Title of Invention	A PROCESS OF PREPARATION OF ANTI-A1 LECTIN FROM THE SEEDS OF DOLICHOS BIFLORUS
Abstract	The present invention relates to an improved process of preparation of a blood typing useful for the diagnosis and characterization of blood having an improved stability and purity. More specifically it is an improved process of preparation of high titer plant agglutinin known as Anti A1 lectin from the Dolichos biflorus seeds extracts. Anti- A1 Lectin here in described is suitable for cell typing in medical science.

Title of Invention

A PROCESS OF PREPARATION OF ANTI-A1 LECTIN FROM THE SEEDS OF DOLICHOS BIFLORUS

Abstract

The present invention relates to an improved process of preparation of a blood typing useful for the diagnosis and characterization of blood having an improved stability and purity. More specifically it is an improved process of preparation of high titer plant agglutinin known as Anti A1 lectin from the Dolichos biflorus seeds extracts. Anti- A1 Lectin here in described is suitable for cell typing in medical science.

Full Text	field of the Invention The present invention relates to an improved process of preparation of a blood typing reagent useful for the diagnosis and characterization of blood having an improved stability and purity. More specifically it is an improved process of preparation of high titer plant agglutinin known as Anti AI lectin from the Dolichos biflorus seeds extracts. Anti AI Lectin here in described is designed for use in agglutination tests for the detection of AI antigen in human red cells. Erythrocytes possessing the A antigen can be sub divided in to At and A2 cells. Group A red blood cells which are agglutinated with anti AI lectin are said to be sub group AI and those which are not agglutinated by anti AI lectin are classified as A2. Background Various plant seed extracts have been shown to influence metabolic process of human and animal lymphocytes. Phytohemagglutinin is a typical example of lymphocyte stimulating material useful in the analysis of lymphocyte growth. Dolichos biflorus were found to contain an extract, which is capable of agglutining erythrocytes containing AI blood group substance. The present invention is an improved method of the earlier invention. The earlier method of preparation of the Anti AI Lectin was by subjecting the seeds to various process like grinding the seeds in normal saline water and decanting and collecting the concentrated solution. To this solution, add NaN3 solution and filter the solution to get the final product. Prior Art: US: 3053739: It describes the process of preparation of Anti H Lectins, which is used for the differentiation of type A\ from the type A2 blood. These extracts have also been used in the separation of individuals of any blood grouping to secretors and non-secretors for ABO(H) substances. The separation of A group into At and A2 can be important in avoiding transfusion reaction as well as in ascertaining the paternity where this problem is involved. US Patent: 3035986: It describes a method and preparation of Anti M lectins derived from the seeds of Cruciferae Iberis as a diagnostic reagent for the identification of human erythrocytes of types M and MN. There is a need for an improved method for the preparation of Anti H which can be prepared readily by subjecting the seeds of Ulex europeus to various processes and can be used for the detection of Bombay Group blood in Forensic science. US Patent No. 20060093686: Lectin for checking a blood type is extracted from seeds of bitter gourd, balsam pear, or Momordica charantia. A blood type checking reagent includes the lectin. The lectin extracted from seeds of bitter gourd preferably has a molecular weight between 100,000 and 170,000 measured with poly-acrylamide gel electrophoresis in existence of sodium dodecyl sulfate (SDS-PAGE). A method of checking a blood type includes the steps of: extracting lectin from seeds of bitter gourd, balsam pear, or Momordica charantia; preparing a blood type checking reagent containing the lectin; and checking a blood type using the blood type checking reagent. Anti-Al lectin in the seeds of Amphicarpaea bracteata. 1978 Feb 15:532(2):225-31. An anti-Al lectin has been isolated from the extract of Amphicarpaea bracteata seeds by affinity chromatography on Epoxy-activated Sepharose 6B coupled to N- acetyl-D-galactosamine. The yield of the purified lectin was 86 microgram/g of seeds. The purified lectin shows one main band on electrophoresis in sodium dodecyl sulfate-polyacrylamide. The amino acid and neutral sugar composition indicate that this lectin is an acidic glycoprotein with a neutral sugar content of approx. 2%. The composition of the lectin is different from that of the Dolichos biflorus lectin but the two lectins have some common characteristics. The most powerful inhibitors of the agglutination of Al red blood cells by the A. bracteata lectin is N-acetyl-D-galactosamine. Much weaker inhibitors of the agglutination are alpha-lactose, D-fucose, and five other sugars. The conventional method of checking the blood group has the following disadvantages. First, it is difficult to determine the blood type sampled from a blood stain using the lectin. It is necessary to use a large amount of raw materials there by increasing the cost. There is a need for an improved method for the preparation of Anti At lecin which can be prepared readily by subjecting the seeds of Dolichos biflorus seeds to various processes and can be used for the detection of AI group in red blood cells. The object of the present invention is to provide a lectin for checking the blood type in which it is possible to accurately and quickly determine a blood type even from a small bloodstain. Another object of the present invention is to provide a reagent for checking a blood type containing the AI lectin. Yet another object of the present invention is to provide a novel procedure for the isolation and preparation of Anti AI Lectin. Summary of the invention Anti Ailectin is designed for use in agglutination tests for the detection of AI antigen on human red cells. This reagent is a purified extract of the seeds of Dolichos biflorus containing a phytohemagglutinin (lectin) which agglutinates red blood cells of the sub group AI or A1B. In order to attain the objects of the present invention, AI lectin for checking the blood type is extracted from seeds of Dolichos biflorus (Horse gram). According to the present invention, AI blood type checking reagent contains the lectin AI. According to the present invention, a method of checking the blood type AI includes the steps of extracting AI lectin from the seeds of Dolichos biflorus and preparing a blood type checking reagent containing the anti AI lectin. With the blood type checking reagent containing anti AI lectin, it is possible to accurately and quickly determine the Aj blood type even from the small bloodstain. The present invention includes a method of producing lectin AI from the Dolichos biflorus which is a low cost material. Accordingly it is possible to produce the AI lectin from the low cost material as compared to the conventional methods of producing the AI lectin for checking the AI blood type. It is possible to determine the AI blood type accurately and quickly even from the small blood stains there by obtaining the reliable data for forensic investigation. Test procedure 1. Place one drop of the Anti-AI Lectin on a glass slide. 2. Add one drop of the whole blood to be tested on the slide and mix well by rotating the slide. 3. Observe the agglutination . Agglutination is a positive test result and indicates the presence of AI antigen. No agglutination is a negative test and indicates the absence of AI antigen. Detailed description Process of Preparation Prepare Anti- AI Lectin by subjecting the Dolichos biflorus seeds to grinding to get the finest powder and then subject the powder to an aqueous medium like distilled water. Collect the supernatant by decanting. To this solution add suitable solvents such as ether or ethyl alcohol or methyl alcohol or acetone or chloroform or benzene or phenol or ethanol amine or Isopropyl alcohol preferably acetone. Mix the solution and centrifuge and collect the pellet. Dry the pellet in air. To this pellet, add any of the buffer solution such as PBS or TRIS Buffer or normal saline and do the sterile filtration of the solution in micron paper. To this solution, add the preservative. Example Take 5-20 gm of Dolichos biflorus seed and ground it mechanically for 30 minutes to obtain the fine seed powder. 5- 20 gm of the seed powder is added to the 30 ml-250 ml of the cold distilled water and vortex it at room temperature for 2 hr to 5 hrs. Centrifuge the solution at 5000- 8000 rpm at cold temperature say 2 to 8°C for 30 to 60 minutes. The supernatant is collected by decanting. To this add suitable solvent such as ether or ethyl alcohol or methyl alcohol or acetone or chloroform or benzene or Phenol or ethanol amine or Iso propyl alcohol preferably acetone in the ratio 1: 1 to 1: 10. Mix it for 1 hr- 2 hrs at room temperature in a magnetic stirrer and keep still for 20 to 30 minutes at room temperature. Then centrifuge the solution at 2000-8000 rpm for 1 hr for getting the pellets. The supernatant is thrown away and the pellet is collected and air dried for 5to 7 hrs. Then dissolve the pellet in 1 to 10 ml of any of the buffer solution such as PBS of pH 7.4 or TRIS buffer of pH 7.5 or normal saline of pH 7 . After that do the sterile filtration of the solution in 0.45 fj, to filter paper and collect the solution. To this solution, add 0.1 % of the Sodium Azide as a preservative. I Claim 1. A method of preparation of Anti Al Lectin from the seeds of dolichos biflorus comprising the steps of subjecting the seeds for the following processes: a. ground the seeds for 30 minutes to get a fine powder; b. 5 to 20 gm of the seed powder is added to 30 to 250ml of the cold distilled water. c. vortex the solution at room temperature for 2 hour to 5 hours; d. centrifuge the solution at 2000 to 8000 rpm at cold temperature 2 to 8°C for 30 to 60 minutes; e. collect the supernatant and to it add the solvent acetone in the ratio 1:1 to 1: 100; f. mix it in a magnetic stirrer to 1 hour to 2 hour at room temperature and keep still for 20 to 30 minutes at room temperature; g. centrifuge the solution at 2000 to 8000 rpm for one hour for getting the pellet; h. collect the pellet and air dry for 5 to 7 hours; i. dissolve the pellet in 1 to 10 ml buffer solution such as PBS of p 7.4 or TRIS buffer of pH 7.5 or normal saline of pH b 7; j. do the sterile filtration of the solution in 0.45 µ to 1.2 µ; k. collect the solution and add 0.1% of the Sodium Azide as the preservative. 2. A process as claimed in claiml where in the solvent is preferably acetone.

Full Text

field of the Invention
The present invention relates to an improved process of preparation of a blood typing reagent useful for the diagnosis and characterization of blood having an improved stability and purity. More specifically it is an improved process of preparation of high titer plant agglutinin known as Anti AI lectin from the Dolichos biflorus seeds extracts. Anti AI Lectin here in described is designed for use in agglutination tests for the detection of AI antigen in human red cells. Erythrocytes possessing the A antigen can be sub divided in to At and A2 cells. Group A red blood cells which are agglutinated with anti AI lectin are said to be sub group AI and those which are not agglutinated by anti AI lectin are classified as A2.
Background
Various plant seed extracts have been shown to influence metabolic process of human and animal lymphocytes. Phytohemagglutinin is a typical example of lymphocyte stimulating material useful in the analysis of lymphocyte growth. Dolichos biflorus were found to contain an extract, which is capable of agglutining erythrocytes containing AI blood group substance.
The present invention is an improved method of the earlier invention. The earlier method of preparation of the Anti AI Lectin was by subjecting the seeds to various process like grinding the seeds in normal saline water and decanting and collecting the concentrated solution. To this solution, add NaN3 solution and filter the solution to get the final product.
Prior Art:
US: 3053739: It describes the process of preparation of Anti H Lectins, which is used for the differentiation of type A\ from the type A2 blood. These extracts have also been used in the separation of individuals of any blood grouping to secretors and non-secretors for ABO(H) substances. The separation of A group into At and A2 can be important in avoiding transfusion reaction as well as in ascertaining the paternity where this problem is involved.
US Patent: 3035986: It describes a method and preparation of Anti M lectins derived from the seeds of Cruciferae Iberis as a diagnostic reagent for the identification of human erythrocytes of types M and MN.
There is a need for an improved method for the preparation of Anti H which can be prepared readily by subjecting the seeds of Ulex europeus to various processes and can be used for the detection of Bombay Group blood in Forensic science.
US Patent No. 20060093686: Lectin for checking a blood type is extracted from seeds of bitter gourd, balsam pear, or Momordica charantia. A blood type checking reagent includes the lectin. The lectin extracted from seeds of bitter gourd preferably has a molecular weight between 100,000 and 170,000 measured with poly-acrylamide gel electrophoresis in existence of sodium dodecyl sulfate (SDS-PAGE). A method of checking a blood type includes the steps of: extracting lectin from seeds of bitter gourd, balsam pear, or Momordica charantia; preparing a blood type checking reagent containing the lectin; and checking a blood type using the blood type checking reagent.
Anti-Al lectin in the seeds of Amphicarpaea bracteata. 1978 Feb 15:532(2):225-31.
An anti-Al lectin has been isolated from the extract of Amphicarpaea bracteata
seeds by affinity chromatography on Epoxy-activated Sepharose 6B coupled to N-
acetyl-D-galactosamine. The yield of the purified lectin was 86 microgram/g of
seeds. The purified lectin shows one main band on electrophoresis in sodium
dodecyl sulfate-polyacrylamide. The amino acid and neutral sugar composition
indicate that this lectin is an acidic glycoprotein with a neutral sugar content of
approx. 2%. The composition of the lectin is different from that of the Dolichos
biflorus lectin but the two lectins have some common characteristics. The most
powerful inhibitors of the agglutination of Al red blood cells by the A. bracteata
lectin is N-acetyl-D-galactosamine. Much weaker inhibitors of the agglutination are
alpha-lactose, D-fucose, and five other sugars.
The conventional method of checking the blood group has the following
disadvantages.
First, it is difficult to determine the blood type sampled from a blood stain using the
lectin. It is necessary to use a large amount of raw materials there by increasing the
cost.
There is a need for an improved method for the preparation of Anti At lecin which
can be prepared readily by subjecting the seeds of Dolichos biflorus seeds to various
processes and can be used for the detection of AI group in red blood cells.
The object of the present invention is to provide a lectin for checking the blood type
in which it is possible to accurately and quickly determine a blood type even from a
small bloodstain.
Another object of the present invention is to provide a reagent for checking a blood
type containing the AI lectin.
Yet another object of the present invention is to provide a novel procedure for the
isolation and preparation of Anti AI Lectin.
Summary of the invention
Anti Ailectin is designed for use in agglutination tests for the detection of AI antigen on human red cells. This reagent is a purified extract of the seeds of Dolichos biflorus containing a phytohemagglutinin (lectin) which agglutinates red blood cells of the sub group AI or A1B.
In order to attain the objects of the present invention, AI lectin for checking the blood type is extracted from seeds of Dolichos biflorus (Horse gram). According to the present invention, AI blood type checking reagent contains the lectin AI.
According to the present invention, a method of checking the blood type AI includes
the steps of extracting AI lectin from the seeds of Dolichos biflorus and preparing
a blood type checking reagent containing the anti AI lectin.
With the blood type checking reagent containing anti AI lectin, it is possible to
accurately and quickly determine the Aj blood type even from the small bloodstain.
The present invention includes a method of producing lectin AI from the Dolichos
biflorus which is a low cost material. Accordingly it is possible to produce the AI
lectin from the low cost material as compared to the conventional methods of
producing the AI lectin for checking the AI blood type.
It is possible to determine the AI blood type accurately and quickly even from the
small blood stains there by obtaining the reliable data for forensic investigation.
Test procedure
1. Place one drop of the Anti-AI Lectin on a glass slide.
2. Add one drop of the whole blood to be tested on the slide and mix well by
rotating the slide.
3. Observe the agglutination .
Agglutination is a positive test result and indicates the presence of AI antigen. No agglutination is a negative test and indicates the absence of AI antigen.
Detailed description Process of Preparation
Prepare Anti- AI Lectin by subjecting the Dolichos biflorus seeds to grinding to get the finest powder and then subject the powder to an aqueous medium like distilled water. Collect the supernatant by decanting. To this solution add suitable solvents such as ether or ethyl alcohol or methyl alcohol or acetone or chloroform or benzene or phenol or ethanol amine or Isopropyl alcohol preferably acetone. Mix the solution and centrifuge and collect the pellet. Dry the pellet in air. To this pellet, add any of the buffer solution such as PBS or TRIS Buffer or normal saline and do the sterile filtration of the solution in micron paper. To this solution, add the preservative.
Example
Take 5-20 gm of Dolichos biflorus seed and ground it mechanically for 30 minutes to obtain the fine seed powder. 5- 20 gm of the seed powder is added to the 30 ml-250 ml of the cold distilled water and vortex it at room temperature for 2 hr to 5 hrs. Centrifuge the solution at 5000- 8000 rpm at cold temperature say 2 to 8°C for 30 to 60 minutes. The supernatant is collected by decanting. To this add suitable solvent such as ether or ethyl alcohol or methyl alcohol or acetone or chloroform or benzene or Phenol or ethanol amine or Iso propyl alcohol preferably acetone in the ratio 1: 1 to 1: 10. Mix it for 1 hr- 2 hrs at room temperature in a magnetic stirrer and keep still
for 20 to 30 minutes at room temperature. Then centrifuge the solution at 2000-8000 rpm for 1 hr for getting the pellets. The supernatant is thrown away and the pellet is collected and air dried for 5to 7 hrs. Then dissolve the pellet in 1 to 10 ml of any of the buffer solution such as PBS of pH 7.4 or TRIS buffer of pH 7.5 or normal saline of pH 7 . After that do the sterile filtration of the solution in 0.45 fj, to filter paper and collect the solution. To this solution, add 0.1 % of the Sodium Azide as a preservative.

I Claim
1. A method of preparation of Anti Al Lectin from the seeds of dolichos biflorus
comprising the steps of subjecting the seeds for the following processes:
a. ground the seeds for 30 minutes to get a fine powder;
b. 5 to 20 gm of the seed powder is added to 30 to 250ml of the cold distilled water.
c. vortex the solution at room temperature for 2 hour to 5 hours;
d. centrifuge the solution at 2000 to 8000 rpm at cold temperature 2 to 8°C for 30 to 60
minutes;
e. collect the supernatant and to it add the solvent acetone in the ratio 1:1 to 1: 100;
f. mix it in a magnetic stirrer to 1 hour to 2 hour at room temperature and keep still for 20
to 30 minutes at room temperature;
g. centrifuge the solution at 2000 to 8000 rpm for one hour for getting the pellet;
h. collect the pellet and air dry for 5 to 7 hours;
i. dissolve the pellet in 1 to 10 ml buffer solution such as PBS of p 7.4 or TRIS buffer of
pH 7.5 or normal saline of pH b 7;
j. do the sterile filtration of the solution in 0.45 µ to 1.2 µ;
k. collect the solution and add 0.1% of the Sodium Azide as the preservative.
2. A process as claimed in claiml where in the solvent is preferably acetone.

Documents:

2480-DEL-2007-1-Post-Grant Opposition-(09-02-2011).pdf

2480-del-2007-abstract.pdf

2480-DEL-2007-Claims-(08-10-2008).pdf

2480-del-2007-claims.pdf

2480-DEL-2007-Correspondence-Others-(08-10-2008).pdf

2480-del-2007-correspondence-others-1.pdf

2480-del-2007-correspondence-others.pdf

2480-del-2007-description (complete).pdf

2480-del-2007-form-1.pdf

2480-del-2007-form-18.pdf

2480-DEL-2007-Form-2-(08-10-2008).pdf

2480-del-2007-form-2.pdf

2480-del-2007-form-3.pdf

2480-del-2007-form-5.pdf

2480-del-2007-form-9.pdf

2480-DEL-2007-GPA-(09-02-2011).pdf

2480-del-2007-Petition-137-(18-01-2011).pdf

2480-DEL-2007-Post Grant Opposition-(18-01-2011).pdf

2480-DEL-2007-Post-Grant -(26-11-2010).pdf

2480-DEL-2007-Post-Grant Opposition-(03-12-2010)-.pdf

2480-DEL-2007-Post-Grant Opposition-(03-12-2010).pdf

2480-DEL-2007-Post-Grant Opposition-(09-02-2011).pdf

2480-DEL-2007-Post-Grant-Opposition-(04-01-2011).pdf

2480-del-2007-post-grant-reply statement evidence.pdf

2480-del-2007-post-grant-representation.pdf

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Patent Number

225354

Indian Patent Application Number

2480/DEL/2007

PG Journal Number

48/2008

Publication Date

28-Nov-2008

Grant Date

11-Nov-2008

Date of Filing

28-Nov-2007

Name of Patentee

MAHAJAN LALIT

Applicant Address

N-118, GREATER KAILASH, PART-I NEW DELHI, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	MAHAJAN; LALIT	N-118, GREATER KAILASH, PART-NEW DELHI, INDIA.

PCT International Classification Number

A61K

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA