lYIET~ODS

C. 1 GENERATION OF POLYCLONAL ANTIBODY TO LPG

Each rabbit was bled before immunization and this prebleed serum was

used as control antibody. After one week, purified LPG in PBS was emulsified with equal

volume of complete Freund's adjuvant. Routinely 20-30 J,lg of LPG was used to

immunize one rabbit each time. For booster immunization, same amount of LPG in . incomplete Freund's adjuvant was used at fifteen days interval. The rabbits were bled on

day 14 and 17 after the third and final immunization. The serum was stored in aliquots

at -70°C.

C. 2 METABOLIC LABELLING AND EXTRACTION OF LPG

Actively growing cells (2-5 X 105cells/ml) were incubated with

radiolabelled precursors [1I] galactose (50-100J,lCilml), [~ mannose (50-100J,lCilml),

[3H] fucose (50-100 J,lCilml), eH] palmitic acid (1.0 mCi Iml) and [32p] orthophosphate

(0.5 mCilml) in TYI - S- 33 medium for 1.5 to 3 h at 36°C. Forpulse/chase experiments,

cells were metabolically labeled with eH] galactose (200-300 J,lCilml) in glucose free

TYI-33 (TYI-S-33 minus serum and vitamin mix), with [3H] palmitic acid (1.5 mCilml)

in TYI -S-33 and [32p] orthophosphate (ImCilml) in phosphate free TYI -S-33 (buffered

with 20mM HEPES) for 5 or 15 min. After incubation the cells were normally collected

by chilling followed by centrifugation at 280 x g for 7 min and washed twice with

PB S # 8 before use.

LPG was purified by extracting the cells sequentially with 5 parts of

chloroform/methanol (3:2) and one part of 4 ruM MgCI2, 5 parts of

chloroform/methanol/water (10: 10:3) and one part of chloroform/methanol (1: 1) and

chloroform/methanol/water (10:10:3). The pellet was then extracted with solvent E

[waterl ethanol/ diethylether/pyridinel ammonium hydroxide ( 15: 15: 5: 1: 0.017)] three

times. The extracted material was dried under nitrogen to reduce the volume and finally

in a centrifugal evaporator under reduced pressure for complete drying. The dried

material was resuspended in a small volume of 0.1 M NaCl, 0.1 M acetic acid, briefly

sonicated and passed through a phenyl-Sepharose CL 4B column for further purification.

C. 3 PHENYL SEPHAROSE CHROMATOGRAPHY

Phenyl-Sepharose chromatography was carried out as described by

25

Bhattacharaya et al.(1992b). The solvent E extracted material was first dried under

nitrogen, dissolved in 0.1 M NaCl, 0.1 M acetic acid and was passed through

phenyl-Sepharose CL 4B (1m1 column volume) (Pharmacia, Sweden). The columns

were washed sequentially with 0.1 M acetic acid containing 0.1 M NaCI, 0.1 M acetic

acid, water and solvent E. 0.5 m1 fractions were collected and radioactivity determined

by liquid scintillation counting. The material eluted with solvent E was dried in a

centrifugal evaporator and used for further experiments. Ocassionally, material bound to

phenyl-Sepharose was eluted with a linear gradient ofn-Propanol (5-50%) in 0.1 MNaCl

and 0.1 M acetic acid.

C.4 METHANOL PRECIPITATION

LPG containing fraction eluted from phenyl-Sepharose CL4B column was

further purified by methanol precipitation (Singh et al., 1993a). Breifly, the LPG

containing sample was dissolved in 0.3 m1 of water. To this was added 0.3 m1 ethanol, 0.1

m1 diethylether and 0:02 m1 pyridine (solvent E minus NH.tOH). The LPG was then

precipitated by the addition of an equal volume of methanol followed by incubation at

-30°C for 48 h. The precipitated LPG was recovered by centrifugation and dried under

N2•

C. 5 CHEMICAL AND ENZYMATIC TREATMENT OF LPG

Typically the amount of LPG used for carrying out the following

treatments varied from 0.5-1.0 J,lg based on total hexoses.

C. 5.1 MILD ACID TREATMENT

LPG was hydrolyzed with mild acid as described by Turco et al. (1984).

Mild acid conditions of 0.02 M HCI, 5 min at 100°C were used to depolymerize the

molecule as phosphorylated saccharide repeat units from the carbohydrate core region.

The reaction was stopped by rapid cooling in ice water and neutralization of pH with 1 M

NaOH. The samples were dried under a stream of nitrogen. In some cases mild acid

treatment was carried out with 0.04M trifluoroacetic acid at 100°C.

26

C. 5.2 PI-PLC TREATMENT

PI-PLC digestion of LPG was carried out as per Low et al. (1987). The

LPG was resuspended in 200 J,llof 25 ruM HEPES, pH 7.4, 0.1 % CHAPS, 5 ruM EDTA

and incubated with 0.1 unit of PI-PLC from Bacillus thuringiensis

(Boehringer-Mannheim, Germany) at 37°C for 16 h.

C. 5.3 NITROUS ACID DEAMINATION

Deamination of LPG was carried out as described by Zamze et al. (1988).

The LPG was resuspended in 0.1 M sodium acetate (PH 4.0) containing 0.25 M sodium

nitrite and incubated either at 42°C for 2-4 h or overnight at room temperature. The

reaction was stopped by rapid cooling in ice water and neutralization of pH with 1 M

NaOH. The samples were dried under a stream of nitrogen.

C. 5.4 STRONG ACID HYDROLYSIS

hnl of 2N TF A was added to LPG, mixed thoroughly by vortexing and

was then placed in a boiling water bath for 3-4 h. After treatment, the sample was dried

under a stream of nitrogen. To remove TFA completely, sample was redried after

dissolving in 0.5 m1 of water.

C.5.5 PERIODATE OXIDATION

Periodate treatment was carried out by the method of Woodward et al.

(1985). LPG was incubated with 50 ruM sodium periodate in O.lM sodium acetate pH

4.5 for 30 min at room temperature in the dark. The excess periodate was neutralized by

incubating with 50 J,ll of 0.2 M ethylene glycol for 15 min at room temperature. The

reaction was stopped by adding 5-10 u1 of 1M Glycine to the reaction mix.

C. 5.6 PRONASE DIGESTION

LPG was digested with 35 J,lg of immobilized pronase CB (Pierce, USA)

for 30 min at 37°C and the enzyme was removed by brief centrifugation in a microfuge.

The supernatant was analyzed by Western blotting as described later.

27

C.S.7 PROTEINASE K DIGESTION

To remove tightly associated polypeptides attached to LPG, it was

incubated with proteinase K (l/8th volume of20 mg/ml stock) in IX SDS- PAGE sample

buffer at SSoC for 3 h before being subjected to SDS PAGE analysis.

C. 5.8 ALKALINE PHOSPHATASE DIGESTION

Dephosphorylation of LPG was performed either enzymatically or

chemically. Breifly, LPG fraction was digested with S units of Alkaline phosphatase

(Sigma Type VIII - N) in 100 III of 100 mM NRtHC03, pH 8.0 for 16 h at 37°C.

Dephosphorylation was also carried out by resuspending dried LPG in SO% HF and

incubation at OOC for 40 h.

C.5.9 PHASE SEPARATION OF LPG

Phase separation was carried out according to published procedure

(Bhattacharya et al., I990b). Briefly, purified LPG was incubated at 4°C for 30 min in

phase separation buffer (10mM Tris Cl pH 7.S, ISO mM NaCl and 1% Triton X - 114).

The samples were then warmed at 37°C for 3 min and centrifuged to separate the two

phases. Both the detergent and aqueous phases were separately washed with wash buffer

by repeating the steps described above and analyzed by SDS-PAGE. In some experiments,

Triton X-114 phase extraction was carried out with CS S] methionine-labelled cells after

detergent extraction of cells with TNT buffer (10mM Tris- Cl pH 7.S, IS0mM NaCl,

1 %(w/v) TritonX-114, 2mM Phenyl methyl sulfonyl fluoride, SmM Parahydroxy mercuric

benzoate, SO Ilg/mlleupeptin, ImM EGTA) for 30 min at 4°C. The lysate was centrifuged

at 40,000 g for 30 min at 4°C. The supernatant solution was incubated at 37°C for 4-S

min and then centrifuged at 12,000 rpm for I min. The pellet was washed with TN (TNT

minus TritonX-114) two times and dissolved in 2 X SDS-PAGE sample buffer and

analyzed by SDS-PAGE.

C. 5.10 CALCIUM TREATMENT OF E. histolytica CELLS

The cells were incubated in phosphate free TYI-S-33 medium (buffered

with 20mM HEPES) for 30 min prior to addition ofSmM CaC12 and SIlg/mlA23187. At

indicated times, labelled precursors were added to the labeling media. A parallel set of

28

cells incubated with only DMSO (solvent for ionophore) were used as controls.

C. 6 TCA PRECIPITATION OF GLYCOPROTEINS

[3H]-galactose labelled cell pellet was resuspended in 1 ml of chilled

PBS # 8 containing 10% (w/v) trichloroacetic acid (TCA), and was incubated on ice for

30 min. Precipitates were collected on glass fibre filter discs (GF/C Whatmann), washed

with 20-30 ml of 5% chilled TCA and 15ml distilled water. To get rid of the lipids from

the TCA precipitate, the filters were first washed sequentially with 2 ml of chloroform :

methanol (1:2, 1:1 and 2: 1), air dried and the level of radioactivity determined by

scintillation counting.

C. 7 EXTRACTION OF TOTAL GLYCOLIPIDS

eH] galactose labelled cell pellet was resuspended in Iml of chilled

PBS # 8 and extracted with 4ml of CHCI3:CH20H (1:2). The layers were separated by

centrifugation at 280 x g for 5 min after vigorous shaking. The top organic layer was

removed and 4ml CHCl3 : CH20H (1:1) was added to the bottom layer. The suspension

was vigourously shaken and centrifuged once more. The procedure was repeated using

4ml of CHCl3 : CH20H (2: 1). Pooled extracts from all the fractions were dried and the

level of radioactivity determined by scintillation counting.

C. 8 FRACTIONATION OF SUBCELLULAR COMPONENTS

Cells were fractionated into different subcellular compartments (plasma

membrane, cytosol and internal vesicles) essentially as described before (Aley et al.,

1980). Briefly, cells (2 x 107 cells/ml) were resuspended in 1 mlPBS # 8 containing 10mM

MgCl2 and rapidly mixed with equal volume of Img/ml concanavalin A. The suspension

was incubated for 5 min and was centrifuged gently at 50 x g for 1 min. The cell pellet

was resuspnded in 4 ml of buffer (10 mM Tris CI pH7.5, 2mM PMSF, ImM MgCI2),

incubated on ice for another 10 min and then homogenized by 18-20 strokes o~ a glass

Dounce homogenizer. The cell homogenate was layered over a two step gradient (8 ml

0.5 M mannitol, 4 ml of 0.58 M sucrose) and was centrifuged at 250 x g for 30 min. The

plasma membrane was collected as the pellet. The supernatant was further centrifuged at

40,000g for 1 h to collect internal vesicles as a pellet. The supernatant obtained after the

29

last centrifugation was used as the cytoplasmic fraction.

C.g IMMUNOFLUORESCENT LOCALIZATION OF LPG

E.histolytica trophozoites were fixed over glass microscopic slides for 10

min in chilled methanol. The slides were then washed thrice, 5 min each, with chilled PBS

# 8. The fixed cells were incubated with polyclonal aLPG HM-l antibody (1: 100 dilution)

for 1 h at room temperature followed by washing with PBS # 8 three times. The antigens

were detected by incubation with 1:40 diluted fluoroscein iso - thiocyanate (FITC)

conjugated goat anti - rabbit IgG (Sigma,USA) and were mounted with 70% glycerol in

PBS # 8 containing 0.1% 2,5 - diphenyl - 1,3,4 - oxadiazole (PPD). Zeiss microscope

with fluorescence attachment was used for visualization.

C. 10 IMMUNOPRECIPITATION

Immunoprecipitation from detergent solubilized amoebae was carried out

exactly as previously described (Bhattacharya et al., 1990b). Radiolabelled and purified

LPG was incubated with monoclonal Ab 2D7.10, polyclonal aLPG HM-l antibody,

human patient sera and their respective control antibodies for 2 h at 4°C in

immunoprecipitation buffer (10 mM NaCI, 0.1% Triton X-I00, 0.1% ovalbumin and

0.05% NaN3). The immune complexes were separated by incubation with Protein

A- Sepharose 4B (Pharmacia, Sweden) and were washed three times with 10mM

Tris - CI pH7.5; 150 mM NaC~ 0.1 % ovalbumin, twice with 10mM Tris -CI pH7.5, 150

mM NaCI and once with 10mM Tris - CI pH 6.8. The bound antigens were eluted from

Sepharose beads by SDS-PAGE sample buffer.

C. 11 SODIUM DODEYCL SULPHATE POLYACRYLAMIDE GEL

ECTROPHORESIS (SDS-PAGE)

Discontinuous SDS-PAGE was carried out under reducing conditions

according to Laemmli (1970). The separating gel was 10% acrylamide in 1.5 M Tris-Cl

pH 8.8, 0.1% (w/v) SDS, 0.04% (w/v) APS and TEMED. The stacking gel was 4%

acrylamide in 0.5 M Tris-Cl pH 6.8, 0.1% (w/v) SDS, 0.04% (w/v) APS and TEMED.

Samples were diluted With an equal volume of 2X SDS-PAGE sample buffer [125 mM

Tris-Cl pH 6.8,4% (w/v) SDS, 10% (v/v) 2-mercaptoethano~ 20% (v/v) glycero~ 0.2%

30

(w/v) bromophenol blue] and placed in a boiling water bath for 4 mm before

electrophoresis.

C. 12 FLUOROGRAPHY AND AUTORADIOGRAPHY

Gels containing [3H] labelled samples were subjected to fluorography

according to Laskey and Mills (1975). The gels were first saturated with DMSO for 1 h

followed by incubation for 3 h with 20% PPO (w/v) in DMSO. Gels were washed with

cold water to remove excess PPO and DMSO. The dried gels were exposed to X-ray

films. Gels containing [32P]_labeled materials were dried and exposed to X-ray films in

presence of intensifying screens without any processing.

C.13 ENZYME - LINKED IMMUNOSORBENT ASSAY (ELISA)

ELISA was carried out according to Bhattacharya et af. (1990a). 0.5~g of

LPG (based on hexose) in PBS was used to coat each well of a microtiter plate (Costar,

. USA) overnight at 4°C. Nonspecific sites were blocked with 3% (w/v) gelatin in TBS

(20 mM Tris-Cl pH 7.5, 500 mM NaCl) for 1 h at room temperature. All incubations

with antibodies were carried out for 2 h at room temperature in 1% gelatin-TBS. The

wells were washed thoroughly with TBS after each treatment. aLPG HM-l or human

patient sera were used as first anitbody. The amount of bound first antibody was detected

with appropriate alkaline phosphatase-labelled second antibody usmg

p-nitrophenylphosphate as substrate. OD was measured at 405 or 411 nm.

C. 14 WESTERN BLOTTING

Prewarmed purified LPG samples were treated with preheated 2X SDS­

PAGE sample buffer (Laemmli, 1970). Polyacrylamide gels (10%) were used for all

separations. Typically 0.5 ~g of LPG (based on hexose) was used for each analysis. After

separation, the molecules were transferred to nitrocellulose membrane electrophoretically

according to Towbin eta!' (1979). The transfer was carried out at 400 mAfor 4 h at 4°C

in Tris-glycine buffer containing 20% methanol. After transfer, the membranes were

incubated overnight in 3% gelatin at room temperature to block the non specific binding

sites. The immunoreactive molecules were located using either anti LPG monoclonal or

polyclonal antibody or human patient sera and alkaline phosphatase-conjugated

31

appropriate second antibody (Bio-Rad, USA). Colour development utilized Nitro Blue

Tetrazolium and 5-Bromo-4-Chloro-3-Indolyl Phosphate as substrates.

C. 15 PAPER CHROMATOGRAPHY

Paper chromatography was performed as described by Turco et al. (1984).

Aliquots of CH]galactose-labelled purified LPG were depolymerized and

dephosphorylated as described before. The digested samples were chromatographed on

paper (Whatman no. 1) for 16 h in n-butyl alcohol : pyridine : water (6:4:3).

Quantification of radioctivity was carried out by cutting 1 cm strips of the paper after

soaking in 0.6 m1 of 0.1 % SDS followed by scintillation counting. Standard sugars were

galactose (monosaccharide), lactose (disaccharide) and raffinose (trisaccharide) and

stachyose (tetrasaccharide). These were detected by staining with orcinol-sulphuric acid

(raffinose and stachyose) and alkaline silver nitrate (galactose and lactose).

C.16 DE-52 CELLULOSE CHROMATOGRAPHY

This was carried out to further purify LPG and performed essentially as

described by Turco et al., (1984). Phenyl- Sepharose CUB purified radiolabelled LPG

was treated with mild acid and neutralized with 40mM ~OH. The digested samples . were dried by evaporation under nitrogen, resuspended in 1 mM Tris-CI, pH 8.0 and

applied to a column of DE-52 cellulose (0.5 cmX 1 cm) equilibrated in 1 mM Tris'-Cl pH

8.0. The column was then washed successively with one column volume of deionised

water, two column volumes of methano~ one column volume of 1% sulphuric acid in

methano~ followed by, one column volume of2.5% sulphuric acid in methanol. LPG was

finally eluted with O.IN NaCl in ImM Tris Cl pH 8.0. Purified LPG was then subjected to

HPLC as described later.

C. 17 BIOGEL P - 4 CHROMATOGRAPHY

The radiolabelled glycans after nitrous acid deamination and end labelling

with NaBCH]4 reduction were separated on a water jacketed Bio-gel P4 column (1 x 100

cm) (BIORAD, USA) in water at 50°C and at a flow rate of 4.5 mlIh. The size of[3H] -

labelled glycans was determined by their elution position relative to the co-injected sugar

standards expressed in glucose units.

32

C. 18 CONCANAVALIN A CHROMATOGRAPHY

Purified LPG was allowed to bind to Con A Agarose column (hnl) in

binding buffer (lOmM Tris CI pH 7.4, O.IM NaC~ ImM MgCI2, ImMCaCI2 , ImM

MnCI2). The column was then washed with 15 column volume binding buffer before

elution with 100mM amethyl D manno-pyranoside. One m1 fractions were collected and

the level of radioactivity was determined by scintillation counting. Bound and eluted

fractions were also desalted by passage through Sephadex G-25 column, freeze dried and

analyzed by SDS-PAGE.

C. 19 HPLC

LPG was also fractionated by size exclusion HPLC (Waters) using a UV

detector set at 215 nm (which detects n to 7t transitions of the phosphodiester bond) and

a 125 SW column (Waters) with a cut offat 30 kDa. The column was eluted for 30 min

in 0.1 M phosphate buffer pH6.8. at a flow rate of Im11min. One m1 fractions were

collected and the position of LPG was identified by scintillation counting.

C.20 HpTLC

Radiolabelled neutral glycans (5,000 cpm) were resuspended in 4 J1,1 of

40% I-propanol. It was applied in 1 J1,1 aliquots to Silica gel 60 precoated aluminium

backed HpTLC plates and dried in between each application. The plate was developed

twice in two different solvents :

Solvent A: 1- propanol:acetone:water (9:6:5)

Solvent B : 1- propanol: acetone: water (5:4:1)

The plate was finally air dried and sprayed with EN3 Hance (NEN) and

exposed to X-Ray film. The carbohydrate standards were developed by spraying with

orcinol sulphuric acid spray.

C. 21 LH] SODIUM BOROHYDRIDE REDUCTION

Purified LPG was delipidified by nitrous acid deamination as described

previously. The deaminated product was dried under vacuum and resuspended in 20J1,1

distilled water and 100J1,1 NaBCH)4 (sp activity - 25.0 mCilm mol).The incubation was

carried out for 2.0 h at 25°C and stopped subsequently by two additions of 68J1,1 of IN

33

acetic acid. The reaction mixture was freeze dried, resuspended in 1O01l1 of H20 and was

desalted by passage through a column of AG50 X 12(H+) (2001l1) over AG3 X 4(OH)

(2001l1). The eluted fraction was concentrated and subjected to Biogel P-4 (Bio Rad,

USA) chromatography and HpTLC(Merck, Germany).

c. 22 IEF (ISOELECTRIC FOCUSSING)

Isoelectric focussing was perfonned according to 'Isoelectric focussing,

principles and methods', published by Phannacia Fine Chemicals AB, Uppasala, Sweden.

IEF was perfonned in the tube gel format and the separation was carried out in a pH

gradient which is established between the two electrodes.

The gel mix prepared consisted of 9M Urea, 4.4% acrylamide, 2.2% Triton

X - 100 (w/v), 0.01% APS, 0.078% TEMED (v/v), 0.8% ofampholine pH 5-7 and 0.1%

of ampholine pH 3.5-10. Sample overlaying buffer consists of 8M Urea, 0.8% of

ampholine pH 5-7 and 0.2% of ampholine pH 3.5 - 10. IEF sample loading buffer

consists of9 M Urea, 2.5% of Triton X 100 (w/v),2f3 - Mercaptoethanol (v/v), 0.8% of

ampholine pH 5-7 and 0.1 % of ampholine pH 3.5-10. Upper tank served as cathode

containing 0.02N NaOH and lower tank serves as anode containing 0.01 M phosphoric

acid. Polymerization was carried out in tubes and were overlayed with deionized water.

After polymerization the water was replaced with 20 III of IEF sample buffer. The gels

were first electrophoresed at 200 V for 15 min, 300 V for 30 min and 400 V for 30 min

followed by electrophoresis with 40111 of sample and 20 III of sample overlay buffer. The

gels were normally run for 12-16 hat 300-400V and at 800V for 1 h before termination.

The pH gradient was checked by cutting one of the gels into 12 pieces of

1 cm each and incubated separately in 5 m1 of deionized and degassed water for 45 min.

They were then mixed well and the pH was determined. The sample tube was similarly

processed and pieces were incubated in 0.6m1 of 1.0% SDS for 1 h before scintillation

counting.

C.23 NEPHGE [NONEQUILIBERATED pH GRADIENT GEL

ELECTROPHORESIS ]

Samples were analyzed by NEPHGE gels as described by O'Farrell

(1975). The gels were polymerized with 20111 of APS and lOll1 of TEMED per 10 m1

34

of gel mix (Gels containing basic ampholines tend not t~ polymerize well). Rest of the

components of the gel mix were same as those of gel mix prepared for IEF, except only

ampholine pH 5-7 (1. 0%) was used. NEPHGE gels were electrophoresed without a pre­

run and with cathode at the bottom and anode on the top (polarity is reversed as

compared to IEF gels) for 4-5 h at 400 V for a total of 1600 - 2000 Volt-hour. The tube

gels, were processed the same way as descnoed for IEF gels.

c. 24 HEXOSE ESTIMATION

Total amount of hexose present in LPG samples was estimated as per

Wingler (1955) using orcinol-sulphuric acid method. 1.6% orcinol in water and

60% sulphuric acid (w/v) were mixed in the ratio of 1:7.5 to make the working reagent

which was added to suitably diluted LPG samples and incubated at 80°C for 20 min,

cooled to room temperature and OD determined at 540 nm. Glucose (0.1-5.0 J.lg) was

used as standard. All measurements were carried out in triplicate.

C.25 PREPARATION OF TOTAL CELL LYSATE

Total lysates of E. histoiytica cells were prepared as described

(Bhattacharya et ai., 1990b). The cells were harvested as described before and washed

three times with PB S # 8 to remove media components. The cells were lysed by

hypotonic shock (1 OmM Tris-CI pH7. 5, 2mM PMSF, 1mM PCMB, 4J.lg/m1leupeptin) for

10 min followed by sonication for 30 seconds to shear the DNA. The lysates were stored

as aliquots at -70°C.

C. 26 PROTEIN ESTIMATION

All protein estimations were carried out using bicinchoninic acid reagent

(Pierce chemical company, U.S.A) (Smith et ai., 1985). The solution B was prepared by

mixing 50 parts of solution A (Bicinchoninic acid reagent) and 1 part of 4% (w/v) CuS04.

In all estimations 100J.lI of sample was mixed with an equal volume of solution B in a

microtitre plate. The absorbance was determined at 542 nm after 30 min. Bovine serum

albumin was used as standard.

35

C. 27 PHOSPHATE ESTIMATION

The level of phosphate in LPG was determined by following the method

of Lindberg and Ernster (1956). Briefly, colour developing reagent was prepared by

mixing 5N H2S04 , 0.25 % ammonium molybdate and 0.01 % reducing reagent which is

a mixture of 1 amino - 2 - naphthol 4 sulfonic acid (0.2g), sodium bisulfite (1.2g) and

sodium sulfite (1.2g). 2.6 mg of this mixture was dissolved in 54 m1 of distilled water.

Potassium dihydrogen phosphate (KH2P04) (0.1 - 1.01lM) was used as standard. In the

final reaction, standard phosphate and LPG suspension were mixed with sulphuric acid,

ammonium molybdate, reducing reagent and water in the ratio of 1:1:1:0:1:6.9. The

mixture was incubated at room temperature till the colour develops, and the O.D was

measured at 66Onm.

C. 28 GROWTH OF E. bistolytica IN PRESENCE OF y- IRRADIATED

E. coli AND GASTRIC MUCIN

E. coli strain DH5a and gastric mucin were used to study their effect on

expression of some key antigens of E. histolytica. DH5a cells were grown overnight in

50 m1 of LB. Cells were harvested, washed with normal saline and finally resuspended in

15m1 of normal saline. These were then irradiated with 500 k Rads ofy-radiation and were

mixed with axenic E. histolytica cultures grown for 24 h. 106 or 107 E. coli cells were

added to each culture tube containing 13m1 of E. histolytica growth medium. Cell density

ofE. coli was determined by measuring the O.D at 600 run. Gastric mucin was added to

the final concentration of 100 mg or 200 mg/IOO m1 to the E. histolytica culture grown

for 24 h. In another case axenic E. histolytica culture adapted with human gut flora for

8-10 generations was used.

36