15. Immunofluorescence


A. Principle :


Target antigens in parasites, tissues or cells against specific antibodies can be localized by incubating them directly with fluorochrome labeled specific antibodies or indirectly using second antibodies labeled with fluorochrome. Fluorochromes emit fluorescence when excited with ultraviolet radiation.

Fluorescein is the most widely used fluorochrome. The protocol describes indirect   fluorescent antibody test (IFAT) on intact filarial parasite.


B. Materials :


a.Brugia malayi infective (L3) larvae/ microfilariae ( mf ) / adult worms.


b.Anti-human IgG   


c.Test sera.


d.Fluorescein isothiocyanate (FITC)


e.PBS, 0.01M, pH 7.4 & 0.05 M pH 7.4 (Appendix-I)


f.0.145 M Sodium chloride solution (850 mg in 100 ml Distilled water).


g.0.2 M Na2 HPO4 2H2O solution (3.5 g in 100 ml Distilled water).


h.0.1 M Na2HPO4 2H2O solution (1.75 g in 100 ml Distilled water).


i.0.1 M Na3PO4 solution (3.8 g in 100 ml Distilled water).


j.Sephadex G-25 column.


k.Mounting solution (50% glycerol, 50% PBS & 0.1% Sodium azide).


l.Evan's Blue (0.1%)  



C. Method :


a.Anti - human  IgG antibody - FITC  Conjugate :


1.ake 250 µl (2.5 mg protein) of anti-human IgG antibodies in a plastic vial and dilute to 1 ml with 0.145 M sodium chloride solution.


2.Add 500 µl of 0.2 M Na2HPO4 dropwise while stirring.


3.Add 1.0 ml FITC solution (12.5 µg FITC/ mg protein), in 0.1 M Na2HPO4


4.Adjust the PH to 9.5 using 0.1 M Na3PO4


5.Incubate the mixture at 250C for 45 min.


6.Apply the conjugate mixture through sephadex G-25 column (2x30cm) and elute with 0.01M  PBS (pH 7.2)  at  a flow rate of about 30 ml/min. Collect the first eluted  yellow colored fractions that contain anti-human IgG antibodies conjugated with FITC.


7.Concentrate  FITC conjugate  by  ultra-membrane  filtration,  add preservative sodium azide (0.1%) and store at 40C.  


b.Immunofluorescence  assay :


1.Incubate about 10 L3 larvae/ 100 microfilariae  ( mf ) /  2 adult  worms  with 200 µl  of optimally diluted (1:10) test sera in PBS (0.05 M) in a conical vial at room temp. for 1 hr.


2.Wash the parasites with PBS two times using a centrifuge at 500 RPM for 2 min.


3.Add 20 µl of optimally diluted (1:10 in 0.05 M PBS) anti-human IgG-FITC conjugate and incubate for 30 min at 40C.


4.Wash the parasites with 0.05 M PBS once. (500 rpm for 2 min)


5.Add 5 µl of 0. 1 % evan's blue.


6.Wash the parasites with 0.05 M PBS once. (500 rpm for 2 min)


7.Add 5 µl mounting buffer, transfer the parasites onto a glass slide, place coverslip  and seal with nail polish.


8.Examine under fluorescence microscope and  compare  the yellow-green  fluorescence with  the parasites  incubated  with positive  sera  compared to  those incubated  with normal sera.