Reversible Binding of a Protein to a Ligand: Oxygen-Binding Proteins: -The Antibody-Antigen Interaction Is the Basis for a Variety of Important Analytical Procedures

The extraordinary binding affinity and specificity of antibodies make them valuable analytical reagents. Two types of antibody preparations are in use: polyclonal and monoclonal. Polyclonal antibodies are those produced by many different B lymphocytes responding to one antigen, such as a protein injected into an animal. Cells in the population of B lymphocytes produce anti bodies that bind specific, different epitopes within the antigen. Thus, polyclonal preparations contain a mix ture of antibodies that recognize different parts of the protein. Monoclonal antibodies, in contrast, are synthesized by a population of identical B cells (a clone) grown in cell culture. These antibodies are homogeneous, all recognizing the same epitope. The techniques for producing monoclonal antibodies were developed by Georges Köhler and Cesar Milstein. The specificity of antibodies has practical uses. A selected antibody can be covalently attached to a resin and used in a chromatography column of the type shown in Figure 3–18c. When a mixture of proteins is added to the column, the antibody specifically binds its target protein and retains it on the column while other proteins are washed through. The target protein can then be eluted from the resin by a salt solution or some other agent. This is a powerful tool for protein purification. In another versatile analytical technique, an antibody is attached to a radioactive label or some other reagent that makes it easy to detect. When the antibody binds the target protein, the label reveals the presence of the protein in a solution or its location in a gel or even a living cell. Several variations of this procedure are illustrated in Figure 5–28.

FIGURE 5–28 Antibody techniques. The specific reaction of an antibody with its antigen is the basis of several techniques that identify and quantify a specific protein in a complex sample. (a) A schematic representation of the general method. (b) An ELISA to test for the presence of herpes simplex virus (HSV) antibodies in blood samples. Wells were coated with an HSV antigen, to which antibodies against HSV will bind. The second antibody is antihuman IgG linked to horseradish peroxidase. Blood samples with greater amounts of HSV antibody turn brighter yellow. (c) An immunoblot. Lanes 1 to 3 are from an SDS gel; samples from successive stages in the purification of a protein kinase have been separated and stained with Coomassie blue. Lanes 4 to 6 show the same samples, but these were electrophoretically transferred to a nitrocellulose membrane after separation on an SDS gel. The membrane was then “probed” with antibody against the protein kinase. The numbers between the SDS gel and the immunoblot (b) indicate Mr in thousands.

An ELISA (enzyme-linked immunosorbent assay) allows for rapid screening and quantification of the presence of an antigen in a sample (Fig. 5–28b). Proteins in a sample are adsorbed to an inert surface, usually a 96 well polystyrene plate. The surface is washed with a solution of an inexpensive nonspecific protein (often ca sein from nonfat dry milk powder) to block proteins introduced in subsequent steps from also adsorbing to these surfaces. The surface is then treated with a solution containing the primary antibody—an antibody against the protein of interest. Unbound antibody is washed away and the surface is treated with a solution containing antibodies against the primary antibody. These secondary antibodies have been linked to an enzyme that catalyzes a reaction that forms a colored product. After unbound secondary antibody is washed away, the substrate of the antibody-linked enzyme is added. Product formation (monitored as color intensity) is proportional to the concentration of the protein of interest in the sample. In an immunoblot assay (Fig. 5–28c), proteins that have been separated by gel electrophoresis are transferred electrophoretically to a nitrocellulose mem brane. The membrane is blocked (as described above for ELISA), then treated successively with primary antibody, secondary antibody linked to enzyme, and substrate. A colored precipitate forms only along the band containing the protein of interest. The immunoblot allows the detection of a minor component in a sample and provides an approximation of its molecular weight.

We will encounter other aspects of antibodies in later chapters. They are extremely important in medicine and can tell us much about the structure of proteins and the action of genes.

اخر الاخبار

اخبار العتبة العباسية المقدسة

قسم شؤون المعارف يباشر أعماله الدورية لجرد إصداراته

العتبة العباسية المقدسة تواصل استلام التبرعات لدعم الشعبين الإيراني واللبناني

قسم الشؤون الفكرية يقدم محاضرة ثقافية لوفد شبابي من بغداد

جمعية العميد تصدر الكتاب الثامن من سلسلة كنوز الآل

المزيد

الآخبار الصحية

عادة نهارية شائعة قد تخفي مشكلات صحية كامنة

الخوف من العيادات الطبية.. أسباب نفسية خفية لدى البالغين

"علكة مبتكرة" تستهدف ميكروبات الفم المسببة للسرطان

دخان حرائق الغابات قد يرفع خطر الإصابة بأنواع من السرطان

المزيد

الآخبار التكنلوجية

منهج جديد باستخدام الساعات الذرية يعيد طرح سؤال: ما طبيعة الزمن؟

اكتشاف آلية خلوية تمنع تكوّن الألياف المرتبطة بألزهايمر

دراسة تكشف أدلة على احتمال وجود محيط قديم بالمريخ

تيم كوك ينهي 15 عاما في قيادة "أبل"

المزيد

مواضيع ذات صلة

Protein Interactions Modulated by Chemical Energy: Actin, Myosin, and Molecular Motors: -Additional Proteins Organize the Thin and Thick Filaments into Ordered Structures

Protein Interactions Modulated by Chemical Energy: Actin, Myosin, and Molecular Motors: - The Major Proteins of Muscle Are Myosin and Actin

Reversible Binding of a Protein to a Ligand: Oxygen-Binding Proteins: -Antibodies Bind Tightly and Specifically to Antigen

Complementary Interactions between Proteins and Ligands: The Immune System and Immunoglobulins: -Antibodies Have Two Identical Antigen-Binding Sites

Complementary Interactions between Proteins and Ligands: The Immune System and Immunoglobulins: -Self Is Distinguished from Nonself by the Display of Peptides on Cell Surfaces

Complementary Interactions between Proteins and Ligands: The Immune System and Immunoglobulins: -The Immune Response Features a Specialized Array of Cells and Proteins

Reversible Binding of a Protein to a Ligand: Oxygen-Binding Proteins: -Sickle-Cell Anemia Is a Molecular Disease of Hemoglobin

Reversible Binding of a Protein to a Ligand: Oxygen-Binding Proteins: -Oxygen Binding to Hemoglobin Is Regulated by 2,3-Bisphosphoglycerate

Protein Denaturation and Folding: - Loss of Protein Structure Results in Loss of Function

Protein Tertiary and Quaternary Structures: -There Are Limits to the Size of Proteins

Protein Tertiary and Quaternary Structures: -Protein Quaternary Structures Range from Simple Dimers to Large Complexes

Reversible Binding of a Protein to a Ligand: Oxygen-Binding Proteins: -Protein-Ligand Interactions Can Be Described Quantitatively