|Title of Invention||
A PROCESS FOR THE PREPARATION OF BRUGIA MALAYI MICROFILARIAL EXCRETORY-SECRETORY (mfES-22) GLYCOPROTEIN.
|Abstract||"A process for the preparation of brugia malayi microfilarial excretory-secretory (mfES-22) glycoprotein" This invention relates to a process for the preparation of brugia malayi microfilarial excretory-secretory (mf ES-22) glycoprotein for use in diagnosis and monitoring of active filarial infection comprising. Collecting brugia malayi microfilariae by lavage of the peritoneal cavities of jirds-with an intraperitoneal filarial infection of 3 months or more duration. Centrifuging peritoneal fluid at lOOOg for 15 mins, suspending the mf pellet in sterile normal saline (0.9% NaC1) and repeated washing with RPMI-1640 medium. Plating the mf at 37°C for 1 hr to remove peritoneal exudate cells. Maintaining about ten lakhs of mf in RPMI- 1640 medium supplemented with malic acid, a keto glutaric acid, D-Succinic acid and fumeric acid at concentrations of 670, 370, 60 and 55 mg per litre respectively and sucrose and fructose at concentrations of 26, 680 and 400 mg per litre respectively, 0.1 mM phenyl methyl sulphonyl fluoride at temperature of 28-37°C in an atmosphere of 5% CO2 for 48 hrs. Centrifuging at 1000g for 20 mins followed by concentrating the supernatant by ultra membrane filtration using Dialysis tubing and dialysing against 0.1M sodium phosphate buffer, pH 7.2. (MONA SAINI) OF L.S. DAVAR & CO., APPLICANTS ATTORNEY|
|Full Text||THE PATENTS ACT. 1970
"A process for the preparation of brugia malayi microfilarial excretory-secretory (mfES-22) glycoprotein"
KASTURBA HEALTH SOCIETY, SEVAGRAM, WARDHA 442 102, MAHARASHTRA, INDIA
THE SECRETARY, DEPARTMENT OF BIOTECHNOLOGY, B-2, 7-8 FLOOR, CGO COMPLEX, LODHI ROAD, NEW DELHI-110 003
The following specification particularly describes the invention and the manner in which it is to be performed.
This invention relates to a process for the preparation of brugia malayi microfilarial excretory - secretory (mf ES-22) glycoprotein for use for diagnosis and monitoring of active filarial infection and morbidity controlled in clinical filariasis.
Human lymphatic filariasis is caused by the infection with major nematode parasites Wuchereria bancrqfti and Brugia malayi. Recent estimates of global prevalence of lymphatic filariasis revealed that there are about 106.2 million people with W. bancrqfti and 12.9 million with B. malayi and B.timori infection. The disease burden amounts to about 850,000 DALYS (Disability Adjusted Life Years) lost. India contributes to 38% of the global disease burden (Report of a WHO/CTD/TDR consultative meeting held at the University Sains Malaysia, Penang, Malaysia, 22-24, Aug. 1994). In India filariasis has been the major vector borne public health problem next only to malaria. As per the estimates made in 1994 about 412 million people are living in bancroftian endemic areas in 13 states and 5 UTS (NICD & NMEP, Revised strategy for the control of lymphatic filariasis in India-Report and Recommendations of the WHO sponsored Workshop, New Delhi 4-5, Jan 1996). Bancroftian filariasis transmitted by Culex quinquefasciatus mosquitoes has been the most predominant infection contributing 99% of the disease problem in the country. About 26.9 million people are estimated to be harboring microfilariae and about 20.4 million suffer from chronic manifestations like hydrocele, lymphoedema, epididymo-orchitis and elephantiasis. Those with acute filarial attacks (recurrent fever associated with acute adenolymphangitis - ADL) and occult filarial infections (e.g. Tropical pulmonary eosinophilia, tenosynovitis, arthritis, acute abdomen, urticaria etc.) were observed to be almost two times more than those with overt manifestations of the disease in filaria endemic area (Harinath et al. Indian J. Clin Biochem 14; 1999).
There are certain disadvantages associated with the conventional diagnostic method based on detection of microfilaraemia.
Another disadvantage is that the microfilaraemia are not usually seen in the peripheral circulation in acute, chronic and occult filariasis (Harinath and Reddy, J Parasitic Dis., 21 :41-51; 1997).
Lack of an established and simple animal model for W. bancrofti infection has necessitated the use of antigens derived from other filarial parasites particularly from its closely related lymphatic filarial parasite B. malayi in immunodiagnostic assays aimed to detect bancroftian filariasis. Recently monoclonal antibody based immunoassays have been developed for filarial antigen detection (Weil et al., J. Infectious Dis., 156:350-355, 1987 and More and Copeman, Trop Med Farasitol., 41 : 403-406; 1990). Though these assays have been found to be quite simple and useful to detect mf carriers, they generally failed to detect most of the clinical cases. The main limitation of the majority of the other immunoassays reported has been the poor sensitivity and specificity arising from not using specific and purified antigens/antibodies.
Microfilariae, one stage of filarial parasite are observed in blood circulation in active filarial infection and hence have been target of detection methods. Secretory proteins are likely to provide the first stimulus in vivo for the humoral and cellular response to filarial parasites and may be valuable in a serological diagnostic test. In our laboratory enzyme immunoassays using whole B. malayi mf ES antigens were shown to be potentially useful to detect microfilaraemia, acute, chronic and occult filarial cases in an endemic community and in immunomonitoring of clinical filarial patients for determining the optimal period of DEC treatment for clinical relief and cure (Harinath and Reddy, J. Parasitic Dis, 21:41-51; 1997).
Therefore the main object of this invention is to provide a process for the preparation of novel brugia malayi microfilarial antigen protein for use for diagnosis and immunomonitoring of microfilariaemic, acute, chronic and occult filarial infections.
According to this invention there is provided a process for the preparation of brugia malayi microfilarial excretory - secretory (mf ES-22) glycoprotein for use for diagnosis and monitoring of active filarial infection comprising collecting brugia malayia microfilariae from peritoneal cavities of jirds, preparing brugia malayi microfilarial excretory-secretory (Bm mf ES) antigen and then subjecting the same to the step of fractionation by fast protein liquid chromatography (FPLC).
The process for the preparation of brugia malayi microfilarial excretory-secretory (mf ES-22) glycoprotein according to a preferred embodiment is herein described in detail.
In accordance with the present invention brugia malayi microfilariae (mf) are obtained by lavage of the peritoneal cavities of jirds (Meriones unguiculatus) with an intra¬peritoneal filarial infection of 3 months or more duration. The peritoneal fluid is centrifuged at lOOOg for 15 min, the supernatant is discarded and the mf pellet is suspended in sterile normal saline (0.9% NaCl) and centrifuged. The procedure is repeated with RPMI-1640 medium. The mf are then plated in sterilized petridishes and incubated at 37°C for 1 hr. to remove peritoneal cells. The non-adherent mf in the medium are collected with out disturbing the peritoneal cells adhered to the petridish.
About ten lakhs of mf are then maintained in RPMI 1640 medium supplemented with organic acids (malic acid, a-keto-glutaric acid, D-succinic acid and fumeric acid at concentrations of 670,370,60 and 55 mg per litre respectively) and sugars (sucrose and fructose at concentrations of 26,680 and 400 mg per litre respectively) and 0.1 mM phenyl methyl sulphonyl floride (PMSF) at temperature of 28 °C (or up to 37°C) in an
atmosphere of 5% CO2 for 48 hrs. The spent medium is collected and centrifuged at lOOOg for 20 min. The supernatant is collected and concentrated by ultra-membrane filtration using Dialysis tubing (Sigma Chemical Co., U.S.A.) and dialyzed against 0.1M sodium phosphate buffer (SPB) pH 7.2. The protein content is measured by Lowry's methods (Lowry et al, J. Biol. Chem.; 265-275. 1951) and the sample is labeled as B.malayi microfilarial excretory and secretory (Bm mf ES) antigen. The antigen is stored at -70°C with a cocktail of protease inhibitors i.e., ImM Ethylene diamine terra acetic acid (EDTA), O.lmM phenyl methyl sulphonyl fluoride (PMSF), 0.2mM tosyl L-lysine chloromethyl ketone (TLCK), O.lmM tosyl L-phenyl alanine chloromethyl ketone (TPCK) and 0.1% sodium azide.
The yield of mf ES antigen by in vitro maintenance of about 10 lakhs microfilariae at 28°C for 48 hrs is about 500 μg.
B. mahyi mf ES antigens are fractionated by FPLC using Superdex 200 HR 10/30-gel filtration column applying preprogrammed parameters as mentioned below in Table 1. Mf ES antigen (250 μg/0.5ml) is applied, eluted using 0.05 M PBS (pH 7.2) at a flow rate of 0.5 ml/min and monitored at 280 nm.
Fractions of 2 ml volume are collected. The mf Es antigen is fractionated into six protein peaks as shown in Fig. 1. The fractions of the six individual protein peaks are pooled and checked for antigenic activity by indirect ELISA using referral pooled filarial positive and negative sera.
Table 1. Pre programmed parameters for FPLC fractionation of mf ES antigen
Volume Function Value
VALVE POSITION 1.2
LEVEL % 5
VALVE POSITION 1.1
PORT SET 6.1
PRINT PEAK 1.10
One of the six fraction of mf ES antigen i.e., ES-22 is significantly more reactive with referral pooled filarial serum than with referral pooled endemic normal serum.
Brugia malayi microfilariae (mf) obtained was evaluated for the detection of filarial IgG antibodies by using ELISA test. For this purpose sixth protein peak of FPLC fractions of Bm mf ES antigen (mf ES-22) as shown in fig.l was used to detect filarial IgG antibodies in different clinical presentations of bancroftian filariasis. The wells in a polyvinyl microtitre plate are sensitized with optimal concentration of mf ES-22 antigen (20 ng/50ul/well) in carbonate buffer (0.06M, pH 9.6) at 37°C for 3 hrs. The wells are then blocked with 1% bovine serum albumin (BSA) in the same buffer at 37°C for 2 hrs and washed with 0.01M phosphate buffer saline with 0.05% tween 20 (PBS/T). The wells are then incubated with optimally diluted sera (1:100) in PBS/T at 37°C for 1 hr. After washing, the wells are further incubated with optimally diluted (1:2000) antihuman IgG peroxidase in PBS/T with 0.5% BSA at 37°C for 1 hr. Followed by final washing the wells are incubated with O-phenelene diamine (OPD) substrate and are read in a Multiscan ELISA Reader at 490 nm.
Test sera showing optical density (O.D) equivalent to or greater than the mean O.D+2 SD of endemic normal sera are considered as positive.
The results of analysis of different groups of sera for filarial IgG antibodies against B.malqyi mf ES-22 antigen by microtitre plate peroxidase ELISA are shown in Table 2.
Table 2 Detection of filarial IgG antibodies in different groups of sera using B.malayi mf ES-22 antigen in indirect ELISA.
SERA No. No.Positive*
Exam (% positivity)
Endemic Normals 30 5 (16.6)
Microfilaraemia 30 20(66.6)
Clinical filariasis 30 24(80)
* Sera showing O.D. of >Mean+2 SD of O.D. of endemic normal sera.
The filarial antigen activity in the FPLC purified diagnostically useful B.malayi mf ES-22 antigen is checked in inhibition ELISA before and after treatment with heat and different bio-chemical agents. The protein concentration of ES-22 antigen (before and after treatment) is adjusted to 10μg/ml, further serially diluted (four fold) and applied in inhibition ELISA to determine the antigen titres.
The results are summarized below in Table 3. B. malayi mf ES-22 antigen showed either complete or significant loss of filarial antigen activity after treatment with heat, proteolytic enzymes, a-amylase, per-iodate and 15% TCA suggesting that it is heat labile and glycoprotein in nature.
Table 3: Effect of treatment with different biochemical agents on FFLC purified B malayi mf ES-22 antigen.
Treatment with Filarial antigen titres in inhibition ELISA
of antigen fraction* mfES-22
Sodium metaperiodate 0
*Bm mf ES-22 antigen before and after treatment is diluted to a protein
concentration of 10μg/ml and then further serial 4 fold dilutions are used in
inhibition ELISA to determine the end titres.
1. A process for the preparation of brugia malayi microfilaria! excretory-secretory (mf ES-22) glycoprotein for use in diagnosis and monitoring of active filarial infection comprising.
(a) collecting brugia malayi microfilariae by lavage of the peritoneal cavities of jirds-with an intraperitoneal filarial infection of 3 months or more duration.
(b) centrifuging peritoneal fluid at 1000g for 15 mins, suspending the mf pellet in sterile normal saline (0.9% NaCl) and repeated washing with RPMI-1640 medium.
(c) plating the mf at 37°C for 1 hr to remove peritoneal exudate cells.
(d) maintaining about ten lakhs of mf in RPMI- 1640 medium supplemented with malic acid, a keto glutaric acid, D-Succinic acid and fumeric acid at concentrations of 670, 370, 60 and 55 mg per litre respectively and sucrose and fructose at concentrations of 26, 680 and 400 mg per litre respectively, 0.1 mM phenyl methyl sulphonyl fluoride at temperature of 28-37°C in an atmosphere of 5% CO2 for 48 hrs.
(e) Centrifuging at 1000g for 20 mins followed by concentrating the supernatant by ultra membrane filtration using Dialysis tubing and dialysing against 0.1M sodium phosphate buffer, pH 7.2.
2. The process as claimed in claim 1, wherein the collection and final storage of microfiiarial excretory-secretory glycoprotein antigen is done with a cocktail of protease inhibitors, ImM ethylene diamine tetra acetic acid, 0.1 mM phenyl methyl sulphonyl fluoride, 0.2 mM tosyl L-lysine chloromethyl ketone, 0.1mM tosyl L phenyl alanine chloromethyl ketone and 0.1% sodium azide.
3. A process as claimed in claims 1 and 2 wherein and said excretory-secretory (Bm mf ES antigen is subjected to the step of fractionation by fast protein liquid chromatography (FPLC) using gel filtration (Superdex 200 HR 10/30) column, applying the parameters as herein described:
Bm mf ES-antigen fraction (250 μg/0.5ml) is applied and eluted using 0.05M PBS (pH 7.2) at a flow rate of 0.5 ml/min and monitored at 280 nm and fractions of 2 ml volume are collected. The 6th protein fraction containing 22 kDa protein (ES-22) showed optimum antigenic activity.
4. A process for the preparation of brugia malayi microfiiarial
excretory-secretory (mf ES-22) glycoprotein substantially as
herein described and illustrated.
Dated this 24th day of December 2002
(MONA SAINI) OF L.S. DAVAR & CO., APPLICANTS ATTORNEY.
|Indian Patent Application Number||1157/MUM/2002|
|PG Journal Number||02/2009|
|Date of Filing||26-Dec-2002|
|Name of Patentee||SECRETARY DEPARTMENT OF BIOTECHNOLOGY|
|Applicant Address||SEVAGRAM, WARDHA 442 102, MAHARASHTRA, INDIA.|
|PCT International Classification Number||A61K|
|PCT International Application Number||N/A|
|PCT International Filing date|