Experimental Systems CHAPTER 23 531 Radioisotopes commonly used may cause denaturation and loss of activity. A convenient la- TABLE23-4in immunology laboratories beling system has been developed which may be used in con- junction with the ELISA and ELISPOT assays described in Radiation type Autoradiography Chapter 6. This labeling technique exploits the high affinity of the reaction between the vitamin biotin and avidin, a large molecule that may be labeled with radioactive isotopes, with 6. 8 da fluorescent molecules, or with enzymes. Biotin is a small 5'Cr 27.8 da molecule(mol wt. 244)that can be coupled to an antibody (or 143da to any protein molecule) by a gentle chemical reaction that causes no loss of antibody activity. After the biotin-coupled 874da 14C5730y 235y the avidin molecule ( figure 23-3). The reaction between bio- tin and avidin is highly specific and of such high affinity that y(gamma)radiation may be detected in a solid scintillation counter. the bond between the two molecules under most assay condi B(beta)radiation is detected in a liquid scintillation counter by its ability to convert energy to photons of light in a solution containing phosphorescent tions is virtually irreversible 可dMm级 Proteins by Size and Charge Gel Electrophoresis Separates normal autoradiographic techniques. When subjected to an electric field in an electrophoresis chamber, a charged molecule will move toward the oppo- sitely charged electrode. The rate at which a charged mole- cule moves in a stable field (its electrophoretic mobility) A general radiolabeling of cell proteins may be carried out depends upon two factors specific to the molecule: one is the by growing the cells in a medium that contains one or more sign and magnitude of its net electrical charge, and the other radiolabeled amino acids. The amino acids selected for this is its size and shape. All other factors being equal, if mole application are those most resistant to metabolic modification cules are of equal size the one with higher net charge will during cell growth so that the radioactive label will appear in move faster in an applied electrical field due to the molecular the cell protein rather than in all cell constituents. Leucine seiving properties of the solid medium. It also follows that marked with"C or H, and cysteine or methionine labeled small molecules will move faster than large ones of the same withS, are the most commonly used amino acids for meta- net charge. Although there are exceptions in which the shape bolic labeling of proteins. Table 23-4 lists some properties of of a molecule may increase or decrease its frictional drag and the radioisotopes used in immunologic research cause atypical migration behavior, these general principles Biotin Labels facilitate detection Most electrophoretic separations are not conducted in of small Amounts of proteins free solution but rather in a stable supporting medium, such as a gel. The most popular in reseach laboratories is a poly- In some instances direct labeling of proteins, especially with merized and crosslinked form of acrylamide. Separation on enzymes or other large molecules, as described in Chapter 6, polyacrylamide gels, commonly referred to as Biotin active ester Labeled avidin biotinylated Ab FIGURE23-3Labeling of antibody with biotin. An antibody prepara- antibody, the bound antibody can be detected with labeled avidin.The tion is mixed with a biotin ester, which reacts with the antibody. The avidin can be radioactively labeled or linked to an enzyme that catalyzes biotin-labeled antibody can be used to detect antigens on a solid sub- a color reaction, as in ELISA procedures(see Figure 6-10) strate such as the well of a microtiter plate. After washing away unboundA general radiolabeling of cell proteins may be carried out by growing the cells in a medium that contains one or more radiolabeled amino acids. The amino acids selected for this application are those most resistant to metabolic modification during cell growth so that the radioactive label will appear in the cell protein rather than in all cell constituents. Leucine marked with 14C or 3 H, and cysteine or methionine labeled with 35S, are the most commonly used amino acids for meta￾bolic labeling of proteins. Table 23-4 lists some properties of the radioisotopes used in immunologic research. Biotin Labels Facilitate Detection of Small Amounts of Proteins In some instances direct labeling of proteins, especially with enzymes or other large molecules, as described in Chapter 6, may cause denaturation and loss of activity. A convenient la￾beling system has been developed which may be used in con￾junction with the ELISA and ELISPOT assays described in Chapter 6. This labeling technique exploits the high affinity of the reaction between the vitamin biotin and avidin, a large molecule that may be labeled with radioactive isotopes, with fluorescent molecules, or with enzymes. Biotin is a small molecule (mol. wt. 244) that can be coupled to an antibody (or to any protein molecule) by a gentle chemical reaction that causes no loss of antibody activity. After the biotin-coupled antibody has reacted in the assay system, the labeled avidin is introduced and binding is measured by detecting the label on the avidin molecule (Figure 23-3). The reaction between bio￾tin and avidin is highly specific and of such high affinity that the bond between the two molecules under most assay condi￾tions is virtually irreversible. Gel Electrophoresis Separates Proteins by Size and Charge When subjected to an electric field in an electrophoresis chamber, a charged molecule will move toward the oppo￾sitely charged electrode. The rate at which a charged mole￾cule moves in a stable field (its electrophoretic mobility) depends upon two factors specific to the molecule: one is the sign and magnitude of its net electrical charge, and the other is its size and shape. All other factors being equal, if mole￾cules are of equal size the one with higher net charge will move faster in an applied electrical field due to the molecular seiving properties of the solid medium. It also follows that small molecules will move faster than large ones of the same net charge. Although there are exceptions in which the shape of a molecule may increase or decrease its frictional drag and cause atypical migration behavior, these general principles underlie all electrophoretic separations. Most electrophoretic separations are not conducted in free solution but rather in a stable supporting medium, such as a gel. The most popular in reseach laboratories is a poly￾merized and crosslinked form of acrylamide. Separation on polyacrylamide gels, commonly referred to as polyacrylamide Experimental Systems CHAPTER 23 531 TABLE 23-4 Radioisotopes commonly used in immunology laboratories Isotope Half-life Radiation type* Autoradiography† 125I 60.0 da
+ 131I 6.8 da
+ 51Cr 27.8 da
– 32P 14.3 da + 35S 87.4 da + 14C 57.30 yrs + 3 H 12.35 yrs – *
(gamma) radiation may be detected in a solid scintillation counter. (beta) radiation is detected in a liquid scintillation counter by its ability to convert energy to photons of light in a solution containing phosphorescent compounds. † Radiation may also be detected by exposure to x-ray film. 35S and 14C must be placed in direct contact with film for detection. 3 H cannot be detected by normal autoradiographic techniques. Labeled avidin Ag Ab Biotin active ester Biotinylated Ab Avidin bound to biotinylated Ab FIGURE 23-3 Labeling of antibody with biotin. An antibody prepara￾tion is mixed with a biotin ester, which reacts with the antibody. The biotin-labeled antibody can be used to detect antigens on a solid sub￾strate such as the well of a microtiter plate. After washing away unbound antibody, the bound antibody can be detected with labeled avidin. The avidin can be radioactively labeled or linked to an enzyme that catalyzes a color reaction, as in ELISA procedures (see Figure 6-10).