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.