Title of Invention	"A PROCESS FOR THE PREPARATION OF ALLERGEN COATED SOLID SUPPORT USEFUL IN ALLERGY DIAGNOSIS"
Abstract	A process for preparation of allergen coated solid support useful in allergy diagnosis by reconstituting the lyophil-ized allergen extracts with buffer or distiled water, dissolving the polyvinyl alcohol (PVA) in water and mixing thoroughly with the above allergen extract(s), coating of the above prepared allergenic mixture on solid support, drying of allergen  coated filter paper at temperature in the range of 25-30 ºC, cross-linking of coated allergens with glutaralde-    hyde, drying the above allergen coated  solid support at    temperature in the range of 25-30º C, punching the  solid support to obtain the desired discs.

Title of Invention

"A PROCESS FOR THE PREPARATION OF ALLERGEN COATED SOLID SUPPORT USEFUL IN ALLERGY DIAGNOSIS"

Abstract

A process for preparation of allergen coated solid support useful in allergy diagnosis by reconstituting the lyophil-ized allergen extracts with buffer or distiled water, dissolving the polyvinyl alcohol (PVA) in water and mixing thoroughly with the above allergen extract(s), coating of the above prepared allergenic mixture on solid support, drying of allergen  coated filter paper at temperature in the range of 25-30 ºC, cross-linking of coated allergens with glutaralde-    hyde, drying the above allergen coated  solid support at    temperature in the range of 25-30º C, punching the  solid support to obtain the desired discs.

Full Text	The present invention relates to a process for preparation of allergen coated solid support useful in allergy diagnosis. The main use of the present invention is to provide allergen coated solid support for diagnosis of allergies by ELISA. The solid support can also be used to find out the potency of allergen extracts by ELISA inhibition. The role of inhalant allergens such as airborne pollen, fungal spores, insects etc. in precipitating type I allergic disorders is well established. As per the estimate, about 15% population of the country suffer from rhinitis, asthma and conjunctivitis. To detect the offending allergen, most of the allergy clinics in India practice skin test with allergy ex tracts. Both the methods (intradermal/prick) of skin tests possess several drawbacks. The discovery of IgE antibody [Ishizaka K. and Ishizaka T. (1967). J.Immunol. 99:1187-98.] led to development of an in-vitro method (RAST) for measuring the circulating IgE antibodies. This has become a valuable tool in diagnosis of type I respiratory allergy diseases [ Wide L. (1973). Clin. Allergy, 3 suppl:583 - 93; Schroder H., Krober A. and Yman L. (1981) . Allergol. Immunopathol. 9:46-50] . and is routinely used in USA, Europe and other developed countries. ELISA is also based on the same principles, but in this procedure enzyme conjugated second antibody is used in place of radiolabelled IgE (RAST). The in-vitro methods of allergy diagnosis have several advantages over in-vivo procedures (Intradermal, Prick & Bronchial Provocation Test) as enumerated below: a. The results of RAST/ELISA show agreement with Bronchial Provocation Test which is considered as the most sensitive test . b. In-vitro test can be performed on symptomatic days of the patients. c. With few millilitres of patients sera, many tests can be performed thus avoiding the painful pricks associated with the skin tests. d. In-vitro tests are less time consuming to patients, since the patients are not required to sit in the clinic for long duration. e. In-vitro tests are devoid of risks (anaphylaxis) to patients. RAST/ELISA are confirmatory tests [ Negrini A.C., Troise C. and Voltolini S. (1985). Allergy, 40:238-41]. However, in India these tests could not be popular because of non-availability of reagents. The imported test kits are expensive and contain only few relevant antigens. The paper discs coated with allergens by cyanogen bromide (CNBr) activation method have been prepared by some workers [Sridhara S .(1985) .Ph.D.thesis, University of Delhi,India]. CNBr activation method is a cumbersome procedure and involves serious health hazards to workers due to inhalation of its vapours. Earlier in our laboratory this method has been worked out in detail [Jaggi K.S. and Gangal S.V. (1987). Int. Arch. Allergy Appl. Immunol., 89:311-13] . However, we could not overcome the problems related to the storage of discs prepared by this method. The Pharmacia, Sweden is known to supply the allergen coated discs for use in ELISA prepared by CNBr activation method. But the discs contain the allergen types prevalent in Europe, USA, and other temperate countries. The commercially available kits for in-vitro diagnostic methods (RAST, ELISA) did not cater the need of Indian patient's as well as of allergy researchers in India. Therefore, the need of developing a ELISA kit was felt since long for quantitation of allergen specific IgE in patient's sera against the allergens prevalent in Indian sub-continent. A novel method of allergen coating on Whatman paper has been evolved and standardized by us with 5 clinically important pollen-Amaranthus spinosus (weed), Imperata cylindrica (grass), Holoptelea integrifolia (tree), Prosopis juliflora (tree), Putranjiva roxburghii (tree) and 5 fungal allergen- Alternaria alternata, Aspergillus fumigatus, Cladosporium herbarum, Curvularia lunata, Epicocum nigrum extracts. The method is simple reliable, cost effective and avoids the use of carcinogenic material as in case of CNBr activation method. The solid support coated allergens prepared by improved method show stability for six months period on storage at the temperature in the range of 4-8°C inside the referigerator and also at room temprature ranging from 25 to 30°C. Unlike CNBr method, allergen coated solid support can be stored in dried form, making convenient to handle the solid support in tests and transport to long distances under variable temperature conditions for use in allergy clinics. The main object of the present invention is to provide a novel process for preparation of allergen coated solid support, useful in allergy diagnosis by ELISA, which obviates the drawbacks as detailed above. Another object of the present invention is to develop allergen coated solid support. Yet another object of the present invention is to use allergen coated solid support for measuring potency of different batches of extracts by ELISA inhibition. Still another object of the present invention is to develop a simple, reliable and cost-effective procedure using polyvinyl alcohol (PVA) and glutaraldehyde for coating of allergenic proteins on solid support. Another object of the present invention is to coat mixed allergen preparations (group allergens) on solid support to detect atopy in individuals exposed to atmospheric allergens . Accordingly, the present invention provides a process for preparation of allergen coated solid support useful in allergy diagnosis which comprises : (a) reconstituting the lyophilized allergen extracts with buffer or distiled water, (b) dissolving the polyvinyl alcohol (PVA) in water and mixing thoroughly with the above allergen extract(s), (c) coating of the above prepared allergenic mixture on solid support, (d) drying of allergen coated filter paper at temperature in o the range of 25-30 C, (e) cross-linking of coated allergens with glutaraldehyde, ! f) drying the above allergen coated solid support: at o temperature in the range of 25-30, C, (g) punching the solid support to obtain the desired discs. The allergen extracts may be selected from different pollens, fungi, insects, dusts, animal scales and mites. In an embodiment of the present invention, the allergenic protein may be reconstituted in PBS or carbonat-bicarbonate buffer or water. In another embodiment of the present invention polyvinyl alcohol may be dissolved in buffer or distilled water. In another embodiment of the present invention, the concentration of the polyvinyl alcohol may be 2% to 5%. In yet another embodiment of the present invention, the glutaraldehyde concentration may be 1% to 4%. In still another embodiment of the present invention, the coating may be affected by immersing the solid support in the allergen protein mixture or incubating the wells of tissue culture plates and or culture tubes with protein mixture. In another embodiment of the present invention,the solid support used may be filter paper, tissue culture plates or culture tubes made of polypropylene/polystyrene. Accordingly the present invention provide an allergen coated solid support useful in allergy diagnosis by the process as described above. A mixture of protein of each extract with polyvinyl alcohol (PVA) AR grade, was coated on solid support. Polyvinyl alcohol forms a thin, uniform and porous membrane on the surface of allergen coated solid support. Later glutaraldehyde was used in a concentration in the range of 1% to 4% for cross linking the amino group of extracted proteins entrapped on the solid support. The discs were punched out from the allergen coated solid support by using a office punch. Preparation of allergen extract/ Collection of patients sera: Polleniferous materials were collected from well identified plants growing in and around Delhi metropolis. The materials were processed carefully to remove floral parts other than pollen. Finally the pollens were separated out by pulverising/ passing the materials through different grades of sieves as per the size of pollen. The samples were subjected to microscopic analysis to check the purity in terms of pollen content. The samples having more than 95% pollen ( 6.H1. , Rosenbrough N.J., Farr A.L. and Randall R.J. (1951) J.Biol. Chem. 193: 265-75]. Phosphotungstic acid reagent (15% PTA in 10% HCl) was used to precipitate the protein from the extracts, [ Singh B.P., Sridhara S., Arora N. and Gangal S.V. (1992). Bio-chem. Int., 27: 477-84]. Lyophilized extracts (1:20 w/v) were reconstituted to 1:500 w/v in phosphate buffer saline (PBS) for skin tests (intradermal) on patients of naso-bronchial allergy. The patient's were selected for intradermal test based on their clinical history. For skin tests, about 0.02 ml of each extract was injected intradermally into the forearm of the patient raising a bleb of 1-2 mm at the site of injection. Simultaneously, as a negative control-PBS and positive control i.e. histamine di-phosphate (100 ug/ml) were also injected. ID test reactions were graded after 20 min in comparison to the wheal size of the negative control. Skin tests with the antigens (pollen and fungi) were also carried out on some healthy non-allergic volunteers and the sera collected. The patients showing positive interadermal test to pollen and/or fungal antigens were persuaded to donate sera for use in ELISA. Enzyme Linked Immunosorbent Assay: ELISA1s were carried out [Voller A., Bidwell D. and Barlett T. In Rose NR, Friedman H. (eds). Manual of Clinical Immunoassay, American Society for Microbiology, Washington, 1980] with allergen coated discs in tissue culture polystyrene plates. Unbound sites of the discs if any, were blocked with 2% bovine serum albumin (BSA, w/v in PBS) for 2 hrs at room temperature in the range of 25-30°C. Washing of the discs was performed (3 changes) with PBS-tween 20 (0.05%). The discs were incubated overnight with patient's sera (1:10 v/v in PBS, 100 ul/well) at 4°C. After washing with PBS-tween 20, anti IgE conjugated to alkaline phosphatase (1:2000, v/v in PBS, 100 ul/well), was added and incubated overnight at 4°C. As a negative control, normal human serum (NHS) was also tested in the same dilution. Washing was performed again as mentioned earlier and the plate was incubated with p-nitrophenyl phosphate (1 mg/ml in glycine buffer) at 37°C for colour development. The reaction was stopped after 40 minutes by adding 3N NaOH (50 ul/well). The discs were removed and the absorbance was read at 405 nm using Dynatech ELISA reader. Initially, solid support (paper discs) coated with Amaranthus spinosus (AS) pollen extract were prepared with a new method as described above. Allergen discs of AS prepared by this method showed good comparison with discs made by CNBr method in measurement of specific IgE by ELISA. Later the discs coated with graded amount of AS protein were prepared by the new procedure. These discs were subjected to specific IgE determinations by ELISA in positive patients sera to Amaranthus spinosus extract. The paper discs coated with approx. 10 ug protein showed better promise for ELISA. The success of above experiments induced us to prepare allergen coated discs from 5 types of clinically important pollen (Amaranthus spinosus, Imperata cylindrica, Holoptelea integrifolia, Prosopis juliflora and Putranjiva roxburghii) extracts by the same procedure. Allergen specific IgE were measured using discs prepared by the improved method in patients sera and also in NHS collected from non allergic volunteers. Interpretation of ELISA results were made based on negative control [Pauwels R., Verschraegen G. and Straeten V.D. (1980), Allergy, 157:665-69; Calenoff E., Me Mahan J.T., Herzon G.D., Kern R.C., Ghadge G.D. and Hanson D.G. (1992) Arch. Otolaryngol, Head Neck Surge. 119 (8) :830-36] or positive control (Pharmacia, Sweden). The analysis of the data revealed that patients sera showing optical densities (O.D.'s) more than, mean +2 SD of normal human serum ( NHS, i.e. negative control, O.D.,s equal to 0.2 or more) may be taken as ELISA positive. Sera showing 4 Phadebas RAST Unit (PRU)/ml i.e. 10 ng/ml (specific IgE) should be designated as ELISA positive [ McSharry C., McKay I.C. and Lewis C. (1993), J. Clin. Immunoassay, 16: 153-58 ]. Validation of Allergen Coated Discs by ELISA: Dilution dependent relationship was worked out taking serialdilutions (11 dilutions e.g.1:1,1:2 1:4 till 1:1024), of two sera positive to Amaranthus spinosus and Prosopis juliflora extracts. The Measurement of allergen specific IgE was carried out by ELISA using the discs coated with respective allergen extract prepared by the improved process. Analysis of the results showed dilution dependent relationship in O.D. values. The assay was found to be working well with these discs upto the sera dilution of 1:1024 (v/v in PBS). Analysis of the results showed coefficient of variations (C.V.'s) in the range of 4-11% for intra-assays, whereas C.V.'s ranged 3-14% in inter-assays with pollen allergen discs prepared by the new procedure. Studies conducted elsewhere with allergen coated discs (mite, timothy & dog allergen) show CV's=2.9% for within assays and CV's= 9-29% for inter assays [Poulsen L.K.,Pedersen M.F.. Mailing H.J., Sondergaard I. and Weeke B. (1989), Allergy 44:173-80; Plebani M, Faggian D. and Borghesan F. (1995),Allergy 50:229-33]. To compare skin test gradings with ELISA, allergen specific IgE was measured in positive patient's sera by ELISA using 5 types of pollen and fungal extracts discs prepared, individually by the new procedure. Analysis of skin tests and ELISA results (25 to 40 patient's sera) showed 50-70% correlation for sera assigned as positive on the basis of intradermal tests. For comparison, determination of specific IgE was carried out in sera samples by ELISA using discs coated allergens and plate binding method. Results indicated that allergen coated discs prepared by our method have better sensitivity in ELISA for estimation of specific IgE compared to plate binding method. Toorenenbergen and his group (1987) have reported [Van Toorenenbergen A.W., Gerth van Wijk R., Dieges P.H. and Leijnse B. (1987), Int. Arch. Allergy Appl. Immunol. 83:436-39] irrelevant IgE binding to cellulose discs (Pharmacia) in RAST. We have carried out ELISA with the discs prepared by the new method and Pharmacia discs with sera of non-IgE mediated diseases. Results show very low non-specific binding with discs prepared by the new procedure (O.D.s 0.088-0.108; normal control O.D. 0.065) compared to the Pharmacia discs (O.D. 0.336-0.441). ELISA's were carried out to compare the allergen coated discs prepared by the improved procedure and the discs procured from Pharmacia, Sweden for Prosopis juliflora and Aspergillus fumigatus extracts. Statistical analysis of the data showed significant correlation with both types of discs (critical values = 0.821 & 0.879 respectively). At 5% probability the critical values of + 0.530 (n=14) and +. 0.599 (n=ll), respectively were recorded significant for 2 tailed analysis [Singh B.P.,Sridhara S. and Gangal S.V. (1996), Project Completion Report (DBT), Centre for Biochemical Technology, Delhi]. For the independent validation of discs prepared by the present method, allergen specific IgE was measured by ELISA in the same sera samples by three persons, independentlly. The optical densities (O.D's) recorded by all three persons were found to be matching for most of the sera samples. Also out of 5 sera tested, the same 4 were assigned as ELISA positive (O.D.> mean +2SD of NHS) by all the three investigators. The invention is described in detail in the examples given below which are described by way of illustrations only and should not be construed to limit the scope of present invention. Example 1: Three gram polyvinyl alcohol (PVA) was dissolved with stirring in one hundred ml distilled water at a temperature of 80°C. The lyophilized allergen extract (1:20 w/v) was reconstituted to 1 ml in 0.05M phosphate buffered saline (PBS), pH 7.8. The PVA and allergenic protein (extract) solutions were mixed together so as to get a concentration of 65 ug protein per square centimeter of the coating area on a filter paper. The mixture of PVA and the allergenic protein was coated by brushing and or immersing both the surfaces of a filter paper (solid support). Protein coated solid support was dried and incubated with 1% glutaraldehyde solution in water. This was followed by washing of the solid support with distilled water and drying at 30°C. Discs were pun'ched out from the solid support as desired. Example 2: 2.5 gram polyvinyl alcohol (PVA) was dissolved with stirring in hundred ml distilled water at the temperature of 80°C. The lyophilized allergen extract (1:20 w/v) was reconstituted to 1 ml in 0. 1M carbonate-bicarbonate buffer, pH 9.6. The PVA and allergenic protein (extract) solutions were mixed together. For coating, 10 ug of protein (200ul) was poured in each well of tissue culture plate or 30 ug protein in 300 ul per culture tube. This was subjected to drying at 30°C for coating. The plate and the tubes were then incubated with 200 ul/lml of 1.5% glutaraldehyde in water. The allergen coated plates/tubes were washed with distilled water and allowed to dry at 30°C. The main advantages of the present invention are: 1. a simple, reliable and cost effective process of allergen coating has been developed, 2. allergen coated discs were prepared with indegenous allergens and standardized by ELISA, 3. the allergen coated solid support (paper discs) prepared by the present procedure showed significant correlation in ELISA with CNBr coated discs,procured from Pharmacia,Sweden, 4. unlike the CNBr method, the allergen coated discs can be stored in dried form at 25-30°C temperature, making it convenient to handle the discs in tests and transport, 5. the product (discs) offers confirmatory diagnosis of respiratory allergies (rhinitis, asthma etc.) by ELISA. We claim : 1. A process for preparation of allergen coated solid support useful in allergy diagnosis which comprises : (a) reconstituting the lyophilized allergen extracts with buffer or distiled water, (b) dissolving the polyvinyl alcohol (PVA) in water and mixing thoroughly with the above allergen extract(s), (c) coating of the above prepared allergenic mixture on solid support, (d) drying of allergen coated filter paper at temperature in the range of 25-30ºC, (e) cross-linking of coated allergens with glutaraldehyde, (f) drying the above allergen coated solid support at temperature in the range of 25-30ºC, (g) punching the solid support to obtain the desired discs. 2. A process as claimed in claim 1 wherein,the allergen extract used is such as from thepollen, fungi, insects, food, animal scales and or mites extracts (whose single or mixed allergen preparations can be coated on solid support). 3. A process as claimed in claim 1 and 2, wherein the buffer used is phosphate buffer saline, carbonate-bicarbonate buffer. 4. A process as claimed in claim 1-3, wherein the concentration of polyvinyl alcohol may be 2% to 5%. 5. A process as claimed in claims 1-4, wherein the concen tration of glutaraldehyde used may be 1% to 4%. 6. A process as claimed in claims 1-5, wherein the coating may be affected by immersing the solid support in allergen mixture or by incubating the wells of the plate and culture tube with protein mixture. 7. A process for preparation of allergen coated solid support useful in diagnosis of allergies substantially as herein described with reference to the examples.

Full Text

The present invention relates to a process for preparation of allergen coated solid support useful in allergy diagnosis.
The main use of the present invention is to provide allergen coated solid support for diagnosis of allergies by ELISA. The solid support can also be used to find out the potency of allergen extracts by ELISA inhibition.
The role of inhalant allergens such as airborne pollen, fungal spores, insects etc. in precipitating type I allergic disorders is well established. As per the estimate, about 15% population of the country suffer from rhinitis, asthma and conjunctivitis. To detect the offending allergen, most of the allergy clinics in India practice skin test with allergy ex tracts. Both the methods (intradermal/prick) of skin tests possess several drawbacks. The discovery of IgE antibody [Ishizaka K. and Ishizaka T. (1967). J.Immunol. 99:1187-98.] led to development of an in-vitro method (RAST) for measuring the circulating IgE antibodies. This has become a valuable tool in diagnosis of type I respiratory allergy diseases [ Wide L. (1973). Clin. Allergy, 3 suppl:583 - 93; Schroder H., Krober A. and Yman L. (1981) . Allergol. Immunopathol. 9:46-50] . and is routinely used in USA, Europe and other developed countries. ELISA is also based on the same principles, but in this procedure enzyme conjugated second antibody is used in place of radiolabelled IgE (RAST). The in-vitro methods of allergy diagnosis have several advantages over in-vivo procedures (Intradermal, Prick & Bronchial Provocation Test) as enumerated below:

a. The results of RAST/ELISA show agreement with Bronchial
Provocation Test which is considered as the most sensitive
test .
b. In-vitro test can be performed on symptomatic days of the
patients.
c. With few millilitres of patients sera, many tests can be
performed thus avoiding the painful pricks associated with
the skin tests.
d. In-vitro tests are less time consuming to patients, since
the patients are not required to sit in the clinic for long duration.
e. In-vitro tests are devoid of risks (anaphylaxis) to
patients.
RAST/ELISA are confirmatory tests [ Negrini A.C., Troise C. and Voltolini S. (1985). Allergy, 40:238-41]. However, in India these tests could not be popular because of non-availability of reagents. The imported test kits are expensive and contain only few relevant antigens. The paper discs coated with allergens by cyanogen bromide (CNBr) activation method have been prepared by some workers [Sridhara S .(1985) .Ph.D.thesis, University of Delhi,India]. CNBr activation method is a cumbersome procedure and involves serious health hazards to workers due to inhalation of its vapours. Earlier in our laboratory this method has been worked out in detail [Jaggi K.S. and Gangal S.V. (1987). Int. Arch. Allergy Appl. Immunol., 89:311-13] . However, we could not overcome the problems related to the storage of discs prepared by this method. The Pharmacia, Sweden is known to supply the allergen coated discs for use in ELISA prepared by CNBr

activation method. But the discs contain the allergen types prevalent in Europe, USA, and other temperate countries. The commercially available kits for in-vitro diagnostic methods (RAST, ELISA) did not cater the need of Indian patient's as well as of allergy researchers in India. Therefore, the need of developing a ELISA kit was felt since long for quantitation of allergen specific IgE in patient's sera against the allergens prevalent in Indian sub-continent. A novel method of allergen coating on Whatman paper has been evolved and standardized by us with 5 clinically important pollen-Amaranthus spinosus (weed), Imperata cylindrica (grass), Holoptelea integrifolia (tree), Prosopis juliflora (tree), Putranjiva roxburghii (tree) and 5 fungal allergen- Alternaria alternata, Aspergillus fumigatus, Cladosporium herbarum, Curvularia lunata, Epicocum nigrum extracts. The method is simple reliable, cost effective and avoids the use of carcinogenic material as in case of CNBr activation method. The solid support coated allergens prepared by improved method show stability for six months period on storage at the temperature in the range of 4-8°C inside the referigerator and also at room temprature ranging from 25 to 30°C. Unlike CNBr method, allergen coated solid support can be stored in dried form, making convenient to handle the solid support in tests and transport to long distances under variable temperature conditions for use in allergy clinics.
The main object of the present invention is to provide a novel process for preparation of allergen coated solid support, useful in allergy diagnosis by ELISA, which obviates the drawbacks as detailed above.

Another object of the present invention is to develop allergen coated solid support.
Yet another object of the present invention is to use allergen coated solid support for measuring potency of different batches of extracts by ELISA inhibition.
Still another object of the present invention is to develop a simple, reliable and cost-effective procedure using polyvinyl alcohol (PVA) and glutaraldehyde for coating of allergenic proteins on solid support.
Another object of the present invention is to coat mixed allergen preparations (group allergens) on solid support to detect atopy in individuals exposed to atmospheric allergens .
Accordingly, the present invention provides a process for preparation of allergen coated solid support useful in allergy diagnosis which comprises :
(a) reconstituting the lyophilized allergen extracts with
buffer or distiled water,
(b) dissolving the polyvinyl alcohol (PVA) in water and
mixing thoroughly with the above allergen extract(s),
(c) coating of the above prepared allergenic mixture on solid
support,
(d) drying of allergen coated filter paper at temperature in
o the range of 25-30 C,
(e) cross-linking of coated allergens with glutaraldehyde,
! f) drying the above allergen coated solid support: at
o temperature in the range of 25-30, C,
(g) punching the solid support to obtain the desired discs.

The allergen extracts may be selected from different pollens,
fungi, insects, dusts, animal scales and mites.
In an embodiment of the present invention, the allergenic protein
may be reconstituted in PBS or carbonat-bicarbonate buffer or
water.
In another embodiment of the present invention polyvinyl alcohol
may be dissolved in buffer or distilled water.
In another embodiment of the present invention, the concentration
of the polyvinyl alcohol may be 2% to 5%.
In yet another embodiment of the present invention, the
glutaraldehyde concentration may be 1% to 4%.
In still another embodiment of the present invention, the coating
may be affected by immersing the solid support in the allergen
protein mixture or incubating the wells of tissue culture plates
and or culture tubes with protein mixture.
In another embodiment of the present invention,the solid support
used may be filter paper, tissue culture plates or culture tubes
made of polypropylene/polystyrene.
Accordingly the present invention provide an allergen coated
solid support useful in allergy diagnosis by the process as
described above.
A mixture of protein of each extract with polyvinyl alcohol (PVA) AR grade, was coated on solid support. Polyvinyl alcohol forms a thin, uniform and porous membrane on the surface of allergen coated solid support. Later glutaraldehyde was used in a concentration in the range of 1% to 4% for cross linking the amino group of extracted proteins entrapped on the solid support.

The discs were punched out from the allergen coated solid support
by using a office punch.
Preparation of allergen extract/ Collection of patients sera:
Polleniferous materials were collected from well identified plants growing in and around Delhi metropolis. The materials were processed carefully to remove floral parts other than pollen. Finally the pollens were separated out by pulverising/ passing the materials through different grades of sieves as per the size of pollen. The samples were subjected to microscopic analysis to check the purity in terms of pollen content. The samples having more than 95% pollen (
6.H1. , Rosenbrough N.J., Farr A.L. and Randall R.J. (1951) J.Biol. Chem. 193: 265-75]. Phosphotungstic acid reagent (15% PTA in 10% HCl) was used to precipitate the protein from the extracts, [ Singh B.P., Sridhara S., Arora N. and Gangal S.V. (1992). Bio-chem. Int., 27: 477-84]. Lyophilized extracts (1:20 w/v) were reconstituted to 1:500 w/v in phosphate buffer saline (PBS) for skin tests (intradermal) on patients of naso-bronchial allergy. The patient's were selected for intradermal test based on their clinical history. For skin tests, about 0.02 ml of each extract was injected intradermally into the forearm of the patient raising a bleb of 1-2 mm at the site of injection. Simultaneously, as a negative control-PBS and positive control i.e. histamine di-phosphate (100 ug/ml) were also injected. ID test reactions were graded after 20 min in comparison to the wheal size of the negative control. Skin tests with the antigens (pollen and fungi) were also carried out on some healthy non-allergic volunteers and the sera collected. The patients showing positive interadermal test to pollen and/or fungal antigens were persuaded to donate sera for use in ELISA. Enzyme Linked Immunosorbent Assay: ELISA1s were carried out
[Voller A., Bidwell D. and Barlett T. In Rose NR, Friedman H.
(eds). Manual of Clinical Immunoassay, American Society for Microbiology, Washington, 1980] with allergen coated discs in tissue culture polystyrene plates. Unbound sites of the discs if any, were blocked with 2% bovine serum albumin (BSA, w/v in PBS) for 2 hrs at room temperature in the range of 25-30°C. Washing of the discs was performed (3 changes) with PBS-tween 20 (0.05%).

The discs were incubated overnight with patient's sera (1:10 v/v in PBS, 100 ul/well) at 4°C. After washing with PBS-tween 20, anti IgE conjugated to alkaline phosphatase (1:2000, v/v in PBS, 100 ul/well), was added and incubated overnight at 4°C. As a
negative control, normal human serum (NHS) was also tested in the same dilution. Washing was performed again as mentioned earlier
and the plate was incubated with p-nitrophenyl phosphate (1 mg/ml in glycine buffer) at 37°C for colour development. The reaction was stopped after 40 minutes by adding 3N NaOH (50 ul/well). The discs were removed and the absorbance was read at 405 nm using Dynatech ELISA reader.
Initially, solid support (paper discs) coated with Amaranthus spinosus (AS) pollen extract were prepared with a new method as described above. Allergen discs of AS prepared by this method showed good comparison with discs made by CNBr method in measurement of specific IgE by ELISA. Later the discs coated with graded amount of AS protein were prepared by the new procedure. These discs were subjected to specific IgE determinations by ELISA in positive patients sera to Amaranthus spinosus extract. The paper discs coated with approx. 10 ug protein showed better promise for ELISA. The success of above experiments induced us to prepare allergen coated discs from 5 types of clinically important pollen (Amaranthus spinosus, Imperata cylindrica, Holoptelea integrifolia, Prosopis juliflora and Putranjiva roxburghii) extracts by the same procedure. Allergen specific IgE were measured using discs prepared by the improved method in patients sera and also in NHS collected from non allergic volunteers.

Interpretation of ELISA results were made based on negative control [Pauwels R., Verschraegen G. and Straeten V.D. (1980), Allergy, 157:665-69; Calenoff E., Me Mahan J.T., Herzon G.D., Kern R.C., Ghadge G.D. and Hanson D.G. (1992) Arch. Otolaryngol,
Head Neck Surge. 119 (8) :830-36] or positive control (Pharmacia, Sweden). The analysis of the data revealed that patients sera
showing optical densities (O.D.'s) more than, mean +2 SD of normal human serum ( NHS, i.e. negative control, O.D.,s equal to 0.2 or more) may be taken as ELISA positive. Sera showing 4 Phadebas RAST Unit (PRU)/ml i.e. 10 ng/ml (specific IgE) should be designated as ELISA positive [ McSharry C., McKay I.C. and Lewis C. (1993), J. Clin. Immunoassay, 16: 153-58 ]. Validation of Allergen Coated Discs by ELISA:
Dilution dependent relationship was worked out taking serialdilutions (11 dilutions e.g.1:1,1:2 1:4 till 1:1024), of two sera positive to Amaranthus spinosus and Prosopis juliflora extracts. The Measurement of allergen specific IgE was carried out by ELISA using the discs coated with respective allergen extract prepared by the improved process. Analysis of the results showed dilution dependent relationship in O.D. values. The assay was found to be working well with these discs upto the sera dilution of 1:1024 (v/v in PBS).
Analysis of the results showed coefficient of variations (C.V.'s) in the range of 4-11% for intra-assays, whereas C.V.'s ranged 3-14% in inter-assays with pollen allergen discs prepared by the new procedure. Studies conducted elsewhere with allergen coated discs (mite, timothy & dog allergen) show CV's=2.9% for within assays and CV's= 9-29% for inter assays [Poulsen L.K.,Pedersen

M.F.. Mailing H.J., Sondergaard I. and Weeke B. (1989), Allergy 44:173-80; Plebani M, Faggian D. and Borghesan F. (1995),Allergy 50:229-33]. To compare skin test gradings with ELISA, allergen specific IgE was measured in positive patient's sera by ELISA using 5 types of pollen and fungal extracts discs prepared, individually by the new procedure. Analysis of skin tests and ELISA results (25 to 40 patient's sera) showed 50-70% correlation for sera assigned as positive on the basis of intradermal tests. For comparison, determination of specific IgE was carried out in sera samples by ELISA using discs coated allergens and plate binding method. Results indicated that allergen coated discs prepared by our method have better sensitivity in ELISA for estimation of specific IgE compared to plate binding method. Toorenenbergen and his group (1987) have reported [Van Toorenenbergen A.W., Gerth van Wijk R., Dieges P.H. and Leijnse B. (1987), Int. Arch. Allergy Appl. Immunol. 83:436-39] irrelevant IgE binding to cellulose discs (Pharmacia) in RAST. We have carried out ELISA with the discs prepared by the new method and Pharmacia discs with sera of non-IgE mediated diseases. Results show very low non-specific binding with discs prepared by the new procedure (O.D.s 0.088-0.108; normal control O.D. 0.065) compared to the Pharmacia discs (O.D. 0.336-0.441). ELISA's were carried out to compare the allergen coated discs prepared by the improved procedure and the discs procured from Pharmacia, Sweden for Prosopis juliflora and Aspergillus fumigatus extracts. Statistical analysis of the data showed significant correlation with both types of discs (critical values = 0.821 & 0.879 respectively). At 5% probability the critical

values of + 0.530 (n=14) and +. 0.599 (n=ll), respectively were recorded significant for 2 tailed analysis [Singh B.P.,Sridhara S. and Gangal S.V. (1996), Project Completion Report (DBT), Centre for Biochemical Technology, Delhi]. For the independent validation of discs prepared by the present method, allergen specific IgE was measured by ELISA in the same sera samples by three persons, independentlly. The optical densities (O.D's) recorded by all three persons were found to be matching for most of the sera samples. Also out of 5 sera tested, the same 4 were assigned as ELISA positive (O.D.> mean +2SD of NHS) by all the three investigators.
The invention is described in detail in the examples given below which are described by way of illustrations only and should not be construed to limit the scope of present invention.
Example 1:
Three gram polyvinyl alcohol (PVA) was dissolved with stirring in one hundred ml distilled water at a temperature of 80°C. The lyophilized allergen extract (1:20 w/v) was reconstituted to 1 ml in 0.05M phosphate buffered saline (PBS), pH 7.8. The PVA and allergenic protein (extract) solutions were mixed together so as to get a concentration of 65 ug protein per square centimeter of the coating area on a filter paper. The mixture of PVA and the allergenic protein was coated by brushing and or immersing both the surfaces of a filter paper (solid support). Protein coated solid support was dried and incubated with 1% glutaraldehyde solution in water. This was followed by washing of the solid support with distilled water and drying at 30°C. Discs were

pun'ched out from the solid support as desired.
Example 2:
2.5 gram polyvinyl alcohol (PVA) was dissolved with stirring in hundred ml distilled water at the temperature of 80°C. The lyophilized allergen extract (1:20 w/v) was reconstituted to 1 ml in 0. 1M carbonate-bicarbonate buffer, pH 9.6. The PVA and allergenic protein (extract) solutions were mixed together. For coating, 10 ug of protein (200ul) was poured in each well of tissue culture plate or 30 ug protein in 300 ul per culture tube. This was subjected to drying at 30°C for coating. The plate and the tubes were then incubated with 200 ul/lml of 1.5% glutaraldehyde in water. The allergen coated plates/tubes were washed with distilled water and allowed to dry at 30°C.
The main advantages of the present invention are:
1. a simple, reliable and cost effective process of allergen
coating has been developed,
2. allergen coated discs were prepared with indegenous
allergens and standardized by ELISA,
3. the allergen coated solid support (paper discs) prepared by
the present procedure showed significant correlation in
ELISA with CNBr coated discs,procured from Pharmacia,Sweden,
4. unlike the CNBr method, the allergen coated discs can be
stored in dried form at 25-30°C temperature, making it
convenient to handle the discs in tests and transport,
5. the product (discs) offers confirmatory diagnosis of
respiratory allergies (rhinitis, asthma etc.) by ELISA.

We claim :
1. A process for preparation of allergen coated solid support
useful in allergy diagnosis which comprises :
(a) reconstituting the lyophilized allergen extracts with
buffer or distiled water,
(b) dissolving the polyvinyl alcohol (PVA) in water and
mixing thoroughly with the above allergen extract(s),
(c) coating of the above prepared allergenic mixture on solid
support,
(d) drying of allergen coated filter paper at temperature in
the range of 25-30ºC,
(e) cross-linking of coated allergens with glutaraldehyde,
(f) drying the above allergen coated solid support at
temperature in the range of 25-30ºC,
(g) punching the solid support to obtain the desired discs.
2. A process as claimed in claim 1 wherein,the allergen
extract used is such as from thepollen, fungi, insects, food,
animal scales and or mites extracts (whose single or mixed
allergen preparations can be coated on solid support).
3. A process as claimed in claim 1 and 2, wherein the
buffer used is phosphate buffer saline, carbonate-bicarbonate
buffer.
4. A process as claimed in claim 1-3, wherein the
concentration of polyvinyl alcohol may be 2% to 5%.
5. A process as claimed in claims 1-4, wherein the concen
tration of glutaraldehyde used may be 1% to 4%.
6. A process as claimed in claims 1-5, wherein the coating

may be affected by immersing the solid support in allergen mixture or by incubating the wells of the plate and culture tube with protein mixture.
7. A process for preparation of allergen coated solid support useful in diagnosis of allergies substantially as herein described with reference to the examples.

Documents:

744-del-1998-abstract.pdf

744-del-1998-claims.pdf

744-del-1998-correspondence-others.pdf

744-del-1998-correspondence-po.pdf

744-del-1998-description (complete).pdf

744-del-1998-form-1.pdf

744-del-1998-form-19.pdf

744-del-1998-form-2.pdf

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Patent Number

215840

Indian Patent Application Number

744/DEL/1998

PG Journal Number

12/2008

Publication Date

21-Mar-2008

Grant Date

04-Mar-2008

Date of Filing

24-Mar-1998

Name of Patentee

COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH

Applicant Address

RAFI MARG, NEW DELHI-110001, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	SUSHEELA SRIDHARA	CENTRE FOR BIOCHEMICAL TECHNOIOGY, MALL ROAD, DELHI, INDIA.
2	NARAYAN BHAGWANDAS TULSANI	CENTRE FOR BIOCHEMICAL TECHNOIOGY, MALL ROAD, DELHI, INDIA.
3	BHANU PRATAP SINGH	CENTRE FOR BIOCHEMICAL TECHNOIOGY, MALL ROAD, DELHI, INDIA.
4	SHARAD VISHWANATH GANGAL	CENTRE FOR BIOCHEMICAL TECHNOIOGY, MALL ROAD, DELHI, INDIA.

PCT International Classification Number

A61K 31/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA