Direct and Indirect Immunofluorescence

Direct immunofluorescence. Immunofluorescence can be used for in-vivo detection of antibodies, complement, viruses, fungi, bacteria, or other im-

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Hemagglutination

Erythrocyte antigen

Erythrocyte antigen

Erythrocyte Antigens
Antigen artificially fixed on erythrocyte
Direct And Indirect Immunofluorescence

Test serum a positivel/32

Test serum b negative

Test serum c positive 1/8 with prozone 1/2

Reciprocal serum dilution 8 16 32 64 128 256 512 1024

Reciprocal serum dilution 8 16 32 64 128 256 512 1024

Fig. 2.25 The hemagglutination test is based on the principle that erythrocytes cross-linked by antibodies settle to the bottom of the microtiter plate wells in matlike aggregates (test sera a and c), whereas non-agglutinated erythrocytes collect at the lowest point of the wells to form a single "button" in the middle (test serum b). The test sera are first pipetted into the wells at the indicated dilutions, then the erythrocyte suspension is added. Non-specific agglutination is prevented by addition of an irrelevant protein. The test can be carried out using erythrocyte antigens (above left). Alternatively, other antigens can be fixed to the erythrocyte surface and the agglutination monitored (above right). The so-called "prozone" phenomenon results from non-specific blocking mechanisms present in sera which has not been sufficiently diluted.

Control pos. neg.

Fig. 2.25 The hemagglutination test is based on the principle that erythrocytes cross-linked by antibodies settle to the bottom of the microtiter plate wells in matlike aggregates (test sera a and c), whereas non-agglutinated erythrocytes collect at the lowest point of the wells to form a single "button" in the middle (test serum b). The test sera are first pipetted into the wells at the indicated dilutions, then the erythrocyte suspension is added. Non-specific agglutination is prevented by addition of an irrelevant protein. The test can be carried out using erythrocyte antigens (above left). Alternatively, other antigens can be fixed to the erythrocyte surface and the agglutination monitored (above right). The so-called "prozone" phenomenon results from non-specific blocking mechanisms present in sera which has not been sufficiently diluted.

mune factors present within patient cells and tissues. For this purpose tissue sections, or cell preparations, are treated with specific antibodies (anti-sera) which have been labeled with a fluorochrome (Fig. 2.26a). Antigen-antibody reactions can thus be detected using a fluorescence microscope. The fluoro-chrome absorbs light of a certain wavelength (e.g., UV light), and emits the light energy in the form of light at a different (visible) wavelength. The fluorochrome fluorescein isothiocyanate (FITC), which absorbs UV light and emits it as green light, is used most frequently (caution: bleaches out quickly!).

Kayser, Medical Microbiology © 2005 Thieme

All rights reserved. Usage subject to terms and conditions of license.

Direct And Indirect Immunofluorescence

Tissue section a Direct b Indirect c Indirect with amplification

Fig. 2.26 Immunofluorescence (a, b) is particularly suitable for the detection of antigens, or specific antibodies, fixed on plastic (solid phase) (ELISA) or present within a tissue section (immunohistology). For direct immunofluorescence (a) the specific primary antibody is labeled with a fluorochrome, or an enzyme (ELISA = enzyme-linked immunosorbent assay). The term indirect immunofluorescence is used when it is not the primary antibody being detected, but a secondary antibody which is directed againstthe unlabeled primary antibodyand has also been labeled with a fluorochrome or enzyme (b). In most cases, this method achieves a certain degree of amplification. However, an even higher level of amplification can be achieved using preformed complexes of secondary antibody and enzyme (c). For the peroxidase method the detector enzyme is bound directly to the secondary antibody (peroxidase catalyzes a color reaction). In the biotin-avidin method the detector enzymes are coupled to either biotin or avidin.

Tissue section a Direct b Indirect c Indirect with amplification

Fig. 2.26 Immunofluorescence (a, b) is particularly suitable for the detection of antigens, or specific antibodies, fixed on plastic (solid phase) (ELISA) or present within a tissue section (immunohistology). For direct immunofluorescence (a) the specific primary antibody is labeled with a fluorochrome, or an enzyme (ELISA = enzyme-linked immunosorbent assay). The term indirect immunofluorescence is used when it is not the primary antibody being detected, but a secondary antibody which is directed againstthe unlabeled primary antibodyand has also been labeled with a fluorochrome or enzyme (b). In most cases, this method achieves a certain degree of amplification. However, an even higher level of amplification can be achieved using preformed complexes of secondary antibody and enzyme (c). For the peroxidase method the detector enzyme is bound directly to the secondary antibody (peroxidase catalyzes a color reaction). In the biotin-avidin method the detector enzymes are coupled to either biotin or avidin.

Indirect immunofluorescence and enzyme histology. In this technique the specific or "first" antibody can be unlabeled. The antigen-antibody complexes that form are then detected using a labeled or "second" antibody, directed against the first antibody (Fig. 2.26b). Instead of fluorochromes, enzyme-labeled antibodies are now frequently used for tissue sections. The enzyme catalyzes the formation of a color signal following addition of a previously colorless detector substance. This color precipitate allows the direct observation of signals using a light microscopic, and exhibits little bleaching.

Indirect immunofluorescence can be used for the qualitative and quantitative analysis of antibodies directed against particular microbial antigens, or self-tissue antigens, within a patients serum. In the quantitative test, the antigen is fixed in a well or to a tissue section on a slide. The patient sample is repeatedly diluted by a factor of two and added to the antigen or section then rendered visible with a labeled anti-antibody.

There are two main methods of amplifying the immunohistological color signal:

Kayser, Medical Microbiology © 2005 Thieme

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■ The direct 'primary' antibody, or the detected 'secondary' antibody, is labeled with peroxidase. Following the antigen-antibody reaction, large preformed peroxidase-antiperoxidase complexes are added to the tissue section; these complexes can attach to the peroxidase-labeled antibodies, which are already specifically bound, thus amplifying the signal considerably (Fig. 2.26c).

■ Similarly, biotinylated antibodies can be used. The vitamin biotin is bound with strong affinity by avidin, a basic glycoprotein. Various colorants or enzymes coupled to avidin thus facilitate the color reactions. Such reactions can be amplified on the tissue section by adding preformed biotin-avidin-perox-idase complexes that bind to those biotin-coupled antibodies which have already been bound.

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Responses

  • carly
    What is direct and indirect immunofluorescence?
    7 years ago
  • tapani piirainen
    What is direct and indirect microbiology?
    7 years ago
  • billy moore
    What is Direct Immunofluorence?
    6 years ago
  • Freddie
    What is direct test in microbiology?
    6 years ago
  • Lorenzo Findlay
    What is indirect and direct in microbilogy?
    6 years ago

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