Precipitin Tests

The dassic method of detecting soluble antigen (i.e., antigen in solution) is Quchterlony double immunodiffusion.

Double Immunodiffusion

In the double immunodiffusion method, small circular wells are cut out of agar or agarose, a gelatin-like matrix from seaweed through which molecules can readily diffuse, in Petri dishes. The patient specimen containing antigen is placed in one well, and antibody directed against the antigen being sought is placed in the adjacent well. Over a period of 18 to 24 hours, the antigen and antibody diffuse toward each other, producing a visible predpitin band at the point at which they meet, known as the zone of equivalence. This method is currently used to detect exoantigens produced by the systemic fungi to confirm their presence in culture (Figure 9-3). However, the technique is too slow

Figure 9-2 Production of a monoclonal antibody.

to be commonly used for antigen detection directly in patient specimens.

Counterimmunoelectrophoresis

Counterimmunoelectrophoresis (CIE) is a modification of the Ouchterlony method that speeds up migration of an antigen and antibody by applying an electrical current. With some exceptions (e.g., Streptococcus pneumoniae serotypes 7 and 14), most bacterial antigens are negatively charged in a slightly alkaline environment, whereas antibodies are neutral. This feature of bacterial antigens is exploited by CIE assays, in which solutions of antibody and sample fluid to be tested are placed in small wells cut into a slab of agarose on a glass surface (Figure 9-4). A paper or fiber wick is used to connect the two opposite sides of the agarose to troughs of slightly alkaline buffer, formulated for each antibody-antigen system. When an electrical current is applied through the buffer, the negatively charged antigen molecules migrate toward the positive electrode and thus toward the wells filled with antibody. The neutrally charged antibodies are carried toward the negative electrode by the flow of the buffer. At some point between the wells, a zone of equivalence occurs, and the antigen-antibody complexes form a visible precipitin band. The entire procedure usually takes about 1 hour.

Any antigen for which antisera are available can be tested by CIE. The sensitivity appears to be less than that of particle agglutination, detecting approximately 0.01 to 0.05 mg/mL of antigen, which translates to about 10* organisms per milliliter of fluid. Bands are often difficult to see, and the agarose gel may require overnight washing in distilled water to remove nonspecific precipitin reactions. Testing positive and negative controls is especially critical, because sera may contain nonspecifically reacting agents that form nonstable

Myeloma cells

Hybridoma cells (one specific antibody per cell)

Antibody produced in culture supernatant

Antibody produced in mcuse ascites fluid

Polyethylene glycol fusion

Polyclonal antiserum

Y Antibody-y producing cells

Polyethylene glycol fusion

Fused cells

Hybridoma cells (one specific antibody per cell)

Antibody produced in culture supernatant

Myeloma cells

Best antibody-producing cell cloned and expanded

Antibody produced in mcuse ascites fluid

Figure 9-3 Exo-Antigen Identification System, Immuno-Mycologics, Inc., Nonnan, Okla. The center well is filled with a 50x concentrate of an unknown mold. The arrow identifies well 1; wells 2 to 6 are shown clockwise. Wells 1,3, and 5 are filled with anti-Histoplasma, anti-Blastomyces, and aali-Cocddioides reference antisera, respectively. Wells 2,4, and 6 are filled with Histoplasma antigen, Blastomyces antigen, and Cocddioides antigen, respectively. The unknown organism can be identified as Histoplasma capsulatum based on the formation of line(s) of identity (arc) linking the control band(s) with one or more bands formed between the unknown extract (center well) and the reference antiserum well (well 1).

Figure 9-3 Exo-Antigen Identification System, Immuno-Mycologics, Inc., Nonnan, Okla. The center well is filled with a 50x concentrate of an unknown mold. The arrow identifies well 1; wells 2 to 6 are shown clockwise. Wells 1,3, and 5 are filled with anti-Histoplasma, anti-Blastomyces, and aali-Cocddioides reference antisera, respectively. Wells 2,4, and 6 are filled with Histoplasma antigen, Blastomyces antigen, and Cocddioides antigen, respectively. The unknown organism can be identified as Histoplasma capsulatum based on the formation of line(s) of identity (arc) linking the control band(s) with one or more bands formed between the unknown extract (center well) and the reference antiserum well (well 1).

Specific antibody

Specific antibody

Particle agglutination Figure 9-5 Alignment of antibody molecules bound to the surface of a latex particle and latex agglutination reaction.

Antigen wells Antibody wells

Antigen wells Antibody wells

Figure 9-4 Apparatus for performing counterimmunoelectrophoresis.

complexes in the gel. Because of the initial capital outlay for the apparatus and the large quantities of antigen and antibody that must be used, CIE is more expensive than agglutination-based tests and is, therefore, no longer widely used for immunodiagnosis.

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Bacterial Vaginosis Facts

Bacterial Vaginosis Facts

This fact sheet is designed to provide you with information on Bacterial Vaginosis. Bacterial vaginosis is an abnormal vaginal condition that is characterized by vaginal discharge and results from an overgrowth of atypical bacteria in the vagina.

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