In this test, RBCs act as carrier particles for polyclonal and monoclonal antibodies for demonstration of specific antigen in the serum, CSF and other body fluids. Reverse passive haemagglutination test (RPHA) is used for demonstration of specific antigens for diagnosis of Hepatitis B infection, Japanese encephalitis, tuberculous meningitis and many other infectious diseases.
KEY FACTS
1 The red blood cells from different sources have been used by various workers in the IHA test.
2 The red blood cells act as carrier particles for the antigen (against which antibodies will be demonstrated).
3 The RBCs sensitized with antigen are added to the patients sera to demonstrate specific antibodies in serum.
4 The IHA test is carried out in a microtiter plate.
5 The lowest amount of antigen which shows the maximum haemagglutination with the known positive control and negative reaction with the negative control is taken as OSD of the antigen for that batch.
6 The serum showing a titre of 1: 128 and above is considered positive for hydatid disease.
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VIVA
1 What is the principle of IHA test?
2 List different RBCs which can be used in the IHA test.
3 List the uses of IHA test.
4 What is reverse passive haemagglutination test?
FURTHER READINGS
1 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworth, London, 94-96, 1995.
2 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC. Color Atlas and Textbook of Diagnostic Microbiology. 5th Edition. Lippincott Williams and Wilkins. 1997; pp. 1395.
3 Stites DP. Terr AI and Parslow TG. Medical Immunology. 10th Edition. 2001. pp. 902.
Indirect Haemagglutination Test
In this experiment you will use the method to demonstrate unknown bacterial antigens. The fluorescein labeled antibody is tagged to alcohol or acetone fixed bacterial smear. This is followed by washing the smear with physiologically buffered saline. During this step any uncombined fluorescent antibody will be washed away. In a positive test, the final reaction will be observed by a green fluorescence when observed under a fluorescent microscope.
Advantages and disadvantages of the immunofluorescence test are summarized in the table 48-1.
REQUIREMENTS
I Equipments
Fluorescent microscope.
II Reagents and lab wares
Acetone, phosphate buffered saline (pH 7.2), buffered glycerol (pH 7.2): 10% glycerol in PBS, Evan’s blue counter stain (1:10000), and bovine serum albumin.
Glass slides, Petri dishes, U-shaped glass rods to fit into petri dishes, filter paper, and Coplin jar and glass marking pencil.
Commercially available fluorescent antibody Streptococcus group A and fluorescent antibody Enterococcus group D.
III Specimen
Broth cultures of Group A Streptococcus pyogenes and Group D Enterococcus faecalis incubated at 35°C for 24 hours.
Unknown mixed broth cultures of Group A S. pyogenes/
Escherichia coli and Group D E.faecalis / E. Coli
PROCEDURE
1 Take three clean glass slides.
2 With glass marking pencil label first slide as S. pyogenes, and second slide as E. faecalis. Divide the third slide in half and label as mixed unknowns.
Immunofluorescence Test
LESSON
48
LEARNING OBJECTIVES
After completing this practical you will be able to:
1 Demonstrate bacterial antigen by immunofluorescence test.
INTRODUCTION
Immunofluorescence assays involve the use of either antigen or antibody labeled with a fluorescent substance. The fluorescent dyes have the property of absorbing the light of one wavelength (e.g., ultra violet light) and reflect back the light of a different wavelength (visible light). These reflected lights are visualized by a fluorescent microscope under ultra violet radiation.. Fluorescein isothiocyanate (FITC) is the most common dye used in the test. The dye emits a greenish/yellow light. Rhodamine (red/orange), dansyl (yellow), and phycoerythrin are the other dyes used in the test.
Direct fluorescent antibody test is employed to detect the specific antigens of bacteria, viruses, parasites or other antigens in the serum, CSF, urine, faeces, tissues and other body fluids.
This test is most frequently used to detect rabies virus antigen in the tissue collected from the skin on the nape of the neck or face. This is also used to detect Corynebacterium diphtheriae, Neisseria gonorrhoeae, measles and mumps in various clinical specimens.
PRINCIPLE
In this test specific antibodies raised against the antigen to be detected (e.g., anti-rabies antibodies to detect rabies antigen) is labeled with a fluorescent dye, and is used to detect unknown antigen (e.g., rabies antigen) in the specimen. If antigen is present in the specimen, the same will bind the fluorescein labeled antibodies, and the antibody-bound fluorescein will emit a fluorescence, which will be observed by a fluorescent microscope using ultra violet radiations.
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3 Prepare alcohol or acetone fixed smears of S. pyogenes on the first slide, of E. faecalis on the second slide and of mixed unknowns on the third slide.
4 Add one drop of fluorescent antibody Streptococcus group A on to the slide one and fluorescent antibody Enterococcus group D to the second slide and spread gently over the surface of the smear.
5 In the third slide, label one side FA-A and the other slide FA-D and add one drop of fluorescent antibody Streptococcus group A to the side FA-A and one drop of fluorescent antibody Enterococcus group D to the side FA-D.
6 Allow to spread gently over the surface of the smear.
7 Place the prepared slides on the U-shaped glass rod kept in a petri dish and incubate at 25°C for 30 minutes.
8 Remove the slides from the petri dish and wash with 1%
buffered saline to remove away excess antibody.
9 Put the slides in a Coplin jar containing 1% buffered saline for 10 minutes at 25°C.
10 Blot dries the slides with paper.
11 Add one drop of buffered glycerol to each slide and cover with a cover slip.
Note: 1-10 µg/ml of phenylene diamine could be added to the mounting medium to prevent the fading of fluorescence of fluorescein.
12 Examine under a fluorescent microscope.
Table 48-1 Advantages and disadvantages of the immunofluorescence test
Advantages Disadvantages
More sensitive. Difficult to quantitate the test.
More specific when fluorescent dye is labeled to Requires UV radiation for visualization of the test result.
monoclonal antibodies. Requires expertise personnel.
It can avoid the danger of radiation-based hazards. Fluorescent dye fades faster.
Rapid method. Improper washing steps cause a very high background.
Useful for the identification of viruses. False positive reactions may occur.
Requires expensive equipment.
KEY FACTS
1 Immunofluorescence assays involve the use of either antigen or antibody labeled with a fluorescent substance.
2 Direct fluorescent antibody test is employed to detect the specific antigens.
3 Slides should be cleaned before use.
4 The smears should not be allowed to dry at any stage.
5 To minimize non-specific binding of proteins to cells or tubes, all dilutions and washings should be done in PBS containing 0.1 – 1% BSA.
6 1-10 µg/ml of phenylene diamine could be added to the mounting medium to prevent the fading of fluorescence of fluorescein.
VIVA
1 What is the principle of the direct fluorescent antibody test?
2 What are the uses of direct fluorescent antibody test?
3 Give examples of fluorescent dyes.
4 What are the advantages and disadvantages of the direct fluorescent antibody test?
QUALITY CONTROL
The slide with S. pyogenes and E. faecalis treated with fluorescent antibody Streptococcus group A and fluorescent antibody Enterococcus group D are used as known positive controls.
OBSERVATIONS
The slides are observed for the absence or presence of fluorescence in all the slides.
First slide with S. pyogenes : Positive for fluorescence.
Second slide with E. faecalis : Positive for fluorescence.
Third slide, the side labeled FA-A : Negative for fluorescence.
Third slide, the side labeled FA-D : Positive for fluorescence.
RESULTS AND INTERPRETATION
The unknown specimen contains the bacteria E. faecalis but does not contain S. pyogenes.
Radial Immunodiffusion Test
FURTHER READINGS
1 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworth, London, 94-96, 1995.
2 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC. Color Atlas and Textbook of Diagnostic Microbiology. 5th Edition. Lippincott Williams and Wilkins. 1997; pp. 1395.
3 Stites DP. Terr AI and Parslow TG. Medical Immunology. 10th Edition. 2001. pp. 902.
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After completing this practical you will be able to:
1 Know the importance of various steps involved in performing