recommended microplate (384-well plate, white, shallow well)