Radiolabeling Techniques Allow Sensitive Detection of Antigens or Antibodies

Radioactive labels on antigen or antibody are extremely sensitive markers for detection and quantification. There are a number of ways to introduce radioactive isotopes into proteins or peptides. For example, tyrosine residues may be labeled with radioiodine by chemical or enzymatic procedures. These reactions attach an iodine atom to the phenol ring of the tyrosine molecule. One of the enzymatic iodina-tion techniques, which uses lactoperoxidase, can label proteins on the plasma membrane of a live cell without labeling proteins in the cytoplasm, allowing the study of cell-surface proteins without isolating them from other cell constituents.

Random chromosomal loss

Hybridoma

(expresses some normal B-cell or T-cell genes but grows indefinitely like a cancer cell)

Hybridoma

(expresses some normal B-cell or T-cell genes but grows indefinitely like a cancer cell)

Monoclonal antibody

Cell Hybridoma

B-cell hybridoma

T-cell hybridoma

FIGURE 23-2

Monoclonal antibody

B-cell hybridoma

T-cell hybridoma

FIGURE 23-2

Production of B-cell and T-cell hybridomas by somatic-cell hybridization. The resulting hybridomas express some of the genes of the original normal B or T cell but also exhibit the immortal-growth properties of the tumor cell. This procedure is used to produce B-cell hybridomas that secrete monoclonal antibody and T-cell hybridomas that secrete various growth factors.

TABLE 23-4

Radioisotopes commonly used in immunology laboratories

Isotope Half-life Radiation type* Autoradiography'

TABLE 23-4

Isotope Half-life Radiation type* Autoradiography'

125|

60.0 da

y

+

1311

6.8 da

y

+

51Cr

27.8 da

y

-

32p

14.3 da

ß

+

35S

87.4da

ß

+

14C

57.30 yrs

ß

+

3H

12.35 yrs

ß

-

* y (gamma) radiation may be detected in a solid scintillation counter. P (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.

* y (gamma) radiation may be detected in a solid scintillation counter. P (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.

T Radiation may also be detected by exposure to x-ray film. 35S and 14C must be placed in direct contact with film for detection. 3H cannot be detected by normal autoradiographic techniques.

A 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 3H, and cysteine or methionine labeled with 35S, are the most commonly used amino acids for metabolic labeling of proteins. Table 23-4 lists some properties of the radioisotopes used in immunologic research.

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Responses

  • melba
    Why to label only those amino acids with radioisotopes which are resistant to metabolic modification?
    8 years ago

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