太鼎生物科技/太鼎食安科技

小鼠分型IgM、IgG1、IgG 2a 、IgG2b、IgG3、IgGA、IgGk、IgGλ Clonotyping Elisa Kits單株抗體, SouthernBiotech貨號5300-05

產品形容: 小鼠分型cloning typing ELISA Kit
分析原理: Horseradish peroxidase (HRP)-based Enzyme-Linked-Immunosorbent-Assay (ELISA) and immunoblotting.
產品規格Components:

內附有IgM、IgG1、IgG 2a 、IgG2b、IgG3、IgGA、IgGk、IgGλ抗體

• 1 mL of affinity purified goat anti-mouse immunoglobulin capture antibody for coating ELISA plates when the antigen to which the antibody is directed is valuable and/or difficult to obtain in pure form
• 1 mL of enzyme-labeled goat anti-mouse immunoglobulin for initial screening of hybridoma-containing wells
• 1 mL each of enzyme-labeled goat anti-mouse IgA, IgM, IgG1, IgG 2a , IgG2b, IgG3, kappa and lambda for the characterization of positive clones
• Enzyme substrate
• Detailed instruction protocol

• 1 mL goat anti-mouse Ig capture antibody ( 2.5 m g/mL)
• 1 mL HRP-labeled goat anti-mouse Ig screening antibody
• 1 mL HRP-labeled goat anti-mouse IgM
• 1 mL HRP-labeled goat anti-mouse IgG1
• 1 mL HRP-labeled goat anti-mouse IgG 2a
• 1 mL HRP-labeled goat anti-mouse IgG2b
• 1 mL HRP-labeled goat anti-mouse IgG3
• 1 mL HRP-labeled goat anti-mouse IgA
• 1 mL HRP-labeled goat anti-mouse κ
• 1 mL HRP-labeled goat anti- mouse λ

ABTS substrate (100 mg)

【SouthernBiotech貨號5300-05操作手冊】

Hybridomas subtyping ELISA 操件流程 (AP), #5300-04, Southern Biotech

1.  準備 substrate buffer (Kit內沒有附) :

400 mL   二次水+24.5 mg  MgCl2.6H2O +48 mL  diethanolamine   (以5N HCl 調整至pH to 9.8)將以上溶液以二次水, 補至總體積為500 mL

2. capture antibody 製備:以BBS或PBS將capture antibody稀釋成濃度5-10 μg/mL (250X~500X)每個well加入0.1mL (100ul) 5~10ug/Ml濃度的capture antibody

註明: 100 mM borate buffered saline (BBS), pH 8.0

         1X phosphate buffered saline (PBS), pH 7.4;

3. 以蓋子或封膜封住plate,  2 -8°C incubate至少12小時

4. Empty wellsà以BBS (or PBS) containing 0.05% Tween清洗3次àempty wellsà fill wells with BBS (or PBS) containing 1% bovine serum albumin (BBS/BSA).

5. Allow antibody-coated plate to stand at room temperature for a minimum of 1 hour to block free binding sites on the plate.

6. Empty plate and wash 3X.

7. 每個well加入0.1mL (100ul) hybridoma supernatant, cover the plate and incubate for 1 hour at room temperature with gentle shaking or overnight at 2 -8°C .

8. Empty plate and wash 3X.

9. Dilute AP-labeled detection antibody(ies) in BBS/BSA, 每個well加入0.1mL (100ul), cover the plate and incubate for 1 hour at room temperature with gentle shaking or overnight at 2 -8°C .

10. Empty the plate and wash 5X.

11.準備1 mg/mL substrate solution (e.g., one 5 mg tablet + 5 mL substrate buffer, 如步驟1.) 每個well加入0.1mL (100ul)

12. Read optical density of each well at 405 nm at 10 minutes and 20 minutes after substrate addition.

13. 記錄data.