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RBC IgG ELISA 试剂盒

RBC IgG 适用: 绵羊 Colorimetric Sandwich ELISA Plasma, Serum
产品编号 ABIN956282
发货至: 中国
  • 抗原
    RBC IgG (Anti-Red Blood Cell IgG (RBC IgG))
    适用
    绵羊
    检测方法
    Colorimetric
    实验类型
    Sandwich ELISA
    应用范围
    ELISA
    样品类型
    Plasma, Serum
    Analytical Method
    Quantitative
    产品特性
    The Rat Anti-SRBC IgG ELISA is based on a solid phase enzyme-linked immunosorbent assay (ELISA). The assay uses detergent solubilized SRBC ghosts for solid phase (microtiter wells) immobilization and horseradish peroxidase (HRP) conjugated anti-rat IgG antibodies for detection. Test serum or plasma samples are diluted and incubated in the microtiter wells for 45 minutes. The microtiter wells are subsequently washed and HRP conjugate is added and incubated for 45 minutes. Anti-SRBC IgG molecules are thus sandwiched between immobilized SRBC antigens and the detection antibody conjugate. The wells are then washed to remove unbound HRP-labeled antibodies and TMB reagent is added and incubated for 20 minutes at room temperature. This results in the development of a blue color. Color development is stopped by the addition of stop solution, changing the color to yellow, and optical density is measured spectrophotometrically at 450 nm. The concentration of anti-SRBC IgG is proportional to the optical density of the test sample.
    组件
    Microtiter Plate: SRBC coated 96-well plate (12 strips of 8 wells)
    Enzyme Conjugate Solution: 11 mL
    Calibrator: Lyoph.
    Diluent Buffer: 30 mL
    TMB Solution: 11 mL
    Stop Solution: 11 mL, 1N HCl
    Wash Buffer (20x): 50 mL.
    试剂未包括
    Plate reader (450 nm)
    Micropipette and tips
    De-ionized water
    Graph paper (PC software is optional)
    Paper towels
    Polypropylene or glass tubes
    Vortex mixer
    Plate shaker/incubator
    Plate washer.
  • 板类型
    Pre-coated
    试剂准备

    Wash Buffer: The wash solution is provided as 20x stock. Prior to use dilute the contents of the bottle (50 mL) with 950 mL of distilled of deionized water. Calibrator
    1. The Rat anti-SRBC IgG calibrator is provided as lyophilized stock. Reconstitute with volume of diluent indicated on the vial label. The reconstituted calibrator is stable at 4°C for one week but should be aliquoted and stored frozen at -20°C after reconstitution if future use is intended.
    2. Label 5 polypropylene or glass tubes as 100, 50, 25, 12.5, and 6.25 u/mL.
    3. Into the tube labeled 100 u/mL prepare a 100 u/mL stock by mixing the volume of reconstituted calibrator stock with the volume of diluent detailed on the vial label.
    4. Dispense 250 µL of diluent into the tubes labelled 50, 25, 12.5, and 6.25 u/mL.
    5. Prepare a 50 u/mL calibrator by diluting and mixing 250 µL of the 100 u/mL calibrator with 250 µL of diluent in the tube labelled 50 u/mL.
    6. Similarly prepare the 25, 12.5, 6.25, and 3.125 u/mL calibrators by serial dilution.

    样品制备

    Note: Studies indicate that anti-SRBC IgG is present in rat serum or plasma at concentrations of ~ 2,000 u/mL (14 days after immunization). In order to obtain values within range of the calibration curve, we suggest samples initially be diluted 100 fold using the following procedure for each sample tested. Optimal dilutions may need to be determined empirically.
    1. For each test sample dispense 247.5 µL of diluent into separate tubes.
    2. Pipette and mix 2.5 µL of the serum/plasma sample into the tube containing 247.5 µL of diluent. This provides a 100 fold diluted sample.
    3. Repeat this procedure for each sample to be tested.
    4. Do not use dilutions lower than 25 fold.

    实验流程
    1. Secure the desired number of coated wells in the holder.
      2. Dispense 100 µL of calibrators, and diluted samples into the wells (we recommend that samples be tested in duplicate).
      3. Incubate on an orbital micro-plate shaker at 100-150 rpm at room temperature (18-25°C) for 45 minutes.
      4. Aspirate the contents of the microtiter wells and wash the wells 5 times with 1x wash solution using a plate washer (400 µL/well). The entire wash procedure should be performed as quickly as possible.
      5. Strike the wells sharply onto absorbent paper or paper towels to remove all residual wash buffer.
      6. Add 100 µL of enzyme conjugate reagent into each well.
      7. Incubate on an orbital micro-plate shaker at 100-150 rpm at room temperature (18-25°C) for 45 minutes.
      8. Wash as detailed in 4 and 5 above.
      9. Dispense 100 µL of TMB reagent into each well.
      10. Gently mix on an orbital micro-plate shaker at 100-150 rpm at room temperature for 20 minutes.
      11. Stop the reaction by adding 100 µL of Stop Solution to each well.
      12. Gently mix. It is important to make sure all the blue color changes to yellow.
      13. Read the optical density at 450 nm with a microtiter plate reader within 5 minutes.
    结果分析
    1. Calculate the average absorbance values for each set of calibrators, and samples.
      2. Construct a calibration curve by plotting the mean absorbance obtained from each calibrator against its concentration in u/mL on linear graph paper, with absorbance values on the vertical or Y axis and concentrations on the horizontal or X axis.
      3. Using the mean absorbance value for each sample, determine the corresponding concentration of anti-SRBC IgG in u/mL from the calibration curve.
      4. Multiply the derived concentrations by the dilution factor to determine the actual concentration for anti-SRBC IgG in the serum/plasma sample.
      5. PC graphing software may be used for the above steps.
      6. If the OD values of samples fall outside the calibration curve when tested at a dilution of 100, samples should be diluted appropriately and re-tested.
    限制
    仅限研究用
  • 注意事项
    Reliable and reproducible results will be obtained when the assay procedure is carried out with a complete understanding of and in accordance with the instructions detailed above.
    The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings.
    储存条件
    4 °C
    储存方法
    Store at 4°C. Microtiter plate should be kept in a sealed bag with desiccant to minimize exposure to damp air. The kit is stable until the expiration date when stored as noted in this section.
    有效期
    The expiry date is stated on the label.
  • 抗原
    RBC IgG (Anti-Red Blood Cell IgG (RBC IgG))
    别名
    Red Blood Cell (SRBC) IgG
    物质类
    Antibody, Antibody
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