On 2015.4.30, our company launched a heated discussion on the technology of peroxidase antibody, and exchanged a series of methods of application and specific methods of this product. The following is a small series of specific meeting content, welcome to inquire and appreciate.
Product introduction:
Peroxidase-labeled antibodies are a class of specific, sensitive and safe immunochemicals widely used in various disciplines of immunology, molecular biology and clinical medicine. At present, it is more common to use horseradish peroxidase (HRP), which is oxidized by sodium iodate and labeled on an antibody IgG molecule to prepare an HRP-antibody conjugate.
All kinds of enzyme standards provided by the company to users are marked according to the modified sodium periodate oxidation method established by the company. A certain concentration of BSA has been added to each finished product as a stabilizer. The working concentration of the product is based on
The method is different, the product needs to be frozen at -20 °C to avoid repeated freezing and thawing.
This product contains HRP about 200ug/branch per 0.1ml liquid, IgG contains about 400ug/branch, BSA300ug/branch. This product can be stored at 4 ° C for about 3 to 6 months, after dilution at 4 ° C can be stored for about 30 days. The dilution was: PBS (pH 7.4).
product
Application range:
Antigenic analysis of antigenic determinants, quantification, characterization and localization of various antigens and antibodies
Analysis and the like.
HRP labeled antibody specific method
There are many methods for cross-linking an enzyme with an antibody, and different methods can be employed depending on the structure of the enzyme. For the preparation of the HRP conjugate, a two-step glutaraldehyde method and a sodium periodate method can be used. Especially the simple sodium periodate method is more commonly used.
Glutaraldehyde two-step method
1. Principle: Glutaraldehyde is a bifunctional reagent that is covalently bound to an enzyme and an amino group on an immunoglobulin by its aldehyde group to form an enzyme-glutaraldehyde-immunoglobulin conjugate.
2. Marking steps:
(1) 25 mg of HRP was weighed and dissolved in a 1.25% glutaraldehyde Solution, and allowed to stand at room temperature overnight.
(2) The enzyme solution after the reaction was subjected to a Sephadex G-25 column and eluted with physiological saline. The flow rate was controlled at 1 ml / 1 minute and the brown effluent was collected. If the volume is greater than 5 ml, concentrate to 5 ml with PEG. Place in a small 25ml beaker and stir slowly.
(3) 12.5 mg of the antibody to be labeled was diluted to 5 ml with physiological saline, and added dropwise to the enzyme solution with stirring.
(4) Using 0.25 ml of 1 MPH 9.5 carbonate buffer, stirring was continued for 3 hours.
(5) Add 0.2 ml of 0.2 M lysine, mix and set at room temperature for 2 hours.
(6) An equal volume of saturated ammonium sulfate was added dropwise with stirring and allowed to stand at 4 ° C for 1 hour.
(7) Centrifuge at 3000 rpm for half an hour and discard the supernatant. The precipitate was washed twice with half-saturated ammonium sulfate, and finally the precipitate was dissolved in a small amount of 0.15 MPH 7.4 in PBS.
(8) The above solution was placed in a dialysis bag, dialyzed against 0.15 MPH7.4 PB buffered saline, and after removal of ammonium ions (detected by naphthalene reagent), the precipitate was removed by centrifugation at 10,000 rpm for 30 minutes, and the supernatant was enzyme-bound. After storage, store it in ice.
3. Result determination:
(1) Qualitative and potency titration: a specific antigen (or antibody) and an enzyme-labeled antibody (or anti-immunoglobulin antibody) are used as a two-way agar diffusion test or an immunoelectrophoresis test. The precipitation arc is then colored with the substrate of the enzyme to initially identify its activity. Finally, the enzyme conjugate is titrated by direct ELISA (or in a formal experimental system) (see the selection of working concentrations in this section (3)).
(2) Quantitative and molar ratio determination: measured by spectrophotometer (1 cm path length).
Enzyme amount (mg/ml) = OD403nm × 0.4
IgG amount (mg/ml) = OD280nm-OD403nm × 0.42) × 0.94 × 0.62
Amount of enzyme (mg/ml) IgG amount (mg/ml)
The molar ratio = ────────÷───────=────×4
40,000 160,000 IgG amount
(3) The marking procedure of this law is relatively simple and reproducible. The disadvantage is that the enzyme utilization rate is low, and generally only 2 to 4% of the enzyme binds to the protein.
4. Reagents and equipment:
(1) 0.1MPH6.8 phosphate buffered saline (PBS): 449 ml of 0.2 M Na2HPO, 451 ml of 0.2 M NaH 2 PO, 1.8 g of NaCl, and distilled water to 200 ml.
(2) 1.25% glutaraldehyde solution: 50 ml of 25% glutaraldehyde was mixed with 1 ml of PBS of pH 6.8.
(3) 1MPH9.5 carbonate buffer: 3 ml of 1 M sodium carbonate was mixed with 7 ml of 1 M sodium hydrogencarbonate.
(4) 0.2 M lysine solution: 29.2 mg of lysine was dissolved in 1 ml of 0.01 MPH9.5 carbonate buffer.
(5) 0.15 MPH 7.4 PBS and physiological saline.
(6) PH7.8 saturated ammonium sulfate solution and semi-saturated ammonium sulfate solution.
(7) Naphthalene reagent and polyethylene glycol (PEG, MW2000).
(8) Purified specific antibody or anti-Ig antibody.
(9) HRP (RZ>3.0).
(10) Sephadex G-25 chromatography column (2 cm x 50 cm).
(11) Stirrer, spectrophotometer, centrifuge.
(12) Dialysis bags, large and small beakers, test tubes, straws, etc.
Shanghai Jinma biological species include: 1. human ELISA test kit; 2. animal ELISA test kit; 3. plant ELISA test kit; 4. food ELISA test kit; 5. agricultural product ELISA test kit; Cell ELISA test kit; 7. Other types of ELISA test kits
For more detailed parameters, please call to order.
Control Cables,Cable Control,Auto Control Cables,Control Wire
TRANCHART Electrical and Machinery Co.,LTD , https://www.tranchart-electrical.com