CHAPTER 4 IMMUNOLOGICAL TECHNIQUES

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1 CHAPTER 4 IMMUNOLOGICAL TECHNIQUES

2 Nitroblue Tetrazolium Chloride (NBT) Reduction test NBT reduction test was evaluated by employing the method described by Hudson and Hay,1989 based upon principle that whenever a particle is ingested by a phagocyte, a respiratory burst is induced. This test is employed to measure the macrophage/neutrophil function. The assay is based on the reaction that the addition of yellow color NBT dye to splenocyte suspension results in the formation of complex which can be phagocytosed by neutrophils/macrophages. The yellow colored NBT is reduced to blue colored formazone which can be extracted in dioxin/pyridine and hence measured spectrophotometrically at 520 nm using dioxan as blank. 1. Phosphate Buffer Saline (PBS) 0.15 M, ph = mm NBT in 0.15 M PBS containing 34 mm sucrose N HCl 4. Dioxin: Commercially available (Ranbaxy Pvt. Ltd.) Briefly, took 1 ml of splenocyte suspension (2 x 10 6 cells/ml) in each test tubes marked as test and a control. Added 0.1 ml NBT in tube marked as test containing different number of cells as 1 x 10 6 cells ml -1 and 1 x 10 9 cells ml -1 for each strain. Incubated both the tubes for 20 minutes at 37 C, centrifuged it and washed the pellets with PBS buffer. Then, added 0.1 N H Cl to stop the reaction, again centrifuged for 10 minutes at 400 g and removed the supernatant and added 5 ml dioxan to both test and control marked tubes. Incubated the tubes for 20 minutes at 70 C and then centrifuged, noted the absorbance of supernatant at 520 nm, using dioxan as blank.

3 % NBT reduction : O.D. of test (T) O.D. of control (C) 100 O.D. of control (C) Inducible Nitric Oxide Synthase activity The inducible nitric oxide Synthase activity was assessed as described by Stuehr and Marletta (1987) based upon the principle that when macrophages are activated they together with T cell derived IFN-gamma begin to express inos which oxidizes L-Arginine to citrulline and nitric oxide. The dark purplish colour so formed is extractable with griess reagent which can be measured spectro-photometrically at 450 nm using griess reagent as blank. 1. L-Arginine:0.2M in normal saline (0.85 % NaCl) 2. Griess reagent: (a) NEDD (Napthyl ethylene dianiline dihydro chloride): Dissolved 0.1g of NEDD in 100 ml sterile distilled water. (b) Sulphanilamide: Dissolved 1 % solution of sulphanilamide (1g in 100 ml distilled water) in 100 ml in 2.5 % orthophospheric acid The Griess reagent was prepared by mixing above solution (a) and (b) in ratio 1:1. Inducible nitric oxide Synthase activity was assessed using Griess reagent. Briefly, splenocytes were incubate with different number of cells as 1 x 10 6 cells ml -1 and 1 x 10 9 cells ml -1 for each strain at 37 C for 24 hour in humidified 5 % CO 2 atmosphere. To the adherent cells mixture added freshly prepared 1 ml MEM and 20 µl of L-arginine solution (0.2M), incubated at 37 C for 24 hour in humidified 5 % CO 2 atmosphere, centrifuged and collected the supernatant.

4 The supernatant was treated with freshly prepared Griess reagent and kept in dark for 10 min. Absorbance was measured spectro-photometrically at 540nm against control. Bactericidal activity (Raghuramulu et al., 1983) of splenocyte. Bactericidal activity is one of the important parameter to measure the Phagocytic function 1. Nutrient medium: 10g Beef extract, 10g peptone and 1 g of NaCl dissolved in distilled water and volume made one litre. 2. Nutrient Agar: 1g Beef extract, 2g yeast extract, 1g NaCl, 1g peptone and 15g agar dissolved in distilled water and final volume made to 1 litre. 3. Kreb s Ringer Phosphate (KRP) buffer: 100 parts of 0.9% NaCl, 4 parts of 1.15 % KCl, 3 parts of 1.22% CaCl 2, 1 part of 3.82 % Mg SO 4.7H 2 O and 20 parts of phosphate buffer dissolved in distilled water. Phosphate buffer was prepared by dissolving 17.8g Na 2 HPO 4 and 20 ml of 1N HCl in 1 litre distilled water. The ph of KRP buffer was 7.4 without further adjustment. E.coli grown in nutrient broth for h at 37 C was collected and centrifuged. The pelleted bacteria was washed thrice in KRP buffer and finally suspended in saline and standardized the number of E.coli ( ) by measuring absorbance at 600 nm. Took 3ml of suspension of splenocytes under sterile conditions, centrifuged at 800 g for 5 min and washed the cells with buffer. Took two test tubes, in one test tube (marked test) added 500 µl of splenocytes /ml and 500 µl of different number of cells as 1 x 10 6 cells ml -1 and 1 x 10 9 cells ml -1 for each strain and 80 µl of E.coli ( cells/ml) and in other test tube (marked control) added only 80 µl E.coli ( cells/ml). Volume was made 2 ml by adding KRP buffer. After incubation at 37 C for 1

5 hour, took 0.2ml of reaction mixture from test tube and added 0.8 ml of distilled water to lyse the splenocytes. Diluted each sample 10 8 times with normal saline, counted number of viable bacteria by plating diluted sample onto agar plates after 24 h at 37 C. Calculated the percent phagocytosis with the following formula: % Bactericidal Activity: CFU/ml in control CFU/ml in test 100 CFU/ml in control Direct Haemagglutination (Haghighi et al., 2005) Reagents 1. Phosphate Buffer Saline 2. Sheep Red Blood Cells Briefly serum sample were incubated at 56 C for 30 min to inactivate the complement. 50µL of PBS containing 0.05% BSA was dispensed into each well of round bottomed 96- well microplate. Serum samples (50 µl) were then added and serially double diluted in the wells from columns 2 to 12. The first column (PBS only) of wells was considered as blank. Then, 50 µl of 1% SRBC in PBS was added to all the wells to make a 100 µl final volume. Subsequently, the plates were shaken for 1 min and incubated for 24 hrs at 37 C and agglutination was monitored visually. Highest dilution capable of visible agglutination was considered as the antibody titer. The results were expressed as mean ± S.E.M log 2 titer o individual animal. Cell mediated immune response Delayed Type Hypersensitivity assay (Titus and Chiller, 1981) Reagents

6 1. Normal saline (0.84% NaCl) 2. Sheep Red Blood Cells All SRBC primed groups were challenged intradermally on day 15 th with SRBC suspension ( /100µl saline) in the hind footpad. The control lateral paw was given equal volume of saline. Paw thickness was measured with micro-caliper at 24h interval up to 72h. The difference in paw thickness compared to control was taken as a measure of DTH and expressed in millimeter. Results are expressed as mean ± S.E.M. of footpad thickness up to 72h expressed in millimeter. Detection of anti BSA antibodies by Indirect ELISA (Haghighi et al., 2005) 1. PBST (PBS with 0.05% Tween 20) 2. Blocking buffer (PBST containing 0.25% gelatin) 3. p-nitrophenylphosphate solution For evaluation of serum antibodies, each well of a flat-bottomed 96-well microplate was coated overnight with 100 μl coating buffer (ph 9.6) containing BSA (30 μg/ml) at 4 C. The wells were washed three times with 200 μl PBST with complete decanting between each wash. Subsequently, 100 μl of blocking buffer was added to each well and incubated for 1 h at 37 C to occupy all unbound sites. Washing was repeated as described above, followed by the addition of 100 μl mouse serum, diluted 1:100 in blocking buffer, to each well. Plates were incubated for 2 h at room temperature and then washed three times, and 100 μl of goat anti-mouse IgG-Fc or goat anti-mouse IgM conjugated with alkaline phosphatase (diluted 1:500 in blocking buffer) was added to each

7 well and incubated for 1 h at room temperature (RT) before the plate was washed three times. 100µL p-nitrophenylphosphate solution was added as the substrate to each well and incubated for 1 h at room temperature in the dark. The absorbance was measured at 405 nm using a microplate reader. Cytokine estimation to study the expression of IL-4 and IFN-gamma by Enzyme Linked Immunosorbent Assay (ELISA) Provided by the manufacturer Cytokine estimation was performed in cell culture suspension as per the instructions of the manufacturer (BD OptEIA set) by ELISA. Briefly, 96-well microtiter ELISA plate were coated overnight with 50µL of capture antibodies in carbonate bicarbonate buffer, ph 9.6 at 4 C. After washing, the wells were incubated with 200µL of blocking buffer for 1 h at room temperature and followed by addition of 100 µl serum samples. The respective cytokine as standard were used in triplicates. Plates were kept at room temperature for 2h, washed five times with wash buffer.biotin labeled secondary antibody was added along with avidin horse radish peroxidase (AV-HRP). The wells were washed and substrate, tetramethylbenzidine and hydrogen peroxide were added to each well and incubated at room temperature for the development of color. The reaction was stopped with 1M H3 PO4 and plates were read at 450 nm. The cytokines were quantified using recommended cytokines standards (BD Opt EIA set).