AU2003290419A1 - A process for preparing egg yolk antibodies - Google Patents

Description

WO 2005/063818 PCT/IN2003/000434 1 A PROCESS FOR PREPARING EGG YOLK ANTIBODIES TECHNICAL FIELD The present invention relates to generating polyclonal antibodies from egg yolk 5 antibodies to specific protein myosin obtained from insects Sitophilus oryzae, Triboliumi castaneumn, Rhizopertha dominica and Acheta domnestica BACKGROUND OF THE INVENTION Insect infestation in stored grain is a universal problem and causes loss to food grain. Insect contamination results in 20-30% loss of food, both in quality and quantity. Pest 10 infestation also reduces the quality of the produce and its marketability. Thus, there is a need to monitor insect infestation to ensure food quality. Detection of insects and insect parts in stored whole grain and milled products is a problem for the cereal and milling industry. The current methods rely on visual examination of grain insects and damaged kernels (Nicholson et al 1953 and Hill 1990). These methods need well-trained 15 personnel and are time consuming besides they are not effective in detecting low level of hidden or internally developing insects, Other experimental techniques include x-ray analysis, nuclear magnetic resonance spectroscopy (Street & Bruce 1976) and chemical techniques (Ashman et al 1969 and Laessig et al 1972). However, none of these methods meet the requirement of an "ideal assay". An ideal assay would be specific, 20 sensitive, inexpensive, easy to perform and adaptable to granaries and grain mills. Biochemical /immunochemical methods meet this requirement. In recent years, techniques of producing antibodies (polyclonal and monoclonal) have been developed which make it possible to obtain homogenous, highly specific antibodies. Generally, polyclonal antibodies are used extensively in diagnostics 25 industry. Most commonly they are raised in mammals such as rabbit, mice, rat, horse and goat. This form of antibody production has several disadvantages - large mammals are expensive to maintain, while small mammals yield small quantities of antibody. In addition there is a requirement for periodic drawing of blood from the animals (Deignan et al 2000). The amount of IgG (antibody) obtained is usually between 3-8 mg/ml of 30 serum. The method also involves bleeding of the rabbit several times to obtain the antibodies, as the titer is highest only between the 8 t h - 10 th day after 2-3 boosters. Monoclonal antibodies are produced by immunizing an animal with a protein, obtaining antibody-producing cells from the animal, and fusing the antibody-producing cells with strains of myeloma cells, i.e., tumor cells, to produce hybridomas, which are isolated WO 2005/063818 PCT/IN2003/000434 2 and cultured as monoclones. The monoclonal hybridomas may either be cultured in vitro or from the cells, ascitic fluid, or serum of a tumor-bearing host animal. Since each antibody-producing cell generates a single unique antibody, each monoclonal culture of hybridomas produces a homogeneous antibody. Not all of the hybridoma 5 clones, which result from fusing myeloma cells with antibody-producing cells, are specific for the desired analyte (or ligand specific), since many of the hybridomas will make antibodies, which the inoculated animal has produced to react with other foreign substances. Even antibodies against the subject antigen will differ from clone to clone, since antibodies produced by different cells may react with different antigenic 10 determinants of the same molecule. From each clone, therefore, it is necessary to obtain the resulting antibody and test its reactivity with the analyte and to test its specificity by determining which particular organochlorines pesticide it recognizes. Further, only certain antibodies or antisera function in specific immunoassay formats or configurations. 15 The present invention relates to the production of egg yolk antibodies for an insect specific protein myosin. The use of antibodies from the egg yolks of hyperimmunized hens (IgY antibody) for immunological procedures overcomes the limitations associated with the polyclonal and monoclonal antibodies, because the present method provides a continuous supply of large quantities of consistent, high titer specific and sensitive 20 antibody which can be easily collected and stored. The manufacture and collection of hen egg antibodies is well understood in the field of clinical medicine. United States. Pat. Nos. 4,387,272 and 4,550,019 to Polson; and Losch, U claimed production of hen egg yolk antibodies and have been used in a number of applications for passive transfer of immunity. 25 United States. Pat No. 4,748,018 to Stolle, et al. discloses a method of passive immunization against bacterial infection comprising a preliminary development of tolerance of IgY by repeated oral ingestion of egg yolk, followed by parenteral injection of IgY antibody to a selected bacterial antigen. United States .Pat.no.DE19737453A1 to Fischer Mattias (1997) for the claims made in 30 the above patent for antibodies developed for elements United States .Pat.no.03784 to Wells ,Jack,N. (1992)discloses production of monoclonal antibodies against radioactive substituted xanthines 8- ( cycloalkyl ) substituted - 1,3- Dipropylxanthines and polyclonal antibodies in Dekalb laying hens.

WO 2005/063818 PCT/IN2003/000434 3 United States. Pat.no.14518 to Mark et al (1994) for antibodies developed for peptides and proteins with weekly boosters United States. Pat No. 5,080,895 to Tokoro discloses prevention of E. Coli diarrhea in newborn piglets by oral administration of anti-bacterial hen egg yolk antibodies. 5 Reference is made to Hamada, S., Infection and Immuinity 59(11): 4161-4167 (1991); and Otake, S., J. Dental Research 70(3): 162-166 (1991) reproduced the results of Beck in protecting rats against dental caries by means of passive immunization with orally administered hen egg yolk antibodies against S. mutans. Reference is made to Bartz, C. et al., J. Infectious Disease 142(3): 439-441 (1980) 10 prevented mnurine rotaviral infection in mice by the oral administration of the water soluble fraction of the eggs of immunized hens. Reference is made to Chen and Kitto, Fd & Agr Immunology (1993) 5, 165 - 175, Species specific immunoassay for S.granaries ,S.oryzae, S. zeaminis in wheat using monoclonal antibodies in mice and polyclonal antibodies in rabbits. 15 Reference is made to Schatzki et al., J. Econ. Entomol (1993) 86 (5): 1584-89 ELISA for Acheta domestica and S. granaries in wheat using polyclonal rabbit antibodies. Reference is made to Yokoyama, H., et al. Infection and Immunity 60(3): 998-1007 (1992) succeeded in passively protecting neonatal piglets from fatal enterotoxigenic E. coli infection by oral administration of a crude yolk immunoglobulin fraction from the 20 eggs of immunized hens. Reference is made to the animal studies of Yolken, R. et al., Pediatrics 81(2): 291-295 (1988); and Journal Clint. Immunol. 10(6): 80S-87S (1990), proposed the oral administration of antiviral IgY immunoglobulin for the prevention and treatment of enteric infections, including rotaviral infection in humans. Method and formulations for 25 the oral administration of immunoglobulin are known (U.S. Pat. No. 4,477,432 to Hardie. Reference is made to Amita rani et al (2001) PCT patent pending, Development of a process for egg yolk antibodies for organochlorine pesticides. Reference is made to Amita rani et al (2001) PCT patent pending, Development of a 30 process for egg yolk antibodies for Cyclodiene pesticides. Reference is made to Amita rani et al (2002) PCT patent pending, Development of a process for egg yolk antibodies for organophosphate pesticides. Reference is made to Amita rani et al (2001) PCT patent pending, Development of a WO 2005/063818 PCT/IN2003/000434 4 test method based on egg yolk antibodies for organochlorine pesticides. However, there are no reports on the production of egg yolk antibodies for an insect specific protein. OBJECT OF THE INVENTION The main object of the present invention relates to a process of producing egg yolk 5 antibodies against insect specific protein. Yet another embodiment of the present invention relates to a process of isolating and purifying insect specific protein from the femur muscle of the grain insects. Another object of the present invention relates to the process of isolating antibodies from the water soluble fraction of the egg yolk 10 DESCRIPTION OF THE ACCOMPANYING FIGURES/DRAWINGS Figure 1. Development of ELISA to detect staes of S.oryzae Figure 2. Development of ELISA using egg yolk antibodies Figure 3. Structure of Egg yolk antibodies and rabbit antibodies SUMMARY OF THE INVENTION 15 The present invention is to develop a process to produce egg yolk antibodies which have high titer, consistent quality of antibody, easy to produce and non invasive. The present invention of egg yolk antibodies thus has very practical economical and important advantages. .Firstly, the present technique gives high yield of antibody. Secondly, production of the titer of the antibody remains high for a longer period of 20 time almost for 60 days, thereby providing a continuous supply of consistent quality of antibody. Thirdly, it is non invasive and hence there is no need to bleed the animal to recover the required antibody as is necessarily in Rabbit antibody. The egg yolk antibody thus produced is more/equally sensitive to the polyclonal/monoclonal antibodies produced using manunals. 25 DETAIL DESCRIPTION OF THE INVENTION The present invention involves a process of developing egg yolk antibodies. The egg yolk antibody thus produced is more/equally sensitive to the polyclonal/monoclonal antibodies produced using mammals. Accordingly, the main embodiment of the present invention relates to a process for the 30 producing egg yolk antibodies against insect specific protein said method comprising the steps of: (a) selecting suitable poultry birds, (b) immunizing the suitable bird of step (a) by injecting insect specific purified WO 2005/063818 PCT/IN2003/000434 5 protein along with Freund's complete adjuvant of about 1000pg in breast muscle of the bird, (c) repeating the immunization by injecting insect specific purified protein along with Freund's incomplete adjuvant at two, three and five weeks intervals and again 5 after another five weeks interval, and. (d) harvesting and isolating the antibodies from the egg yolk of the birds. Another embodiment of the present invention relates to the poultry bird in step (a) wherein the poultry bird is selected is White Leghorn. Still another embodiment of the present invention relates to the insects wherein the 10 insects are selected from group comprising of Acheta domestica, Sitophilus oryzae, Rhizopertha dominica and Tribolium casteneum. One more embodiment of the present invention relates to the injected specific purified protein along with Freund's complete adjuvant of the insect in step (a) wherein said injected specific purified protein is in the range of about 450 to 500 tg. 15 Yet another embodiment of the present invention relates to the injected specific purified protein along with Freund's incomplete adjuvant during the intervals two, three and five weeks and again after another five weeks interval periods in step (c) wherein the range of the injected purified protein is in the range of about 200 to 300 jIg Another embodiment of the present invention relates to a process of isolating and 20 purifying the insect specific myosin protein from femur muscles of the stored grain insects said method comprising the steps of: (a) isolating the myosin protein from the femur of the poultry bird using phosphate buffer of pH 7.5 to obtain of the protein in the range of about 1 to 7 mg/L by convention method, 25 (b) purifying the protein obtained in step (a) using acetonitrile in the range of about 60 to 80%, trifluoroacetic acid in the range of about 0.05 to 2.0%, and (c) identifying the purified protein of step (c) at a wavelength of 2 80nm, with a absorbance unit of full scale (AUFs) of 0.08 in run time of 25 minutes and a peak at 17 minutes. 30 Yet another.embodiment of the present invention relates to the yield of protein from insect grain Acheta doemnstica wherein the said yield of the protein is in the range of about 1.5 to 2.0 mg/L. Still another embodiment of the present invention relates to the yield of protein from insect grain Acheta doemnstica wherein the said protein is about 1.76 mg/L.

WO 2005/063818 PCT/IN2003/000434 6 Still another embodiment of the present invention relates to the yield of protein from insect grain Stiophilus oryzae is wherein the said protein in the range of about 5 to 7 -mg/L Still another embodiment of the present invention relates to the yield of protein from 5 insect grain Stiophilus oryzae is wherein the said protein is about 5.6 mg/L Still another embodiment of the present invention relates to the yield of protein from insect grain Rhizopertha dominica is in the range, of about 1 to 5 mg/L Still another embodiment of the present invention relates to the yield of protein from insect grain Rhizopertha dominica is about 1.6 mg/L 10 Still another embodiment of the present invention relates to the yield of protein from insect grain Tribolium castaneu is in the range of about 1 to Smg/L Still another embodiment of the present invention relates -to the yield of protein from insect grain Tribolium castaneu is about 2mg/. Another embodiment of the present invention relates to the conventional method in step 15 (a) wherein said conventional method is centrifugation and SDS-PAGE. Yet another embodiment of the present invention relates to the purified protein in step (c) wherein the said purified protein from insect Acheta domniestica is about 200 kDa and from insects Stiophilus oryzae, Rhizopertha domninica and Tribolium castaneu is about 45 kDa. 20 Another embodiment of the present invention relates the a process of isolating antibodies wherein antibodies against the specific insect protein are isolated from water soluble fraction of the egg yolk, saidprocess comprising of: (a) rupturing the egg and obtaining to obtain lipid contents of the egg yolk, (b) diluting the lipid contents of egg yolk of step (a) with water followed by 25 addition of kappa carragenan in the range of about 50 to 75 % to obtain water soluble protein fraction antibodies (WSPF), (c) passing the WSPF antibodies from step (b) through sodium hypophosphate in the range of about 0.5 to 2 mM, (d) precipitating the WSPF antibodies obtained from step (c) with PEG 8000 in 30 the range of about 10-20 %, and (e) purifying the WSPF antibodies obtained from step (d) by affinity column chromatography Still another'embodiment of the present invention relates to the kappa carragenan in WO 2005/063818 PCT/IN2003/000434 7 step (b) wherein kappa carragenan is about 60%. One more embodiement of the present invention relates to the sodium hypophosphate in step (c), wherein sodium hypophosphate is about 1mM Yet another -embodiment of the present invention relates to the PEG 8000 in step (d) 5 wherein PEG 8000 is about 14%. Another embodiment of the present invention relates to the yield of antibodies wherein the yield of antibodies from Acheta domnestica, Sitophilus oryzae, Rhizopertha dominica and Triboliumn casteneum protein is in the range of about 80-98 %. Still another embodiment of the present invention relates to yield of antibodies wherein 10 the yield of the antibodies from Acheta domestica, Sitophilus oryzae, Rhizopertha dominica and Tribolium casteneumn protein is about 95 %. One more embodiment of the present invention relates to the production of the antibody wherein the production of the antibody is initiated from the day 7 after immunization and continues upto 60 days. 15 Yet another embodiment of the present invention relates to the antibody produced wherein the antibody produced from insect Acheta domestica is in the range of about 5 15g /hen, from insect Sitophilus otyzae is in the range of about 3 -15g/hen, from insect Rhizopertha dominica is in the range of about 3 -10g /hen and from insect Tribolium castaneum is in the range of about 3 - 15 g /hen. 20 Still another embodiment of the present invention relates to the antibody produced wherein the antibody produced from insect Acheta domestica is in the range of about 6 12g /hen, from insect Sitophilus oryzae is in the range of about 4.3 -10 g/hen, from insect Rhizopertha domininica is in the range of about 4 -8 g /hen and from insect Tribolinum castaneumn is in the range of about 5 - 9 g /hen. 25 One more embodiment of the present invention relates to the egg yolk antibodies wherein the egg yolk antibodies are useful for analysis of insect contamination and filth in foods by ELISA (Fig.1). Another embodiment of the present invention relates to the egg yolk antibodies produced provide the basic material for developing ELISA test methods using plate, 30 tube, dipstick, PVP films and biosensors (Fig.2). Still another embodiment of the present invention relates to the developed ELISA test methods wherein developed ELISA test methods the find application in the detection of insect contamination /filth in food, milled products and processed foods. (Fig.1 and WO 2005/063818 PCT/IN2003/000434 8 Table 3) Table 1: Purification of antibodies from egg yolk S1.No. Method Percentage yield Sensitivity 1 CHANGE OF pH 80 +++ 2 CHLOROFORM 75 ++ 3 DEAE-SEPHACEL COLUMN 80 ++ 4 DEXTRAN SULPHATE 40 + 5 GUMS 80 +++ 6 MAGNESIUM CHLORIDE AND 60 ++ PHOSPHOTUNGSTIC ACID 7 POLYETHYLENE GLYCOL 75 +++ 8 PATENTED METHOD "95 ++++ 5 Table 2: Comparison between present invention and Prior Arts SL.No. Present Invention Chen and Kitto Schalzi et al 1 Antibodies against insects Antibodies against Antibodies against Acheta domestica, Sitophilus S.granaris S.granaries oryzae, Rhizopertha domininica and Triboliumn castaneum 2 Egg yolk antibodies from hen Antibodies from Antibodies from -IgY rabbit-IgG rabbit-IgG 3 Antibodies purified from Antigen purified Antigen purified from insects, Acheta domestica, from S.granaris Acheta-domnestica Sitophilus oryzae, Rhizopertha domninica and Triboliumn castaneum 4 Whole IgY-antibody molecule Whole IgG-antibody Fragment of antibody molecule -Fab 5 Indirect ELISA format Sandwitch ELISA Sandwitch ELISA format format 6 Extraction buffer-0.2M Extraction Buffer - Extraction buffer- 150 50 mM mM Table 3: Spike and Recovery Number of Insect Spiking in Number of insect recovery Percentage 100 g of food 1 1.2 120 5 3.6 72 50 27.0 54 10 The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention.

EXAMPLES

WO 2005/063818 PCT/IN2003/000434 9 EXAMPLE -1 Accordingly the present invention provides a process for the production of egg yolk antibodies for an insect specific protein, which comprises of periodic intra muscular immunization of the poultry birds with the insect specific protein (500 pxg of the 5 purified protein) in the breast muscle at 3-4 sites. Individual poultry (White Leghorn birds) were immunized with the immunogen (insect protein myosin). The initial immunization using 50% Freund's complete adjuvant which is an emulsifier and helps in diffusion of the antigen and made up to one mL with normal saline was followed by 250pig per bird in Freund's incomplete adjuvant, two, three and five weeks later and at 10 five weeks interval thereafter. Antibodies were harvested from egg yolk. EXAMPLE -2 The insect protein was obtained from the femur muscle of the field insect cricket Acheta domestica. The protein was purified by the modified method of Chaplain and Tregear (1996) and Woods et al (1963). The femurs (10g) were separated from the field 15 insect cricket and used to isolate the protein myosin. The femurs were ground with cold extraction phosphate buffer pH at 7.5. The extract was centrifuged at 3000xg for 5 min. The supernatant collected and stored as crude extract. Cold deionised water was added to the pellet and centrifuged at 20,000x g for 30 min. The pellet was resuspended in 0.2 M phosphate buffer + NaC1, at 4 0 for 1 hour, centrifuged at 16,000 x g for 25 min. The 20 supenatant was collected and stored at - 80 0 C. Yield - 1.76mg/mL. EXAMPLE-3 The insect protein was obtained from the stored grain insect Sitophilus oryzae. Whole insects were used to obtain the protein. The protein was purified by the modified method of Chaplain and Tregear (1996) and Woods et al (1963). 25 Whole insects (50g) were used to isolate the protein myosin. The insects were ground with cold extraction phosphate buffer pH at 7.5. The extract was centrifuged at 3000xg for 5 min. The supernatant collected and stored as crude extract. Cold deionised water was added to the pellet and centrifuged at 20,000x g for 30 min. The pellet was resuspended in 0.2 M phosphate buffer + NaCl, at 4 0 for 1 hour, centrifuged at 16,000 x 30 g for 25 min. The supenatant was collected and stored at - 80 0 C. Yield - 5.6 mg/mL or 112mg/20mL. EXAMPLE -4 The insect protein was obtained from the insect Rhizopertha dominica (2g). The protein WO 2005/063818 PCT/IN2003/000434 10 was purified by the modified method of Chaplain and Tregear (1996) and Woods et al (1963). Whole insects (2g) were used to isolate the protein myosin. The insects were ground with cold extraction phosphate buffer pH at 7.5. The extract was centrifuged at 3000xg 5 for 5 min. The supernatant collected and stored as crude extract. Cold deionised water was added to the pellet and centrifuged at 20,000x g for 30 min. The pellet was resuspended in 0.2 M phosphate buffer + NaC1, at 4 0 for 1 hour, centrifuged at 16,000 x g for 25 min. The supenatant was collected and stored at - 80 0 C. Yield - 1.6mg /mL or 20.8mg/13mL 10 EXAMPLE -5 The insect protein was obtained from the femur muscle of the stored grain pest Tribolium castaneum. The protein was purified by the modified method of Chaplain and Tregear (1996) and Woods et al (1963). 15 Whole insects (2g) were used to isolate the protein myosin. The insects were ground with cold extraction phosphate buffer pH at 7.5. The extract was centrifuged at 3000xg for 5 min. The supernatant collected and stored as crude extract. Cold deionised water was added to the pellet and centrifuged at 20,000x g for 30 min. The pellet was resuspended in 0.2 M phosphate buffer + NaC1, at 4 0 for 1 hour, centrifuged at 16,000 x 20 g for 25 rain. The supenatant was collected and stored at - 80 0 C. Yield - 2 mg/m EXAMPLE -6 The insect specific protein myosin purified from cricket Acheta domestica was checked for its purity using SDS - PAGE. A resolving gel of 8%, stacking gel - 5% at 100 V was found to be most suitable. The purified protein from Acheta domestica was 25 analysed for its purity using Poly Acrylamide Gel Electrophoresis. 30% acrylamide solution was poured into the glass plates assembled upright and a clean Teflon comb inserted. The gel was allowed to solidify at room temperature. Tris glycine electrophoresis buffer (pH 8.3) was added to the reservoirs. The gel was mounted in the electrophoresis apparatus. 30 50ug of sample and 50ug standard molecular weight marker (myosin 20 KD) were loaded on the gel in a predetermined order. A power supply of 100V for 6-7 hrs was supplied to the electrophoresis apparatus. After the completion of the run, the gel was transferred into a staining trough and stained with Coomassie brilliant blue for 4 hr at WO 2005/063818 PCT/IN2003/000434 11 room temperature on a rocker. The gel was then destained for 5-8hrs at room temperature in a destaining solution consisting of Methanol and glacial acetic acid. The destaining solution was changed 3 or 4 times. The bands seen were two distinct bands were detected in the standard. One major band corresponded to myosin heavy 5 chain (MHC) (200KD) and the other could be paramyosin subunit (41 KD) . EXAMPLE -7 The insect specific protein myosin purified from stored grain insect Sitophilus oryzae was checked for its purity using SDS - PAGE. A resolving gel of 8%, stacking gel 5% at 100 V was found to be most suitable. 10 The purified protein from Sitophilus oryzae was analysed for its purity using Poly Acrylamide Gel Electrophoresis. 30% acrylamide solution was poured into the glass plates assembled upright and a clean Teflon comb inserted. The gel was allowed to solidify at room temperature. Tris glycine electrophoresis buffer (PH 8.3) was added to the reservoirs. The gel was mounted in the electrophoresis apparatus. 15 50ug of sample and 50ug standard molecular weight marker (myosin 20 KD) were loaded on the gel in a predetermined order. A power supply of 100V for 6-7 hrs was supplied to the electrophoresis apparatus. After the completion of the run, the gel was transferred into a staining trough and stained with Coomassie brilliant blue for 4 hr at room temperature on a rocker. The gel was then destained for 5-Shrs at room 20 temperature in a destaining solution consisting Methanol and 8 mL of glacial acetic acid. The destaining solution changed 3 or 4 times. The bands seen were two distinct bands were detected in the standard. One major band corresponded to myosin heavy chain (MHC) (200KD) and other band could be paramyosin subunit (41 KD). EXAMPLE -8 25 The insect specific protein myosin purified from the insect Rhizopertha Dominica was checked for its purity using SDS - PAGE. A resolving gel of 8%, stacking gel - 5% at 100 V was found to be most suitable. The purified protein from Rhizopertha dominica was analysed for its purity using Poly Acrylamide Gel Electrophoresis. 30% acrylamide solution was poured into the glass 30 plates assembled upright and a clean Teflon comb inserted. The gel was allowed to solidified at room temperature. Tris glycine electrophoresis buffer (PH 8.3) was added to the reservoirs. The gel was mounted in the electrophoresis apparatus. 50ug of sample and 50ug standard molecular weight marker(myosin 20 KD) were loadedon the gel in a WO 2005/063818 PCT/IN2003/000434 12 predetermined order. A power supply of 100V for 6-7 hrs was supplied to the electrophoresis. apparatus. After the completion of the run, the gel was transferred into a staining trough and stained with Coomassie brilliant blue for 4 hr at room temperature on a rocker. The gel was then destained for 5-8hrs at room temperature in a destaining 5 solution consisting methanol and glacial acetic acid. The destaining solution changed 3 or 4 times. The bands seen were two distinct bands were detected in the standard. One major band.corresponded to myosin heavy chanin (MHC) (200KD) the other band could be paramyosin subunit (41 KD). EXAMPLE -9 10 The insect specific protein myosin purified from the insect Triobolium castaneum was checked for its purity using SDS - PAGE. A resolving gel of 8%, stacking gel - 5% at 100 V was found to be most suitable. The purified protein from Tribolium castaneum was analysed for its purity using Poly Acrylamide Gel Electrophoresis. 30% acrylamide solution was poured into the glass 15 plates assembled upright and a clean Teflon comb inserted. The gel was allowed to solidify at room temperature. Tris glycine electrophoresis buffer (PH 8.3) was added to the reservoirs. The gel was mounted in the electrophoresis apparatus. 50ug of sample and 50ug standard molecular weight marker(myosin 20 KD) were loaded on the gel in a predetermined order. A power supply of 100V for 6-7 hrs was supplied to the 20 electrophoresis apparatus. After the completion of the run, the gel was transferred into a staining trough and stained with Coomassie brilliant blue for. 4 hr at room temperature on a rocker. The gel was then destained for 5-8hrs at room temperature in a destaining solution consisting of methanol and glacial acetic acid. The destaining solution changed 3 or 4 times. The bands seen were two distinct bands were detected in the standard. One 25 major band corresponded to myosin heavy chain (MHC) (200KD). The other band could be paramyosin subunit (41 KD). EXAMPLE -10 The insect specific protein myosin purified from cricket Acheta domestica was checked for its purity using HPLC with a mobile phase comprising of 70 % acetonitrile, and 30 0.1% trifluroacetic acid at a wavelength of 280 nm, flow rate lml/min and Aufs of 0.08 and run time of 75 mins. The peaks were not resolved well EXAMPLE -11 The insect specific protein myosin purified from cricket Acheta domestica was checked WO 2005/063818 PCT/IN2003/000434 13 for its purity using HPLC with the mobile phase of 70 % acetonitrile, 0.1% trifluroacetic acid at a wavelength of 230 rnm, flow rate lmI/min and Aufs of 0.08 and run time of 45 mins. The peaks were not resolved well EXAMPLE -12 5 The insect specific protein myosin purified from cricket Acheta domestica was checked for its purity using HPLC with the mobile phase of 70 % acetonitrile, 0.1% trifluroacetic acid at a wavelength of 230 rnm, flow rate lml/min and Aufs of 0.08 and run time of 25 mins. Three major peaks were detected at 2.96, 9.65 and 17.0 minutes. The peak at 2.96 was 10 the solvent peak, peak at 9.65 was the paramyosin and the protein (Myosin) peak was detected at 17 th minute EXAMPLE -13 The insect specific protein myosin purified from stored grain pest - Sitophilus oryzae was checked for its purity using HPLC with the mobile phase of 70 % acetonitrile, 0.1% 15 trifluroacetic acid at a wavelength of 280 rn, flow rate lml/min and Aufs of 0.08 and run time of 75 mins. The peaks were not resolved well EXAMPLE -14 The insect specific protein myosin purified from stored grain pest -Sitophilus oryzae was checked for its purity using HPLC with the mobile phase of 70 % acetonitrile, 0.1% 20 trifluroacetic acid at a wavelength of 230 urn, flow rate Iml/min and Aufs of 0.08 and run time of 45 mins. The peaks were not resolved well EXAMPLE -15 The insect specific protein myosin purified from stored grain pest - Sitophilus oryzae was checked for its purity using HPLC with the mobile phase of 70 % acetonitrile, 0.1% 25 trifluroacetic acid at a wavelength of 230 nm, flow rate iml/min and Aufs of 0.08 and run time of 25 mins. Three major peaks were detected at 2.96, 9.65 and 17.0 minute. The peak at 2.96 was the solvent peak, 9.65 was the paramyosin peak and the protein (Myosin) peak was detected at 17 t h i minute. 30 EXAMPLE -16 The insect specific protein myosin purified from stored grain pest - Rhizopertha dominica as checked for its purity using HPLC with the mobile phase of 70 % acetonitrile, 0.1% trifluroacetic acid at a wavelength of 280.nm, flow rate lml/min and WO 2005/063818 PCT/IN2003/000434 14 Aufs of 0.08 and run time of 75 mins. The peaks were not resolved well EXAMPLE -17 The insect specific protein myosin purified from stored grain pest - Rhizopertha dominica was checked for its purity using HPLC with the mobile phase of 70 % 5 acetonitrile, 041% trifluroacetic acid at a wavelength of 230 nm, flow rate iml/min and Aufs of 0.08 and run time of 45 mins. The peaks were not resolved well EXAMPLE -18 The insect specific protein myosin purified from stored grain pest - Rhizopertha dominica was checked for its purity using HPLC with the mobile phase of 70 % 10 acetonitrile, 0.1% trifluroacetic acid at a wavelength of 230 nm, flow rate 1ml/min and Aufs of 0.08 and run time of 25 mins. Three major peaks were detected at 2.96, 9.65 and 17.0 minute. The peak at 2.96 was the solvent peak,' 9.65 was the paramyosin peak and the protein (myosin) peak was detected at 17 ' minute. 15 EXAMPLE -19 The insect specific protein myosin purified from stored grain pest - Tribolium castaneum as checked for its purity using HPLC with the mobile phase of 70 % acetonitrile, 0.1% trifluroacetic acid at a wavelength of 280 nm, flow rate Iml/min and Aufs of 0.08 and run time of 75 mins. The peaks were not resolved well 20 EXAMPLE -20 The insect specific protein myosin purified from stored grain pest - Tribolium castaneum was checked for its purity using HPLC with the mobile phase of 70 % acetonitrile, 0:1% trifluroacetic acid at a wavelength of 230 nm, flow rate Iml/min and Aufs of 0.08 and run time of 45 mins. The peaks were not resolved well 25 EXAMPLE -21 The insect specific protein myosin purified from stored grain pest - Tribolium castaneum was checked for its purity using HPLC with the mobile phase of 70 % acetonitrile, 0.1% trifluroacetic acid at a wavelength of 230 nm, flow rate Iml/min and Aufs of 0.08 and run time of 25 mins. 30 Three major peaks were detected at 2.96, 9.65 and 17.0 minute. The peak at 2.96 was the solvent peak, 9.65 was the paramyosin peak and the protein (Myosin) peak was detected at 17 th minute. EXAMPLE -22 WO 2005/063818 PCT/IN2003/000434 15 The purified protein from cricket femur muscle was injected into white Leghorn bird's poultry. The initial immunization using 50% Freund's complete adjuvant (500 gtg of the purified protein) was followed by 250kg per bird in Freund's incomplete adjuvant, two, three and five weeks later and at five weeks interval thereafter. 5 EXAMPLE -23 The purified protein from the stored grain pest - Sitophilus oryzae was injected into white Leghorn bird's poultry. The initial immunization using 50% Freund's complete adjuvant (500 ptg of the purified protein) was followed by 250ptg per bird in Freund's incomplete adjuvant, two, three and five weeks later and at five weeks interval 10 thereafter. EXAMPLE -24 The purified protein from the stored grain pest - Rhizopertha Dominica was injected into white Leghorn bird's poultry. The initial inumunization using 50% Freund's complete adjuvant (500 pg of the purified protein) was followed by 250pg per bird in 15 Freund's incomplete adjuvant, two, three and five weeks later and at five weeks interval thereafter. EXAMPLE -25 The purified protein from stored grain pest - Tribolium castaneum was injected into white Leghorn bird's poultry. The initial immunization using 50% Freund's complete 20 adjuvant (500 gg of the purified protein) was followed by 250ig per bird in Freund's incomplete adjuvant, two, three and five weeks later and at five weeks interval thereafter. EXAMPLE -26 The production of the antibody (Acheta domestica, Sitophilus Oryzae, Rhizopertha 25 dominica and Tribolium castaneum) started on day 7 after immunization and was continuous for 60 days. EXAMPLE -27 The eggs from the birds immunised with protein myosin obtained from Acheta domestica were collected from the 7 th day after the first booster the eggs were used to 30 purify the antibodies from the yolk. The egg yolk was carefully removed from the eggshell without rupturing the yolk membrane. The entire albumin adhering to the membrane was washed off. And the egg yolk membrane was then ruptured and the contents transferred into a measuring cylinder through a funnel. For every 10 ml of WO 2005/063818 PCT/IN2003/000434 16 yolk, 100ml of Tris buffer saline, pH 7.4 was added. The precipitate formed was removed by centrifugation at 2 000g for 30 minutes. To the supernatant the precipitating solution of magnesium chloride (3 M) and phosphotungstic acid (4.8%) were added and centrifuged again. The pellet 5 was discarded. To the supernatant now called the watersoluble protein fraction, 12%. polyethylene glycol (PEG 6000) was added and incubated for 10 minutes and then centrifuged. The antibody precipitated out. 10 ml of 10mM phosphate buffer was added and the precipitate dissolved. The antibody solution is then cooled to 0 0 C and 10 ml of pre cooled ethanol added. The solution was centrifuged at 4 0 C and the sediment was 10 dissolved in 10 mM phosphate buffer and dialysed against 3 changes of phosphate buffer for 24 h at 4 OC.Yield - 60%. The eggs from the birds immunised with protein myosin obtained from Sitophilus Oryzae were collected from the 7 th day after the first booster the eggs were used to purify the antibodies from the yolk. The egg yolk was carefully removed from the 15 eggshell without rupturing the yolk membrane. The entire albumin adhering to the membrane was washed off. And the egg yolk membrane was then ruptured and the contents transferred into a measuring cylinder through a funnel. For every 10 ml of yolk, 100ml of Tris buffer saline, pH 7.4 was added. The precipitate formed was removed by centrifugation at 2000g for 30 minutes. To the supernatant the precipitating 20 solution of magnesium chloride (3M) and phosphotungstic acid (4.8%) were added and centrifuged again. The pellet was discarded. To the supernatant now called the watersoluble protein fraction, 12% polyethylene glycol (PEG 6000) was added and incubated for 10 minutes and then centrifuged. The antibody precipitated out. 10 ml of 10mM phosphate buffer was added and the precipitate dissolved. The antibody solution 25 is then cooled to 0 0 C and 10 ml of pre cooled ethanol added. The solution was centrifuged at 4oC and the sediment was dissolved in 10 mM phosphate buffer and dialysed against 3 changes of phosphate buffer.for 24 h at 4 OC.Yield - 60%. The eggs from the birds immunised with protein myosin obtained from Rhizopertha dominica were collected from the 7 th day after .the first booster the eggs were used to 30 purify the antibodies from the yolk. The egg yolk was carefully removed from the eggshell without rupturing the yolk membrane. The entire albumin adhering to the membrane was washed off. And the egg yolk membrane was then ruptured and the contents transferred into a measuring cylinder through a funnel. For every 10 ml of WO 2005/063818 PCT/IN2003/000434 17 yolk, 100ml of Tris buffer saline, pH 7.4 was added. The precipitate formed was removed by centrifugation at 2000g for 30 minutes. To the supernatant the precipitating solution of magnesium chloride (3M) and phosphotungstic acid (4.8%) were added and centrifuged again. The pellet was discarded. To the supernatant now called the 5 watersoluble protein fraction, 12% polyethylene glycol (PEG 6000) was added and incubated for 10 minutes and then centrifuged. The antibody precipitated out. 10 ml of 1OmM phosphate buffer was added and the precipitate dissolved. The antibody solution is then cooled to 0 0 C and 10 ml of pre cooled ethanol added. The solution was centrifuged at 4 0 C and the sediment was dissolved in 10 mM phosphate buffer 10 anddialysed against 3 changes of phosphate buffer for 24 h at 4 oC.Yield - 60%. The eggs from the birds immunised with protein myosin obtained from Tribolium castaneum were collected from the 7 th day after the first booster the eggs were used to purify the antibodies from the yolk. The egg yolk was carefully removed from the eggshell without rupturing the yolk membrane. The entire albumin adhering to the 15 membrane was washed off. And the egg yolk membrane was then ruptured and the contents transferred into a measuring cylinder through a funnel. For every 10 ml of yolk, 100ml of Tris buffer saline, pH 7.4 was added. The precipitate formed was removed by centrifugation, at 2000 g for 30 minutes. To the supernatant the precipitating solution of magnesium chloride (3M) and phosphotungstic acid (4.8%) 20 were added and centrifuged again. The pellet was discarded. To the supernatant now called the waters soluble protein fraction, 12% polyethylene glycol was added and incubated for 10 minutes and then centrifuged. The antibody precipitated out. 10 ml of 10mM phosphate buffer was added and the precipitate dissolved. The antibody solution is then cooled to 0 0 C and 10 ml of pre cooled ethanol added. The solution was 25 centrifuged at 4 0 C and the sediment was dissolved in 10 mM phosphate buffer and dialysed against 3 changes of phosphate buffer for 24 h at 4 OC.Yield - 60%. EXAMPLE -28 The eggs from the birds immunised with protein. myosin obtained from Acheta domestica were collected from the 7" day after the first booster was used to purify the 30 antibodies from the yolk. The egg yolk was carefully removed from the eggshell without rupturing the yolk membrane. The entire albumin adhering to the membrane was washed off. And the egg yolk membrane was then ruptured and transferred the contents into a measuring cylinder through a funnel. For every 10 ml of yolk, 1 00ml of WO 2005/063818 PCT/IN2003/000434 18 Tris buffer saline, pH 7.4 was added. The precipitate formed was removed by centrifugation at 2000g. To the supernatant the precipitating solution of magnesium chloride (3M) and phosphotungstic acid (4.8%) was added and centrifuged again. The pellet was discarded. To the supernatant now called the water soluble protein fraction 5 12% polyethylene glycol was added and incubated for 10 minutes and then centrifuged. The antibody precipitated out. 10 ml of 10mM phosphate buffer were added and the precipitate dissolved. The antibody solution was then cooled to 0 0 C and 10 ml of pre cooled ethanol added. The solution was centrifuged at 4 0 C and the sediment was dissolved in 10 mM phosphate buffer and dialysed against 3 changes of phosphate 10 buffer for 24 h at 4 oC. To increase the yield of antibody, the lipid from the egg yolk was precipitated out twice using the precipitating solution of phosphotungstic acid and magnesium chloride to the supernatant and centrifuged. Yield - 75%. The eggs from the birds immunised with protein myosin obtained from Sitophilus oryzae were collected from the 7 th day after the first booster were used to purify the . 15 antibodies from the yolk. The egg yolk was carefully removed from the eggshell without rupturing the yolk membrane. The entire albumin adhering to the membrane was washed off. And the egg yolk membrane was then ruptured and transferred the contents into a measuring cylinder through a funnel. For every 10 ml of yolk, 1 00ml of Tris buffer saline, pH 7.4 was added. The precipitate formed was removed by 20 centrifugationi,-at 2000g for 30 minutes. To the supematant the precipitating solution of magnesium chloride (3M) and phosphotungstic acid (4.8%) was added and centrifuged again. The pellet was discarded. To the supernatant now called the water soluble protein fraction 12% polyethylene glycol (PEG 6000) was added and incubated for 10 minutes and then centrifuged. The antibody precipitated out. 10 ml of 10mM phosphate buffer 25 were added and the precipitate dissolved. The antibody solution was then cooled to 0 0 C and 10 ml of pre cooled ethanol added. The solution was centrifuged at 4 0 C and the sediment was dissolved in 10 mM phosphate buffer and dialysed against 3 changes of phosphate buffer for 24 h at 4 oC. To increase the yield of antibody, the lipid from the egg yolk was precipitated out twice using the precipitating solution of phosphotungstic 30 acid and magnesium chloride to the supernatant and centrifuged. Yield - 75%. The eggs from the birds immunised with protein myosin obtained from Rhizopertha domininica were collected from the 7 th day after the first booster were used to purify the antibodies from the yolk. The egg yolk was carefully removed from the eggshell WO 2005/063818 PCT/IN2003/000434 19 without rupturing the yolk membrane. The entire albumin adhering to the membrane was washed off. And the egg yolk membrane was then ruptured and transferred the contents into a measuring cylinder through a funnel. For every 10 ml of yolk, 100ml of Tris buffer saline, pH 7.4 was added. The precipitate formed was removed by 5 centrifugation at 2000g for 30 minutes. To the supernatant the precipitating solution of magnesium chloride (3M) and phosphotungstic acid (4.8%) was added and centrifuged again. The pellet was discarded. To the supernatant now called the water soluble protein fraction 12% polyethylene glycol was added and incubated for 10 minutes and then centrifuged. The antibody precipitated out. 10 ml of 10mM phosphate buffer were 10 added and the precipitate dissolved. The antibody solution was then cooled to 0 0 C and 10 ml of pre cooled ethanol added. The solution was centrifuged at 4 0 C and the sediment was dissolved in 10 mnM phosphate buffer and dialysed against 3 changes of phosphate buffer for 24 h at 4 OC. To increase the yield of antibody, the lipid from the egg yolk was precipitated out twice using the precipitating solution of phosphotungstic 15 acid and magnesium chloride to the supernatant and centrifuged.Yield - 75%. The eggs from the birds immunised with protein myosin obtained from Tribolium castaneum were collected from the 7 th day after the first booster were used to purify the antibodies from the yolk. The egg yolk was carefully removed from the eggshell without rupturing the yolk membrane. The entire albumin adhering to the membrane 20 was washed off. And the egg yolk membrane was then ruptured and transferred the contents into a measuring cylinder through a funnel. For every 10 ml of yolk, 1 00ml of Tris buffer saline, pH 7.4 was added. The precipitate formed was removed by centrifugation at 2000g for 30 minutes. To the supernatant the precipitating solution of magnesium chloride (3M) and phosphotungstic acid (4.8%) was added and centrifuged 25 again. The pellet was discarded. To the supernatant now called the water soluble protein fraction 12% polyethylene glycol (PEG 6000) was added and incubated for 10 minutes and then centrifuged. The antibody precipitated out. 10 ml of 1OmM phosphate buffer were added and the precipitate dissolved. The antibody solution was then cooled to 0 0 C and 10 ml of pre cooled ethanol added. The solution was centrifuged at 4 0 C and the 30 sediment was dissolved in 10 mM phosphate buffer and dialysed against 3 changes of phosphate buffer for 24 h at 4 oC. To increase the yield of antibody, the lipid from the egg yolk was precipitated out twice using the precipitating solution of phosphotungstic acid and magnesium chloride to the supernatant and centrifuged. Yield - 75%.

WO 2005/063818 PCT/IN2003/000434 20 EXAMPLE -29 The eggs from the birds immunised with protein myosin obtained from Acheta domestica were collected and from the 7 t day after the first booster the eggs were used to purify the antibodies from the yolk. The egg yolk was carefully removed from the 5 eggshell without rupturing the yolk membrane. The entire albumin adhering to the membrane was washed off. And the egg yolk membrane was ruptured and transferred into a measuring cylinder through a funnel. For every 10 ml of yolk, 100ml of Tris buffer saline, pH 7.4 was added. The precipitate formed was removed by centrifugation at 2000g for 30 minutes. To the supernatant the precipitating solution of magnesium 10 . chloride (3M) and phosphotungstic acid (4.8%) was added and centrifuged again. The pellet discarded and to the supernatant now called the water soluble protein fraction 12% polyethylene glycol (PEG 6000) was added and incubated for 10 minutes and then centrifuged. The antibody precipitates out. 10 ml of 10mM phosphate buffer is added and the precipitate dissolved. The antibody solution is then cooled to 0 0 C and 10 ml of 15 pre cooled ethanol added. The solution is centrifuged at 4 0 C and the sediment is dissolved in 10 mM phosphate buffer and dialysed against phosphate buffer for 24 h at 4 oC. The lipid from the egg yolk was precipitated out twice using the precipitating solution of phosphotungstic acid and magnesium chloride to the supernatant and centrifuged.The pH of the water soluble protein fraction obtained after the removal of 20 the lipids was checked and adjusted to pH 5.0 in order to further precipitate out the antibodies.Yield - 80 -90%. The eggs from the birds innunised with protein myosin obtained from Sitophilus oryzae were collected and from the 7 th day after the first booster the eggs were used to purify the antibodies from the yolk. The egg yolk was carefully removed from the 25 eggshell without rupturing the yolk membrane. The entire albumin adhering to the membrane was washed off. And the egg yolk membrane was ruptured and transferred into a measuring cylinder through a funnel. For every 10 ml of yolk, 100ml of Tris buffer saline, pH 7.4 was added. The precipitate formed was removed by centrifugation at 2000g for 30 minutes. To the supernatant the precipitating solution of magnesium 30 chloride (3M) and phosphotungstic acid (4.8%) was added and centrifuged again. The pellet discarded and to the supernatant now called the water soluble protein fraction 12% polyethylene glycol (PEG 6000) was added and incubated for 10 minutes and then centrifuged. The antibody precipitates out. 10 ml of 10mM phosphate buffer is added WO 2005/063818 PCT/IN2003/000434 21 and the precipitate dissolved. The antibody solution is then cooled to 0 0 C and 10 ml of pre cooled ethanol added. The solution is centrifuged at 4 0 C and the sediment is dissolved in 10 mM phosphate buffer and dialysed against phosphate buffer for 24 h at 4 oC. The lipid from the egg yolk was precipitated out twice using the precipitating 5 solution of phosphotungstic acid (4.8%)and magnesium chloride (3M) to the supernatant and centrifuged.The pH of the water soluble protein fraction obtained after the removal of the lipids was checked .and adjusted to pH 5.0 in order to further precipitate out the antibodies.Yield - 80 -90%. The eggs from the birds immunised with protein myosin obtained from Rhizopertha 10 dominica were collected and from the 7t h day after the first booster the eggs were used to purify the antibodies from the yolk. The egg yolk was carefully removed from the eggshell without rupturing the yolk membrane. The entire albumin adhering to the membrane was washed off. And the egg yolk membrane was ruptured and transferred into a measuring cylinder through a funnel. For every 10 ml of yolk, 1 00m1 of Tris 15 buffer saline, pH 7.4 was added. The precipitate formed was removed by centrifugation at 2000g for 30. To the supernatant the precipitating solution of magnesium chloride (3M) and phosphotungstic acid (4.8%) was added and centrifuged again. The pellet discarded and to the supernatant now called the water soluble protein fraction 12% polyethylene glycol (PEG 6000) was added and incubated for 10 minutes and then 20 centrifuged. The antibody precipitates out. 10 ml of 10mM phosphate buffer is added and the precipitate dissolved. The antibody solution is then cooled to 0 0 C and 10 ml of pre cooled ethanol added. The solution is centrifuged at 4 0 C and the sediment is dissolved in 10 mM phosphate buffer and dialysed against phosphate buffer for 24 h at 4 oC. The lipid from the egg yolk was precipitated out twice using the precipitating 25 solution of phosphotungstic acid and magnesiutm chloride to the supernatant and centrifuged. The pH of the water soluble protein fraction obtained after the removal of the lipids was checked and adjusted to pH 5.0 in order to further precipitate out the antibodies. Yield - 80 -90%. The eggs from the birds immunised with protein myosin obtained from Tribolium 30 castaneum were collected and from the 7 t day after the first booster the eggs were used to purify the antibodies from the yolk. The egg yolk was carefully removed from the eggshell without rupturing the yolk membrane. The entire albumin adhering to the membrane was washed off. And the egg yolk membrane was ruptured and transferred WO 2005/063818 PCT/IN2003/000434 22 into a measuring cylinder through a funnel. For every 10 ml of yolk, 100ml of Tris buffer saline, pH 7.4 was added. The precipitate formed was removed by centrifugation at 2000g for 30 minutes. To the supernatant the precipitating solution of magnesium chloride (3M) and phosphotungstic acid (4.8%) was added and centrifuged again. The 5 pellet discarded and to the supernatant now called the water soluble protein fraction 12% polyethylene glycol was added and incubated for 10 minutes and then centrifuged. The antibody precipitates out. 10 ml of 10mM phosphate buffer is added and the precipitate dissolved. The antibody solution is then cooled to 0 0 C and 10 ml of pre cooled ethanol added. The solution is centrifuged at 4 0 C and the sediment is dissolved 10 in 10 mM phosphate buffer and dialysed against phosphate buffer for 24 h at 4 oC. The lipid from the egg yolk was precipitated out twice using the precipitating solution of phosphotungstic acid and magnesium chloride to the supernatant and centrifuged. The pH of the water soluble protein fraction obtained after the removal of the lipids was checked and adjusted to pH 5.0 in order to further precipitate out the antibodies.Yield 15 80 -90%. EXAMPLE -30 The eggs from the birds immunised with protein myosin obtained from Acheta domestica were collected and from the 7 t h day after the first booster the eggs were used to purify the antibodies from the yolk. The eggyolk was carefully removed from the 20 eggshell without rupturing the yolk membrane.The entire albumin adhering to the membrane was washed off. And the egg yolk membrane was ruptured and transferred into a measuring cylinder through a funnel. The egg yolk was separated and diluted with distilled water and pH adjusted to 7.4. The pH of the water-soluble protein fraction (WSPF) obtained after centrifugation was adjusted to 5.0. 14% (w/v) polyethylene 25 glycol 8000 was added to the WSPF and incubated at room temperature and the precipitate collected after centrifugation. The precipitate was dissolved in 10 ml of 10 mM phosphate buffer and pre-cooled ethanol added and centrifuged. The sediment thus obtained was dissolved in phosphate buffer and dialysed against phosphate buffer for 24 h at 4 OC.Yield -75%. 30 The eggs from the birds immunised with protein myosin obtained fi.om Sitophilus oryzae were collected and from the 7 t ' day after the first booster the eggs were used to purify the antibodies from the yolk. The eggyolk was carefully removed from the eggshell without rupturing the yolk membrane.The entire albumin adhering to the WO 2005/063818 PCT/IN2003/000434 23 membrane was washed off. And the egg yolk membrane was ruptured and transferred into a measuring cylinder through a funnel. The egg yolk was separated and diluted with distilled water and pH adjusted to 7.4. The pH of the water-soluble protein fraction (WSPF) obtained after centrifugation was adjusted to 5.0. 14% (w/v) polyethylene 5 glycol 8000 was added to the WSPF and incubated at room temperature and the precipitate collected after centrifugation. The precipitate was dissolved in 10 ml of 10 mM phosphate buffer and pre-cooled ethanol added and centrifuged. The sediment thus obtained was dissolved in phosphate buffer and dialysed against phosphate buffer for 24 h at 4 oC.Yield -75%. 10 The eggs from the birds immunised with protein myosin obtained from Rhizopertha dominica were collected and from the 7 t h day after the first booster the eggs were used to purify the antibodies from the yolk. The eggyolk was carefully removed from the eggshell without rupturing the yolk membrane. The entire albumin adhering to the membrane was washed off. And the egg yolk membrane was ruptured and transferred 15 into a measuring cylinder through a funnel. The egg yolk was separated and diluted with distilled water and pH adjusted to 7.4. The pH of the water-soluble protein fraction (WSPF) obtained after centrifugation was adjusted to 5.0. 14% (w/v) polyethylene glycol 8000 was added to the WSPF and incubated at room temperature and the precipitate collected after centrifugation. The precipitate was dissolved in 10 ml of 10 20 mM phosphate buffer and pre-cooled ethanol added and centrifuged. The sediment thus obtained was dissolved in phosphate buffer and dialysed against phosphate buffer for 24 h at 4 OC.Yield -75%. The eggs from the birds immunised with protein myosin obtained from Tribolium castaneum were collected and from the 7 th day after the first booster the eggs were used 25 to purify the antibodies from the yolk. The eggyolk was carefully removed from the eggshell without rupturing the yolk membrane.The entire albumin adhering to the membrane was washed off. And the egg yolk membrane was ruptured and transferred into a measuring cylinder through a funnel. The egg yolk was separated and diluted with distilled water and pH adjusted to 7.4. The pH of the water-soluble protein fraction 30 (WSPF) obtained after centrifugation was adjusted to 5.0. 14% (w/v) polyethylene glycol 8000 was added to the WSPF and incubated at room temperature and the precipitate collected after centrifugation. The precipitate was dissolved in 10 ml of 10 mnM phosphate buffer and pre-cooled ethanol added and centrifuged. The sediment thus WO 2005/063818 PCT/IN2003/000434 24 obtained was dissolved in phosphate buffer and dialysed against phosphate buffer for 24 h at 4 oC.Yield -75%. EXAMPLE -31 The eggs from the birds immunised with protein myosin obtained from Acheta 5 domestica were collected and from the 7 t i day after the first booster the eggs were used to purify the antibodies from the yolk. The egg yolk was carefully removed from the eggshell without rupturing the yolk membrane. The entire albumin adhering to the membrane was washed off. And the egg yolk membrane was ruptured and transferred into a measuring cylinder through a funnel. The egg yolk was diluted with water and 10 filtered and kappa carrageenan (60%) added. The WSPF obtained after centrifugation was passed through whatmann filter paper and 1mM Sodium hypophosphate added and filtered again. Antibody purity was 75-80 %. The eggs from the birds immunised with protein myosin obtained from Sitophilus oryzae were collected and from the 7 th day after the first booster the eggs were used to 15 purify the antibodies from the yolk. The egg yolk was carefully removed from the eggshell without rupturing the yolk membrane. The entire albumin adhering to the membrane was washed off. And the egg yolk membrane was ruptured and transferred into a measuring cylinder through a funnel. The egg yolk was diluted with water and filtered and kappa carrageenan (60%) added. The WSPF obtained after centrifugation 20 was passed through whatmann filter paper and ImM Sodium hypophosphate added and filtered again. Antibody purity was 75-80 %. The eggs from the birds immunised with protein myosin obtained from Rhizopertha dominica were collected and from the 7 t i day after the first booster the eggs were used to purify the antibodies from the yolk. The egg yolk was carefully removed from the 25 eggshell without rupturing the yolk membrane. The entire albumin adhering to the membrane was washed off. And the egg yolk membrane was ruptured and transferred into a measuring cylinder through a funnel. The egg yolk was diluted with water and filtered and kappa carrageenan (60%) added. The WSPF obtained after centrifugation was passed through whatmann filter paper and 1mM Sodium hypophosphate added and 30 filtered again. Antibody purity was 75-80 %. The eggs from the birds immunised with protein myosin obtained from Tribolium castaneum were collected and from the 7 th day after the first-booster the eggs were used to purify the antibodies from the yolk. The egg yolk was carefully removed from the WO 2005/063818 PCT/IN2003/000434 25 eggshell without rupturing the yolk membrane. The entire albumin adhering to the membrane was washed off And the egg yolk membrane was ruptured and transferred into a measuring cylinder through a funnel. The egg yolk was diluted with water and filtered and kappa carrageenan (60%) added. The WSPF obtained after centrifugation 5 was passed through whatmann filter paper and lmM Sodium hypophosphate added and filtered again. Antibody purity was 75-80 %. EXAMPLE- 32 The eggs from the birds inununised with protein myosin obtained from Acheta domestica were collected and from the 7 th day after the first booster the eggs were used 10 to purify the antibodies from the yolk. The egg yolk was carefully removed from the eggshell without rupturing the yolk membrane. The entire albumin adhering to the membrane was washed off. And the egg yolk membrane was ruptured and transferred into a measuring cylinder through a funnel. The egg yolk was diluted with water and filtered and kappa carrageenan added. The WSPF obtained after centrifugation was 15 passed through Whatmann filter paper and 1mM Sodium hypophosphate added and filtered again. The WSPF obtained was passed through a DEAE - Sephacel column and 20 mM phosphate buffer pH 8.0passed through it along with the. WSPF. The column was washed- and the antibody eluted out with 0.2M phosphate buffer at pH 8.0. 15% (w/v) of sodium sulphate (anhydrous) was added at 20 oC and centrifuged. The 20 precipitate thus obtained was diluted in 100mM phosphate buffer, centrifuged and subsequent precipitate was made up in 10 mM phosphate buffer and dialysed against 10 mM phosphate buffer pH 8.0, then filtered through 0.45 pm membrane. Antibody purity -80%. The eggs from the birds immunised with protein myosin obtained from Sitophilus 25 oryzae were collected and from the 7 th day after the first booster the eggs were used to purify the antibodies from the yolk. The egg yolk was carefully removed from the eggshell without rupturing the yolk membrane. The entire albumin adhering to the membrane was washed off. And the egg yolk membrane was ruptured and transferred into a measuring cylinder through a funnel. The egg yolk was diluted with water and 30 filtered and kappa carrageenan added. The WSPF obtained after centrifugation was passed through whatmann filter paper and 1mM Sodium hypophosphate added and filtered again. The WSPF obtained was passed through a DEAE - Sephacel column and 20 mM phosphate buffer pH 8.0passed through it along with the WSPF. The column WO 2005/063818 PCT/IN2003/000434 26 was washed and the antibody eluted out with 0.2M phosphate buffer at pH 8.0. 15% (w/v) of sodium sulphate (anhydrous) was added at 20 oC and centrifuged. The precipitate thus obtained was diluted in 100mM phosphate buffer, centrifuged and subsequent precipitate was made up in 10 mM phosphate buffer and dialysed against 10 5 mnM phosphate buffer pH 8.0, then filtered through 0.45 pgm membrane. Antibody purity -80%. The eggs from the birds immunised with protein myosin obtained from Rhizopertha dominica were collected and from the 7 th day after the first booster the eggs were used to purify the antibodies from the yolk. The egg yolk was carefully removed from the 10 eggshell without rupturing the yolk membrane. The entire albumin adhering to the membrane was washed off. And the egg yolk membrane was ruptured and transferred into a measuring cylinder through a funnel. The egg yolk was diluted with water and filtered and kappa carrageenan added. The WSPF obtained after centrifugation was passed through whatmann filter paper and 1mM Sodium hypophosphate added and 15 filtered again. The WSPF obtained was passed through a DEAE - Sephacel column and 20 mM phosphate buffer pH 8.0passed through it along with the WSPF. The column was washed and the antibody eluted out with 0.2M phosphate buffer at pH 8.0. 15% (w/v) of sodium sulphate (anhydrous) was added at 20 oC and centrifiuged. The precipitate thus obtained was diluted in 100mM phosphate buffer, centrifuged and 20 subsequent precipitate was made up in 10 mM phosphate buffer and dialyzed against 10 mM phosphate buffer pH 8.0, then filtered through 0.45 pin membrane. Antibody purity -80%. The eggs from the birds immunised with protein myosin obtained from Tribolium castaneum were collected and from the 7 th day after the first booster the eggs were used 25 to purify the antibodies from the yolk. The egg yolk was carefully removed from the eggshell without rupturing the yolk membrane. The entire albumin adhering to the membrane was washed off. And the egg yolk membrane was ruptured and transferred into a measuring cylinder through a funnel. The egg yolk was diluted with water and filtered and kappa carrageenan added. The WSPF obtained after centrifugation was 30 passed through whatmann filter paper and 1mM Sodium hypophosphate added and filtered again. The WSPF obtained was passed through a DEAE - Sephacel column and 20 mM phosphate buffer pH 8.0passed through it along with the WSPF. The column was washed and the antibody eluted out with 0.2M phosphate buffer at pH 8.0. 15% WO 2005/063818 PCT/IN2003/000434 27 (w/v) of sodium sulphate (anhydrous) was added at 20 oC and centrifuged. The precipitate thus obtained was diluted in 100mM phosphate buffer, centrifuged and subsequent precipitate was made up in 10 rM phosphate buffer and dialysed against 10 mM phosphate buffer pH 8.0, then filtered through 0.45 pm membrane. Antibody purity 5 -80%. EXAMPLE -33 The eggs from the birds immunised with protein myosin obtained from Acheta domestica were collected and from the 7 th day after the first booster the eggs were used to purify the antibodies from the yolk. The egg yolk was carefully removed from the 10 eggshell without rupturing the yolk membrane. The entire albumin adhering to the membrane was washed off. And the egg yolk membrane was ruptured and transferred into a measuring cylinder through a funnel. One volume of egg yolk was diluted with 4 volumes of 0.1 M phosphate buffered saline and the lipid precipitated using 1 volume of chloroform in 4 volume of PBS. The 15 aqueous layer obtained after centrifugation was treated with 14% (w/w) of polyethylene glycol 8000 and the precipitate containing the antibodies was reconstituted in PBS containing azide and glycerol. Antibody purity - 75%. The eggs from the birds immunised with protein myosin obtained from Sitophilus oryzae were collected and from the 7 t" day after the first booster the eggs were used to 20 . purify the antibodies from the yolk. The egg yolk was carefully removed from the eggshell without rupturing the yolk membrane. The entire albumin adhering to the membrane was washed off. And the egg yolk membrane was ruptured and transferred into a measuring cylinder through a funnel. One volume. of egg yolk was diluted with 4 volumes of 0.1 M phosphate buffered saline 25 and the lipid precipitated using 1 volume of chloroform in 4 volume of PBS. The aqueous layer obtained after centrifugation was treated with 14% (w/w) of polyethylene glycol 8000 and the precipitate containing the antibodies was reconstituted in PBS containing azide and glycerol. Antibody purity- 75%. The eggs from the birds immunised with protein myosin obtained from Rhizopertha 30 dominica were collected and from the 7"1 day after the first booster the eggs were used to purify the antibodies from the yolk. The egg yolk was carefully removed from the eggshell without rupturing the yolk membrane. The entire albumin adhering to the membrane was washed off. And the egg yolk membrane was ruptured and transferred WO 2005/063818 PCT/IN2003/000434 28 into a measuring cylinder through a funnel. One volume of egg yolk was diluted with 4 volumes of 0.1 M phosphate buffered saline and the lipid precipitated using 1 volume of chloroform in 4 volume of PBS. The aqueous layer obtained after centrifugation was treated with 14% (w/w) of polyethylene 5 glycol 8000 and the precipitate containing the antibodies was reconstituted in PBS containing azide and glycerol. Antibody purity - 75%. The eggs from the birds immunised with protein myosin obtained from Tribolium castaneum were collected and from the 7 th day after the first booster the eggs were used to purify the antibodies from the yolk. The egg yolk was carefully removed from the 10 eggshell without rupturing the yolk membrane. The entire albumin adhering to the membrane was washed off. And the egg yolk membrane was ruptured and transferred into a measuring cylinder through a funnel. One volume of egg yolk was diluted with 4 volumes of 0.1 M phosphate buffered saline and the lipid precipitated using 1 volume of chloroform in 4 volume of PBS. The 15 aqueous layer obtained after centrifugation was treated with 14% (w/w) of polyethylene glycol 8000 and the precipitate containing the antibodies was reconstituted in PBS containing azide and glycerol. Antibody purity - 75%. EXAMPLE -34 The eggs from the birds immunised with protein myosin obtained from Acheta 20 domestica were collected and from the 7 th day after the first booster the eggs. were used to purify the antibodies from the yolk. The egg yolk was carefully removed from the eggshell without rupturing the yolk membrane. The entire albumin adhering to the membrane was washed off. And the egg yolk membrane was ruptured and transferred into a measuring cylinder through a funnel. The egg yolk was diluted with water and 25 filtered and kappa carrageenan (60%) added. The WSPF obtained after centrifugation was passed through whatmnann filter paper and 1mM Sodium hypophosphate added and filtered again. The WSPF obtained was treated with 14% (w/v) PEG 8000 and the antibody precipitated out. Antibody purity- 90 -95% 30 The eggs from the birds immunised with protein myosin obtained from Sitophilus oryzae were collected and from the 7 th day after the first booster the eggs were used to purify the antibodies from the yolk. The egg yolk was carefully removed from the eggshell without rupturing the yolk membrane. The entire albumin adhering to the WO 2005/063818 PCT/IN2003/000434 29 membrane was washed off. And the egg yolk membrane was ruptured and transferred into a measuring cylinder through a funnel. The egg yolk was diluted with water and filtered and kappa carrageenan (60%)added. The WSPF obtained after centrifugation was passed through whatmanm filter paper and ImM Sodium hypophosphate added and 5 filtered again. The WSPF obtained was treated with 14% (w/v) PEG 8000 and the antibody precipitated out. Antibody purity - 90 -95% The eggs from the birds immunised with protein myosin obtained from Rhizopertha dominica were collected and from the 7 th day after the first booster the eggs were used 10 to purify the antibodies from the yolk. The egg yolk was carefully removed from the eggshell without rupturing the yolk membrane. The entire albumin adhering to the membrane was washed off. And the egg yolk membrane was ruptured and transferred into a measuring cylinder through a funnel. The egg yolk was diluted with water and filtered and kappa carrageenan (60%) added. The WSPF obtained after centrifugation 15 was passed through whatmann filter paper and 1mM Sodium hypophosphate added and filtered again. The WSPF obtained was treated with 14% (w/v) PEG 8000 and the antibody precipitated out. Antibody purity- 90 -95% The eggs from the birds immunised with protein myosin obtained from Tribolium 20 castneum were collected and from the 7 th day after the first booster the eggs were used to purify the antibodies from the yolk. The egg yolk was carefully removed from the eggshell without rupturing the yolk membrane. The entire albumin adhering to the membrane was washed off. And the egg yolk membrane was ruptured and transferred into a measuring cylinder through a funnel. The egg yolk was diluted with water and 25 filtered and kappa carrageenan (60%) added. The WSPF obtained after centrifugation was passed through whatmanm filter paper and 1mM Sodium hypophosphate added and filtered again. The WSPF obtained was treated with 14% (w/v) PEG 8000 and the antibody precipitated out. Antibody purity - 90 -95% 30 EXAMPLE.-35 The eggs from the birds immunised with protein myosin obtained from Acheta domestica were collected and from the 7 t h day after the first booster the eggs were used to purify the antibodies from the yolk. The egg yolk was carefully removed from the WO 2005/063818 PCT/IN2003/000434 30 eggshell without rupturing the yolk membrane. The entire albumin adhering to the membrane was washed off. And the egg yolk membrane was ruptured and transferred into a measuring cylinder through a funnel. The egg yolk was diluted with water and filtered and kappa carrageenan (60%) added. The WSPF obtained after centrifugation 5 was passed through whatmann filter paper and 1mM Sodium hypophosphate added and filtered again. The WSPF obtained was treated with 14% (w/v) PEG 8000 and the antibody precipitated out. The purified antibody obtained was further purified using ligand specific affinity column. The affinity column were prepared as follows: The activation of sepharose gel (4B) was done by taking 50ml of Sepharose (4B) gel 10 ,washed with distilled water 3 times (100 ml each time).To 50 ml of the gel, 50 ml of distilled water and 2M Sodium carbonate (1:1:1 ratio) was added. The contents were vortexed on a magnetic stirrer. 10 ml of Cyanogen bromide was added, kept in ice bath for 5 minutes with constant stirring. Washed the gel with ice-cold distilled water 4 times (100 ml each time). Washed with 0.01 M HCI 4 times (100ml each time). Added 0.1 M 15 Sodium carbonate, containing 0.5 M of Sodium chloride (100 ml) and use for coupling reaction. It is important that all steps should be carried out under a fume hood. Cyanogen bromide (CNBr) reagent should be handled using gloves and no mouth pipetting. The activated gel thus obtained was conjugated to the Protein or IgG as follows. Estimated the amount of protein content. All steps to be carried out in cold 20 conditions. 5ml of the activated gel at 25mg of protein (IgG/protein) in 5 ml of 0.1 M Sodium bicarbonate, containing 0.5 M Sodium chloride (pH-9). Shake gently for 10- 12 hours. Add 0.25 ml of Ethanolamine to the reactants and shake gently for 2 hours. Pack the gel in a glass or a plastic column and wash with 10 bed volumes (50x10) of 0.1 M Sodium bicarbonate, containing 0.5 M Sodium chloride (pH-9), followed by 10 mM 25 PBS (pH-7.4). Store the gel- coupled protein in 10mM PBS, containing 0.02% Sodium Azide at 40 C. The protocol for purification of the antibody using the column was as follows: Matrix (bed volume -500 1). Wash with 3 bed volumes of the binding buffer. 500ul of the sample, diluted 1:1 with the binding buffer is loaded. The outflow was collected. The column was washed with 10 ml of the binding buffer. Aliquots of 1 ml 30 each were collected and absorbance was read at 280 nm. The column was eluted with 10 ml of Lysine- HCI buffer (pH-2.8). Aliquots of 1 ml each .were collected. Absorbance was read at 280 nm. The antibodies thus obtained had better sensitivity. The eggs from the birds immunised with protein myosin obtained Sitophilus Oryzae WO 2005/063818 PCT/IN2003/000434 31 were collected and from the 7 th day after the first booster the eggs were used to purify the antibodies from the yolk. The egg yolk was carefully removed from the eggshell without rupturing the yolk membrane. The entire albumin adhering to the membrane was washed off. And the egg yolk'membrane was ruptured and transferred into a 5 measuring cylinder through a funnel. The egg yolk was diluted with water and filtered and kappa carrageenan added. The WSPF obtained after centrifugation was passed through whatmann filter paper and 1mM Sodium hypophosphate added and filtered again. The WSPF obtained was treated with 14% (w/v) PEG 8000 and the antibody precipitated out. The purified antibody obtained was further purified using ligand 10 specific affinity column. The affinity column were prepared as follows: The activation of sepharose gel (4B13) was done by taking 50ml of Sepharose (4B) gel ,washed with distilled water 3 times (100 ml each time).To 50 ml of the gel, 50 ml of distilled water and 2M Sodium carbonate (1:1:1 ratio) was added. The contents were vortexed on a magnetic stirrer. 10 ml of Cyanogen bromide was added, kept in ice bath for 5 minutes 15 with constant stirring. Washed the gel with ice-cold distilled water 4 times (100 ml each time). Washed with 0.01 M HCl 4 times (100ml each time). Added 0.1 M Sodium carbonate, containing 0.5 M of Sodium chloride (100 ml) and use for coupling reaction. It is important that all steps should be carried out under a fume hood. CNBr reagent should be handled using gloves and no mouth pipetting. The activated gel thus obtained 20 was conjugated to the Protein or IgG as follows. All steps were carried out in cold conditions. 5ml of the activated gel at 25mg of protein (IgG/protein) in 5 ml of 0.1 M Sodium bicarbonate, containing 0.5 M Sodium chloride (pH-9). Shake gently for 10- 12 hours. Add 0.25 ml of ethanolamine to the reactants and shake gently for 2 hours. Pack the gel in a glass or a plastic column and wash with 10 bed volumes (50x10) of 0.1 M 25 Sodium bicarbonate, containing 0.5 M Sodium chloride (pH-9), followed by 10 mM PBS (pH-7.4). Store the gel- coupled protein in 10mM PBS, containing 0.02% Sodium Azide at 40 C. The protocol for purification of the antibody using the column was as follows: Matrix (bed volume -500 1). Wash with 3 bed volumes of the binding buffer. 500ul of the sample, diluted 1:1 with the binding buffer is loaded. The outflow was 30 collected. The column was washed with 10 ml of the binding buffer. Aliquots of 1 ml each were collected and absorbance was read at 280 nm. The column was eluted with 10 ml of Lysine- HCI buffer (pH-2.8). Aliquots of 1 ml each were collected. Absorbance was read at 280 nm. The antibodies thus obtained had WO 2005/063818 PCT/IN2003/000434 32 90 - 95 % sensitivity. The eggs from the birds ininmmunised with protein myosin obtained Rhizopertha dominica were collected and from the 7 t h day after the first booster the eggs were used to purify the antibodies from the yolk. The egg yolk was carefully removed from the eggshell 5 without rupturing the yolk membrane. The entire albumin adhering to the membrane was washed off. And the egg yolk membrane was ruptured and transferred into a measuring cylinder through a funnel. The egg yolk was diluted with water and filtered and kappa carrageenan (60%) added. The WSPF obtained after centrifugation was passed through whatmann filter paper and ImM Sodium hypophosphate added and 10 filtered again. The WSPF obtained was treated with 14% (w/v) PEG 8000 and the antibody precipitated out. The purified antibody obtained was further purified using ligand specific affinity column. The affinity column were prepared as follows: The activation of sepharose gel (4B) was done by taking 50ml of Sepharose (4B) gel, washed with distilled water 3 times (100 ml each time). To 50 ml of the gel, distilled 15 water and 2M Sodium carbonate (1:1:1 ratio) was added. The contents were vortexed on a magnetic stirrer. 10 ml of Cyanogen bromide was added, kept in ice bath for 5 minutes with constant stirring. Washed the gel with ice-cold distilled water 4 times (100 ml each time). Washed with 0.01 M HCI 4 times (100ml each time). Added 0.1 M Sodium carbonate, containing 0.5 M of Sodium chloride (100 ml) and use for coupling 20 reaction. It is important that all steps should be carried out under a fume hood. CNBr reagent should be handled using gloves and no mouth pipetting. The 'activated gel thus obtained was conjugated to the Protein or IgG as follows. Estimated the amount of protein content. All steps to be carried out in cold conditions. 5ml of the activated gel at 25mg of protein (IgG/protein) in 5 ml of 0.1 M Sodium 25 bicarbonate, containing 0.5 M Sodium chloride (pH-9). Shake gently for 10- 12 hours. Add 0.25 ml of Ethanolamine to the reactants and shake gently for 2 hours. Pack the gel in a glass or a plastic column and wash with 10 bed volumes (50x10) of 0.1 M Sodium bicarbonate, containing 0.5 M Sodium chloride (pH-9), followed by 10 mM PBS (pH 7.4). Store the gel- coupled protein in 10mM PBS, containing 0.02% Sodium Azide at 30 40 C. The protocol for purification of the antibody using the column was as follows: Matrix (bed volume -500 1). Wash with 3 bed volumes of the binding buffer. 500ul of the sample, diluted 1:1 with the binding buffer is loaded. The outflow was collected. The column was washed with 10 ml of the binding buffer. Aliquots of 1 ml each were WO 2005/063818 PCT/IN2003/000434 33 collected and absorbance was read at 280 am. The column was eluted with 10 ml of Lysine- HC1 buffer (pH-2.8). Aliquots of 1 ml each were collected. Absorbance was read at 280 nm. The antibodies thus obtained had better sensitivity. 90 -95% binding. The eggs from the birds immunised with protein myosin obtained Tribolium castaneum 5 were collected and from the 7 t h day after the first booster the eggs were used to purify the antibodies from the yolk. The egg yolk was carefully removed from the eggshell without rupturing the yolk membrane. The entire albumin adhering to the membrane was washed off. And the egg yolk membrane was ruptured and transferred into a measuring cylinder through a funnel. The egg yolk was diluted with water and filtered 10 and kappa carrageenan (60%) added. The WSPF obtained after centrifugation was passed through whatmann filter paper and ImM Sodium hypophosphate added and filtered again. The WSPF obtained was treated with 14% (w/v) PEG 8000 and the antibody precipitated out. The purified antibody obtained was further purified using ligand specific affinity column. The affinity column were prepared as follows: The 15 activation of sepharose gel (4B) was done by taking 50ml of Sepharose (4B) gel, washed with distilled water 3 times (100 ml each time). To 50 ml of the gel, 50 ml of distilled water and 2M Sodium carbonate (1:1:1 ratio) was added. The contents were Vortexed on a magnetic stirrer. 10 ml of Cyanogen bromide was added, kept in ice bath for 5 minutes with constant stirring. Washed the gel with ice-cold distilled water 4 times 20 (100 ml each time). Washed with 0.01 M HCI 4 times (100ml each time). Added 0.1 M Sodium carbonate, containing 0.5 M of Sodium chloride (100 ml) and use for coupling reaction. It is important that all steps should be carried out under a fume hood. CNBr reagent should be handled using gloves and no mouth pipetting. The activated gel thus obtained was conjugated to the Protein or IgG as follows. Estimated the amount of 25 protein content. All steps to be carried out in cold conditions. Sml of the activated gel at 25mg of protein (IgG/protein) in 5 ml of 0.1 M Sodium bicarbonate, containing 0.5 M Sodium chloride (pH-9). Shake gently for 10- 12 hours. Add 0.25 ml of Ethanolamine to the reactants and shake gently for 2 hours. Pack the gel in a glass or a plastic column and wash with 10 bed volumes (50x10) of 0.1 M Sodium bicarbonate, containing 0.5 M 30 Sodium chloride (pH-9), followed by 10 mM PBS (pH-7.4). Store the gel- coupled protein in 1.0mM PBS, containing 0.02% Sodium Azide at 40 C. The protocol for purification of the antibody using the column was as follows: Matrix (bed volume -500 1). Wash with 3 bed volumes of the binding buffer. 500ul of the sample, diluted 1:1 with WO 2005/063818 PCT/IN2003/000434 34 the binding buffer is loaded. The outflow was collected. The column was washed with 10 ml of the binding buffer. Aliquots of 1 ml each were collected and absorbance was read at 280 nm. The column was eluted with 10 ml of Lysine- HC1 buffer (pH-2.8). Aliquots of 1 ml each were collected. Absorbance was read at 280 mn. The antibodies 5 thus obtained had better sensitivity. 90 - 95 % binding. EXAMPLE -36 The eggs from the birds immunised with protein myosin obtained from Acheta domestica were collected and from the 7 th day after the first booster the eggs were used to purify the antibodies from the yolk. The egg yolk was carefully removed from the 10 eggshell without rupturing the yolk membrane. The entire albumin adhering to the membrane was washed off. And the egg yolk membrane was ruptured and transferred into a measuring cylinder through a funnel. The egg yolk was diluted with water and filtered and kappa carrageenan (60%) added. The WSPF obtained after centrifugation was passed through whatmann filter paper and ImM Sodium hypophosphate added and 15 filtered again. The WSPF obtained was treated with 14% (w/v) PEG 8000 and the antibody precipitated out. The purified antibody was passed through mercaptopyridine sepharose column purchased locally. The column was equilibrated with 2 bed volumes of binding buffer. Iml of the yolk extract was added (after it had been passed through a 0.45 m filter), along with 1 ml of the binding buffer. The binding buffer was passed at a 20 rate of 5ml/minute. Passing the elution buffer eluted the binding buffer. The eluted solution was collected in 2ml fractions. Passing the cleaning buffer at a rate of 5ml/minute then cleaned the column. The absorbance was read at 280 mn of the 2ml fractions was taken. The fraction giving the peak absorbance was used as the purified IgG fraction. The sensitivity of the antibody did not improve. 25 The eggs from the birds immunised with protein myosin obtained Sitophilus Oryzae were collected and from the 7 th day after the first booster the eggs were used to purify the antibodies from the yolk. The egg yolk was carefully removed from the eggshell without rupturing the yolk membrane. The entire albumin adhering to the membrane was washed off. And the egg yolk membrane was ruptured and transferred into a 30 - measuring cylinder through a funnel. The egg yolk was diluted with water and filtered and kappa carrageenan added(60%) . The WSPF obtained after centrifugation was passed through whatmann filter paper and ImM Sodium hypophosphate added and filtered again. The WSPF obtained was treated with 14% (w/v) PEG 8000 and the WO 2005/063818 PCT/IN2003/000434 35 antibody precipitated out. The purified antibody was passed through mercaptopyridine sepharose column purchased locally. The column was equilibrated with 2 bed volumes of binding buffer, Iml of the yolk extract was added (after it had been passed through a 0.45 m filter) along with 1 ml of the binding buffer. The binding buffer was passed at a 5 rate of 5nl/minute. Passing the elution buffer eluted the binding buffer. The eluted solution was collected in 2ml fractions. Passing the cleaning buffer at a rate of 5ml/minute then cleaned the column. The absorbance was read at 280 nm of the 2m1 fractions was taken. The fraction giving the peak absorbance was used as the purified IgG fraction. The sensitivity of the antibody did not improve. 10 The eggs from the birds immunised with protein myosin obtained Rhizopertha dominica were collected and from the 7 th day after the first booster the eggs were used to purify the antibodies from the yolk. The egg yolk was carefully removed from the eggshell without rupturing the yolk membrane. The entire albumin adhering to the membrane was washed off. And the egg yolk membrane was ruptured and transferred into a 15 measuring cylinder through a funnel. The egg yolk was diluted with water and filtered and kappa carrageenan added (60%). The WSPF obtained after centrifugation was passed through whatmam filter paper and 1mM Sodium hypophosphate added and filtered again. The WSPF obtained was treated with 14% (w/v) PEG 8000 and the antibody precipitated out. The purified antibody was passed through mercaptopyridine 20 sepharose column purchased locally. The column was equilibrated with 2 bed volumes ,of binding buffer.Iml of the yolk extract was added (after it had been passed through a 0.45 m filter) along with 1 ml of the binding buffer. The binding buffer was passed at a rate of 5ml/minute. Passing the elution buffer eluted the binding buffer. The eluted solution was collected in 2ml fractions. Passing the cleaning buffer at a rate of 25 5ml/minute then cleaned the column. The absorbance was read at 280 nm of the 2ml fractions was taken. The fraction giving the peak absorbance was used as the purified IgG fraction. The sensitivity of the antibody did not improve. The eggs from the birds imanunised with protein myosin obtained Tribolium castaneum were collected and from the 7t" day after the first booster the eggs were used to purify 30 the antibodies from the yolk. The egg yolk was carefully removed from the eggshell without rupturing -the yolk membrane. The entire albumin adhering to the membrane was washed off. And the egg yolk membrane was ruptured and transferred into a measuring cylinder through a funnel. The egg yolk was diluted with water and filtered WO 2005/063818 PCT/IN2003/000434 36 and kappa carrageenan added (60%). The WSPF obtained after centrifugation was passed through whatmann filter paper and ImM Sodium hypophosphate added and filtered again. The WSPF obtained was treated with 14% (w/v) PEG 8000 and the antibody precipitated out. The purified antibody was passed through mercaptopyridine 5 sepharose column purchased locally. The column was equilibrated with 2 bed volumes of binding buffer. ml of the yolk extract was added (after it had been passed through a 0.45 min filter) along with 1 ml of the binding buffer. The binding buffer was passed at a rate of 5ml/minute. The binding buffer was eluted by passing the elution buffer. The eluted solution was collected in 2ml fractions. Passing the cleaning buffer at a rate of 10 5ml/minute then cleaned the column. The absorbance was read at 280 nm of the 2ml fractions was taken. The fraction giving the peak absorbance was used as the purified IgG fraction. The sensitivity of the antibody did not improve. EXAMPLE -37 The eggs were collected and from the 7 t day after the first booster the eggs were used 15 to purify the antibodies from the yolk. The egg yolk was carefully removed from the eggshell without rupturing the yolk membrane. The entire albumin adhering to the membrane was washed off. The egg yolk membrane was ruptured and transferred into a measuring cylinder through a funnel. The egg yolk was diluted with water and filtered and kappa carrageenan added (60%). The WSPF obtained after centrifugation was 20 passed through whatmann filter paper and 1mM Sodium hypophosphate added and filtered again. The WSPF obtained was treated with 14% (w/v) PEG 8000 and, the antibody precipitated out. The precipitated antibody was used to see the titer by two different methods. The antibody titer was found by reading the absorbance of the purified serum at 280 nm. The Antibody titer ranged from 3 - 12 mg/mL and was 7 mg 25 /mL on average. EXAMPLE -38 The eggs were collected and from the 7 th day after the first booster the eggs were used to purify the antibodies from the yolk. The egg yolk was carefully removed from the eggshell without rupturing the yolk membrane. The entire albumin adhering to the 30 membrane was washed off. And the egg yolk membrane was ruptured and transferred into a measuring cylinder through a funnel. The egg yolk was diluted with water and filtered and kappa carrageenan added (60%). The WSPF obtained after centrifugation was passed through whatmann filter paper and ImM Sodium hypophosphate added and WO 2005/063818 PCT/IN2003/000434 37 filtered again. The WSPF obtained was treated with 14% (w/v) PEG 8000 and the antibody precipitated out.- Protein estimation by Bradford method using BSA as the standard. Coomassie Brilliant blue G - 250 was dissolved in 95% ethanol and mixed with twice the volume of 85% Orthophosphoric acid and made the volume to 1000mL 5 with water. This reagent is used to estimate the antibody titer using BSA as a standard and read the absorbance at 595nm. It can be stored at 40 C for almost a month. The titer of the antibody (Acheta domestica) produced was 6 - 12g /hen. The titer of the antibody (Sitophilus oryzae) produced was 4.3 -10g/hen. The titer of the antibody (Rhizopertha dominica) produced was 4 - 8g /hen. The titer of the antibody (Tribolium 10 castaneum) produced was 5 - 9g /hen. ADVANTAGES The main advantages of the present invention are 1. Antibodies kits not available commercially. 2. High yield/titer of the antibody 15 3. Consistent quality of the antibody 4. Sensitivity of the assay equal/better than rabbit antibodies 5. Non-invasive 6. Good affinity to the analyte

Claims (28)

1. A process for the producing egg yolk antibodies against insect specific protein said method comprising the steps of: 5 (a) selecting suitable poultry birds, (b) immunizing the suitable bird of step (a) by injecting insect specific purified protein along with Freund's complete adjuvant of about 1000[g in breast muscle of the bird, (c) repeating the immunization by injecting insect specific purified protein along 10 with Freund's incomplete adjuvant at two, three and five weeks intervals and again after another five weeks interval, and (d) harvesting and isolating the antibodies from the egg yolk of the birds.

2. A process as claimed in claim 1, wherein the poultry bird in step (a) selected is White Leghorn. 15

3. A process as. claimed in claim 1, wherein the insects are selected from group comprising of Acheta domnestica, Sitophilus oryzae, Rhizopertha domninica and Tribolium casteneum.

4. A process as claimed in claim 1, wherein injected specific purified protein along with Freund's complete adjuvant of the insect in step (a) is in the range of about 20 450 to 500ptg.

5. A process as claimed in claim 1, wherein the injected specific purified protein along with Freund's incomplete adjuvant during the intervals two, three and five weeks and again after another five weeks interval periods in step (c) is in the range of about 200 to 300 tg. 25

6. A process as claimed in claim 1, wherein step (b) the insect specific myosin protein is isolated and purified from femur muscles of the stored grain insects said method comprising the steps of: (a) isolating the myosin protein from the femur of the stored grain insects using phosphate buffer of pH 7.5 to obtain of the protein by convention 30 method, and (b) purifying the protein obtained in step (a) using acetonitrile in the range of about 60 to 80%, trifluoroacetic acid in the range of about 0.05 to 2.0%, and (c) identifying the purified protein of step (c) at a wavelength of 280nm, WO 2005/063818 PCT/IN2003/000434 39 with a absorbance unit of full scale (AUFs) of 0.08 in run time of 25 minutes and a peak at 17 minutes.

7. A process as claimed in claim 6, wherein yield of protein from grain insect Acheta doemstica is in the range of about 1.5 to 2.0 mg/L. 5

8. A process as claimed in claim 7, wherein yield of protein from grain insect Acheta doemstica is about 1.76 mg/L.

9. A process as claimed in claim 6, wherein yield of protein from grain insect Stiophilus oryzae is in the range of about 5 to 7 mg/L

10. A process as claimed in claim 9, wherein yield of protein from grain insect 10 Stiophilus oryzae is about 5.6 mg/L

11. A process as claimed in claim 6, wherein yield of protein from grain insect Rhizoperthla dominica is in the range of about 1 to 5 mg/L

12. A process as claimed in claim 11, wherein yield of protein from grain insect Rhizopertha dominica is about 1.6 mg/L 15

13. A process as claimed in claim 6, wherein yield of protein from grain insect Tribolium castaneu is in the range of about 1 to 5mg/L

14. A process as claimed in claim 13, wherein yield of protein from grain insect Tribolium castaneu is in the range of about 2 mg/.

15. A process as claimed in claim 6, wherein conventional method in step (a) is 20 centrifugation and SDS-PAGE.

16. A process as claimed in claim 6, wherein purified protein in step (c) from grain insect Acheta domestica is about 200 kDa and from grain insects Stiophilus oryzae, Rhizopertha dominica and Triboliumn castaneu is about 45 kDa.

17. A process as claimed in claim 1, wherein antibodies against the specific grain 25 insect protein are isolated from water-soluble fraction of the egg yolk, said process comprising of: S(a) rupturing the egg and obtaining to obtain lipid contents of the egg yolk, (b), diluting the lipid contents of egg yolk of step (a) with water followed by addition of kappa carragenan in the range of about 50 to 75 % to obtain 30 water soluble protein fraction antibodies (WSPF), (c) passing the WSPF antibodies from step (b) through sodium hypophosphate-in the range of about 0.5 to 2 mM, (d) precipitating the WSPF antibodies obtained from step (c) with PEG 8000 in the range of about 10-20 %, and WO 2005/063818 PCT/IN2003/000434 40 (e) purifying the WSPF antibodies obtained from step (d) by affinity column chromatography

18. A process as claimed in claim 17, wherein kappa carragenan in step (b) is about 5 60%.

19. A process as claimed in claim 17, wherein sodium hypophosphate is about ImM

20. A process as claimed in claim 17, wherein PEG 8000 in step (d) is about 14%.

21. A process as claimed in claim 17, wherein yield of antibodies from grain insects Acheta domnestica, Sitophilus oryzae, Rhizopertha dominica and Triboliumn 10 casteneum protein is in the range of about 80-98 %.

22. A process as claimed in claim 17, wherein yield of antibodies from grain insects Acheta domestica, Sitophilus oryzae, Rhizopertha dominica and Triboliumn casteneum protein is in the range of about 95 %.

23. A process as claimed in claim 21, wherein the production of the antibody is 15 initiated from the day 7 after immunization and continues upto 60 days.

24. A process as claimed in claims 1 and 17,wherein antibody produced from grain insect Acheta domestica is in the range of about 5 -1 Sg /hen, from insect Sitophilus oryzae is in the range of about 3 -15g/hen, from insect Rhizopertha domninica is in the range of about 3 -10g /hen and from insect Triboliumn castaneum is in the range 20 of about 3 - 15 g/hen.

25. A process as claimed in claim 24, wherein antibody produced from insect Acheta domestica is in the range of about 6 - 12g /hen, from insect Sitophilus oryzae is in the range of about 4.3 -10 g/hen, from insect Rhizopertha dominica is in the range of about 4 -8 g /hen and from insect Tribolium castaneum is in the range of about 25 5 - 9 g /hen.

26. A process as claimed in claims 1 and 17, wherein the egg yolk antibodies are useful for analysis of insect contamination and filth in foods by ELISA (Fig. 1).

27. A process as claimed in claims 1 and 17, wherein the egg yolk antibodies produced provide the basic material for developing ELISA test methods using plate, tube, 30 dipstick, PVP films and biosensors (Fig.2).

28. A process as claimed in claims 25 and 26, wherein the developed ELISA test methods find application in the detection of insect contamination /filth in food, milled products and processed foods. (Fig. 1 and Table 3) WO 2005/063818 PCT/IN2003/000434 41 AMENDED CLAIMS [(received by the International Bureau on 18 January 2005 (18.01.05); original claims 1-28 replaced by new claims 1-28 (4 pages)] + STATEMENT 1. A process for the producing egg yolk antibodies against insect protein said method comprising the steps of: (a) selecting suitable poultry birds, (b) immunizing the suitable bird of step (a) by injecting insect specific purified protein along with Freund's complete adjuvant of about 1000 Lg in breast muscle of the bird, (c) repeating the immunization by injecting insect specific purified protein along with Freund's incomplete adjuvant at two, three and five weeks intervals and again after another five weeks interval, and (d) harvesting and isolating the antibodies from the egg yolk of the birds. 2. A process as claimed in claim 1, wherein the poultry bird in step (a) selected is White Leghorn. 3. A process as claimed in claim 1, wherein the insects are selected from group comprising of Acheta domestica, Sitophilus oryzae, Rhizopertha dominica and Triboliumn casteneum. 4. A process as claimed in claim 1, wherein injected purified protein along adjuvant of the insect in step (a) is in the range of about 450 to 5004g. 5. A process as claimed in claim 1, wherein the injected purified protein during the intervals two, three and five weeks and again after another five weeks interval periods in step (c) is in the range of about 200 to 300 [g. 6. A process as claimed in claim 1, wherein step (b) the insect specific myosin protein is isolated and purified from femur muscles of the stored grain insects said method comprising the steps of: (a) isolating the myosin protein from the femur of the stored grain insects using phosphate buffer of pH 7.5 to obtain of the protein by convention method, and (b) purifying the protein obtained in step (a) using acetonitrile in the range of about 60 to 80%, trifluoroacetic acid in the range of about 0.05 to 2.0%, and WO 2005/063818 PCT/IN2003/000434 42 (c) identifying the purified protein of step (c) at a wavelength of 280nmn, with a absorbance unit of full scale (AUFs) of 0.08 in run time of 25 minutes and a peak at 17 minutes. 7. A process as claimed in claim 6, wherein yield of protein from grain insect Acheta domestica is in the range of about 1.5 to 2.0 mg/L. 8. A process as claimed in claim 7, wherein yield of protein from grain insect Acheta dominestica is about 1.76 mg/L. 9. A process as claimed in claim 6, wherein yield of protein from grain insect Stiophilus oryzae is in the range of about 5 to 7 mg/L 10. A process as claimed in claim 9, wherein yield of protein from grain insect Stiophilus oryzae is about 5.6 mg/L 11. A process as claimed in claim 6, wherein yield of protein from grain insect Rhizopertha dominica is in the range of about 1 to 5 mg/L 12. A process as claimed in claim 11, wherein yield of protein from grain insect Rhizopertha domininica is about 1.6 mg/L 13. A process as claimed in claim 6, wherein yield of protein from grain insect Tribolium castaneu is in the range of about 1 to 5mg/L 14. A process as claimed in claim 13, wherein yield of protein from grain insect Tribolium castaneu is in the range of about 2 mg/. 15. A process as claimed in claim 6, wherein conventional method in step (a) is centrifugation and SDS-PAGE. 16. A process as claimed in claim 6, wherein purified protein in step (c) from grain insect Acheta domestica is about 200 kDa and from grain insects Stiophilus oryzae, Rhizopertha dominica and Triboliumn castaneu is about 45 kDa. 17. A process as claimed in claim 1, wherein antibodies against the specific grain insect protein are isolated from water-soluble fraction of the egg yolk, said process comprising of: (a) rupturing the egg and obtaining to obtain lipid contents of the egg yolk, (b) diluting the lipid contents of egg yolk of step (a) with water followed by addition of kappa carragenan in the range of about 50 to 75 % to obtain water soluble protein fraction antibodies (WSPF), AMENDED SHEET (ARTICLE 19) WO 2005/063818 PCT/IN2003/000434 43 (c) passing the WSPF antibodies from step (b) through sodium hypophosphate in the range of about 0.5 to 2 mM, (d) precipitating the WSPF antibodies obtained from step (c) with PEG 8000 in the range of about 10-20 %, and (e) purifying the WSPF antibodies obtained from step (d) by affinity colunm chromatography 18. A process as claimed in claim 17, wherein kappa carragenan in step (b) is about 60%. 19. A process as claimed in claim 17, wherein sodium hypophosphate is about 1mM 20. A process as claimed in claim 17, wherein PEG 8000 in step (d) is about 14%. 21. A process as claimed in claim 17, wherein yield of antibodies from grain insects Acheta domestica, Sitophilus oryzae, Rhizopertha dominica and Tribolium casteneum protein is in the range of about 80-98 %. 22. A process as claimed in claim 17, wherein yield of antibodies from grain insects Acheta domestica, Sitophilus oryzae, Rhizopertha domininica and Tribolium casteneum protein is in the range of about 95 %. 23. A process as claimed in claim 21, wherein the production of the antibody is initiated from the day 7 after immunization and continues upto 60 days. 24. A process as claimed in claims 1 and 17,wherein antibody produced from grain insect Acheta domestica is in the range of about 5 - 15g /hen, from insect Sitophilus oryzae is in the range of about 3 -15g/hen, from insect Rhizopertha dominica is in the range of about 3 -10g /hen and from insect Triboliumn castaneum is in the range of about 3 - 15 g /hen. 25. A process as claimed in claim 24, wherein antibody produced from insect Acheta domnestica is in the range of about 6 - 12g /hen, frominsect Sitophilus oiyzae is in the range of about 4.3 -10 g/hen, from insect Rhizopertha dominica is in the range of about 4 -8 g /hen and from insect Tribolium castaneumn is in the range of about 5 - 9 g /hen. 26. A process as claimed in claims 1 and 17, wherein the egg yolk antibodies are useful for analysis of insect contamination and filth in foods by ELISA (Fig. 1). AMENDED SHEET (ARTICLE 19) WO 2005/063818 PCT/IN2003/000434 44 27. A process as claimed in claims 1 and 17, wherein the egg yolk antibodies produced provide the basic material for developing ELISA test methods using plate, tube, dipstick, PVP films and biosensors (Fig.2). 28. A process as claimed in claims 25 and 26, wherein the developed ELISA test methods find application in the detection of insect contamination /filth in food, milled products and processed foods. (Fig.1 and Table 3) AMENDED SHEET (ARTICLE 19) WO 2005/063818 PCT/IN2003/000434 45 + STATEMENT In the citation D1 antibodies have been raised against reptile venom, whereas in present invention antibodies raised against the stored grain insect, which means chemistry of the two antibodies are entirely different. The citation D2 discloses antibodies raised against Lepidopterans insects whereas the present invention the antibodies have been raised against Coleopteran insects. The citation D3 discloses an obvious and most widely used adjuvant which is usually used in such kind of studies and hence is not obvious. The antibodies raised in the citation D4 are from rabbit and thus chemically different from the antibodies raised in the present study. The antibodies raised in the citation D5 is against the pesticides and not against stored grain pests, hence they are chemically and physiologically different in their actions. The inventors respectfully submit that the chemistry of the antibodies raised in the present invention is different from the prior arts. Since the chemistry of the antibodies is different therefore the person skilled in the art cannot envisage from the prior arts.