Human cntn1 antigen, human cntn1 antibody detection kit, preparation method and application thereof
A detection kit and antibody detection technology, applied in the field of biopharmaceuticals, to achieve good stability, strong specificity, and high sensitivity
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Embodiment 1
[0045] Example 1 Construction of recombinant expression plasmid and engineering bacteria
[0046]1. Contact proteins are a subgroup of the immunoglobulin superfamily, and its members include CNTN1, CNTN2, CNTN3, CNTN4, CNTN5 and CNTN6. The contact protein CNTN1 is the first identified contact protein member. The gene is located in the 12q11-q12 region of chromosome with a molecular weight of 135 kDa. Its structure consists of six Ig-like regions and four fibronectin type III-like (fibronectin type III-like, FNⅢ-like) fragment and glycosyl phosphatidylinositol (GPI) linked to the cell membrane in an anchored manner. The full length is 1018 amino acids, of which the amino terminal has a signal peptide sequence consisting of 20 amino acids. In this study, the amino acid sequence of CNTN1 protein was analyzed including hydrophilicity, surface accessibility, etc., and combined with its spatial conformation and the modification characteristics of each domain, the mature CNTN1 prote...
Embodiment 2
[0057] Example 2 Expression and purification of antigen
[0058] 1. Use the constructed recombinant protein expression engineering bacteria to conduct the induction expression experiment. Inoculate the 4 recombinant bacteria in 600ml of LB culture solution (components: 10g sodium chloride / liter, 10g peptone / liter and 5g yeast extract / liter), shake the bacteria at 37 degrees and 200RPM to OD600 to 0.6-0.8, press IPTG at a concentration of 24 mg / ml was added at 1:1000 for induction for 4 hours. Centrifuge, harvest bacteria, and prepare for purification.
[0059] 2. The filler selected for purification is GE's Ni Sepharose (Item No. 17-0729-10), and the following solutions are prepared according to its instructions:
[0060] Loading Buffer A: 0.5M NaCl + 20mM Na 2 HPO 3 +10 mM imidazole.
[0061] Binding buffer B: 0.5M NaCl + 20mM Na 2 HPO 3 +20mM imidazole
[0062] Elution buffer C: 0.5M NaCl, 20mM Na 2 HPO 3 , 500mM imidazole;
[0063] and the solution used to purify...
Embodiment 3
[0069] Example 3 Human CNTN1 antibody detection kit and method of use
[0070] 1. Coated ELISA plate
[0071] (1) Coating solution: NaCl 8.5g, Na 2 HPO 4 ·12H 2 O 30.8g, KH 2 PO 4 2.2g, add ddH 2 O to 1000ml, adjust pH to 7.4.
[0072] (2) Lotion for coating: NaCl 8.0g, KH 2 PO 4 0.24g, Na 2 HPO 4 ·12H 2 O 2.9g, KCl 0.2g, TWEEN200.5ml, add to ddH 2 O to 1000ml, adjust to PH7.4.
[0073] (3) Coating method: 4 CNTN1 antigen fragments were respectively coated with 0.1M PBS (PH7.4) coating buffer in the wells of the ELISA plate, overnight at 4 degrees, among which CNTN1-39, CNTN1-322, The coating concentrations of CNTN1-609 and CNTN1-809 were 200ng / ml, 200ng / ml, 250ng / ml and 150ng / ml, respectively. Add 100 μL to each well of a 96-well microtiter plate, and set it to adsorb at 2-8°C for 24 hours. Empty the coating solution and wash the plate 3 times with washing solution.
[0074] 2. Close:
[0075] (1) Blocking solution for coating: Na 2 HPO 4 ·12H 2 O 3.582g, ...
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