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兔单抗亲和力和灵敏度更高
相对于鼠单抗（解离常数Kd在10-9-10-10M水平），兔单抗的亲和力可提高100-1000倍（Kd在10-10-10-12M水平）。实际使用时，兔单抗的工作浓度更低，灵敏度更高，因此产生的背景更低，大大降低了假阳性的发生。用作免疫组化等用途时，有时甚至可免去抗原修复的步骤。
兔单抗能识别更多表位
很多蛋白在人类和啮齿动物中同源性很高，这些保守表位在小鼠体内免疫原性很弱，不容易产生高质量抗体。而兔做为宿主，可以更好地识别这些表位，产生针对更多表位的抗体。当然，兔也适合生产针对鼠类蛋白的抗体。
Purification of Antiserum or Ascites by Protein A/G Chromatography
(4) To prepare 100 ml of Elution Buffer pH 2.7 (50 mM Glycine-HCl, pH 2.7), add the following to 80 ml of distilled water
0.38 g of Glycine mix thoroughly and adjust the pH to 2.7 with 5N HCl. Add distilled water to a final volume of 100ml.
(5) To prepare 100 ml of Elution Buffer pH 1.9 (50 mM Glycine-HCl, pH 1.9), add the following to 80 ml of distilled water
0.38 g of Glycine mix thoroughly and adjust the pH to 1.9 with 5 N HCl. Add distilled water to a final volume of 100 ml.
3、Procedure
(1) Section I Preparation of a Protein A Agarose or Protein G Agarose Affinity Column
Typically, columns containing 5 ml or 10 ml of packed protein A/G Agarose are prepared. The size of the column is determined by the binding capacity of protein A/G and the amount of antiserum that must be processed. Protein A and protein G bind about 20 mg of IgG per ml of conjugated agarose. Therefore, the binding capacity of a 10 ml (5 ml) column is 200 mg (100 mg) of IgG. A high-titer rabbit antiserum has roughly 5 mg/ml of IgG, mouse ascites has roughly 10 mg/ml of IgG, and goat or sheep antiserum has roughly 20 mg/ml of IgG. Use the guidelines below to choose a column size to avoid exceeding the capacity of the column. Do not exceed 90% of the binding capacity of the column.
10 ml Bead Volume Protein A/G Affinity Column
Source of Antibody Concentration Volume for Capacity Volume for 90% Capacity
Rabbit Antiserum 5 mg/ml 40 ml 36 ml
Mouse Ascites 10 mg/ml 20 ml 18 ml
Goat/Sheep Antiserum 20 mg/ml 10 ml 9 ml
5 ml Bead Volume Protein A/G Affinity Column
Source of Antibody Concentration Volume for Capacity Volume for 90% Capacity
Rabbit Antiserum 5 mg/ml 20 ml 18 ml
Mouse Ascites 10 mg/ml 10 ml 9 ml
Goat/Sheep Antiserum 20 mg/ml 5 ml 4.5 ml
Pouring the Protein A/G Affinity Column
Ⅰa pipet to transfer the desired volume of a 50% protein A/G agarose slurry to a vacuum flask. Remember to transfer enough slurry to prepare a column with the desired bed volume and capacity.
ⅡPlace a stopper in the vacuum flask and apply vacuum for at least 15 minutes at room temperature. This step removes gas from the agarose and is necessary to prevent bubble formation in the column that would reduce the column's capacity and resolution.
Ⅲ While degassing the agarose, chill the TBS on ice. Prepare TBS without azide for antibodies that will be used in vivo or in cellular assays. TBS is used to equilibrate and wash the protein A/G column.
Ⅳ Slowly add the degassed protein A/G agarose to a glass column, e.g., a Biorad Econo Column, using a wide bore pipet to prevent rupturing the beads.
Ⅴ Pack the column at a flow rate of about 1-2 ml per min. Do not let the column run dry! Flow rate may be controlled by using a Mariotte flask or pump.
Ⅵ Wash the column with 10 bed volumes of ice-cold TBS. This step chills the column, which reduces nonspecific binding of proteins and slows the metabolism of any remaining viable microbes. <
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