Fig

Use of bacterial staphylococcus A and streptococcus G proteins to detect and isolate antibody—antigen complexes Pathogenic staphylococci and streptococci express Fc-binding proteins on their surface to bind and inactivate antibody molecules by forcing them to face away from the bacterial cell. This reaction is quite useful in the laboratory, and the A protein of Staphylococcus aureus (staph A protein) and the G protein of group C streptococci (strep G protein) are com mercially available for use as specific reagents to detect the presence of the Fc regions on human, rabbit, and mouse IgG molecules. An example is shown in Fig. 12.6(a). Here, all the proteins from a virus-infected cell were fractionated and blotted (immobilized) onto a membrane to which they tightly stick.

This type of protein transfer blot is called a western blot for a rather amusing reason. In the late 1960s and early 1970s, a scientist named Edward Southern developed a quantitative method for transferring gels of DNA fragments produced by restriction endonuclease digestion onto nitrocellulose filter paper. Such DNA transfer blots have ever since been called Southern blots. Subsequently, RNA transfer technology was developed and such blots were named northern blots both to distinguish them from DNA blots and to establish similarity of the process. Protein blots were then named western blots for comparable reasons.

In the example shown, the membrane and transferred proteins were incubated with antibodies to viral proteins. These antibodies stick only to those proteins that they "recognize." The blot was rinsed and incubated with 35S-methionine-labeled staph A or strep G protein. This protein reacts with the antibody's Fc region and the area of immune complex is revealed.

■4 Viral proteins •4 Cellular proteins

Virus-infected cell proteins fractionated on a denaturing gel.

  • i7 Blot onto filter paper
  • 4 Viral proteins •4 Cellular proteins
  • i7 Blot onto filter paper

Virus-infected cell proteins fractionated on a denaturing gel.

All proteins stick to blot

Incubate with antibodies to viral proteins

Incubate with antibodies to viral proteins

3. Incubate with labeled staph A protein I c

3. Incubate with labeled staph A protein I c

Viral protein bands are visible by virtue of the label on the Staph A protein

Fig. 12.6 Detection and isolation of proteins reactive with a specific antibody by use of immunoaffinity chromatography. (a) western blot. A mixture of viral and cellular proteins from an infected cell extract was fractionated on a sodium dodecyl sulfate gel, and the proteins blotted onto a membrane filter. The filter was then reacted with a specific antibody and washed, and then the antibody located by using radiolabeled staph A protein. (b) The antibody and antigen mixture is incubated so that specific interaction occurs. This is followed by passing the whole mix through a column with staph A protein bound to the column matrix (sepharose). All antibody molecules bind through their Fc regions, and any antigen bound to them can be eluted with a gentle denaturation rinse that does not cause the staph A protein—Fc binding to be disrupted. (c) A similar approach in which the antibody first is bound to the column matrix, and the proteins are washed over the column for binding. Both methods provide essentially equivalent results.

Add antibody J against A

Various proteins

Add antibody J against A

Various proteins

Staph A

Run protein and antibody mix through a column which contains Staph A bound to an insoluble matrix

Staph A

Specific protein binds to antibody on column

bound t> material A

Wash off unbound (7 material

Various proteins

Various proteins

Load onto antibody column

Antibody against ^

Specific protein binds to antibody on column

Load onto antibody column

Antibody against ^

Specific protein binds to antibody on column

Elute bound material

Wash off unbound material

Fig. 12.6 Continued

Immunoaffinity chromatography Two variations on methods utilizing the binding of Fc regions to antibody molecules are frequently used to isolate specific proteins. Some methods using the affinity of staph A protein are shown in Figs 12.6(b) and 12.6(c). In Fig. 12.6(b), an antibody against a protein in a complex mix is incubated with the protein mixture, and then passed through a sepharose column (a high-molecular-weight polysaccharide) to which the Fc-binding protein was chemically bound. All antibody molecules bind to the column, and any proteins that are bound to the antibody molecules also stick. All other proteins are washed off the column and discarded. Finally, the protein is eluted from the antibody, which is itself bound to the column via the Fc-binding region, using conditions that will not disturb the antibody's binding to the column, and the protein can be recovered in pure form. In Fig. 12.6(c), the antibody is first bound to the column. Then it is allowed to react with antigen as the protein mix is washed through the column. It can be eluted later, after unwanted proteins are thoroughly rinsed away.

An example of one use of this method, to characterize a HSV mutant that does not express a specific glycoprotein (glycoprotein C), is shown in Fig. 12.7. Here, a polyclonal antibody against viral envelope proteins was prepared by immunizing rabbits. This antibody was allowed to bind to 35S-labeled membrane proteins synthesized after infection with a wild-type and a gC- mutant of HSV. The total protein mix and the envelope proteins that bound to the antibody preparation then were fractionated on a gel and exposed to x-ray film. Absence of the protein in the mutant virus is quite evident.

These same methods can be used with antibodies against the Fc region of antibodies from a different animal. Use of such antibody-binding methods provides another degree of specificity (just as did its use in immunofluorescence) and allows purification of even very small quantities of protein in a mix.

Total infected cell Anti-env Anti-env protein wt mutant (gC-)

"gA"

gB gC

Fig. 12.7 Use of immunoaffinity chromatography to isolate HSV envelope proteins from infected cells. Total infected cell protein was labeled by incubation with radioactive amino acids. The protein then was mixed with a polyclonal antibody monospecific for viral envelope proteins. The reactive proteins were isolated as described in Fig. 12.6 and fractionated on a denaturing gel. The third column shows the results of a similar experiment where a virus unable to express glycoprotein C was used. wt=wild type.

Total infected cell Anti-env Anti-env protein wt mutant (gC-)

"gA"

gB gC

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