FORM 2
THE PATENT ACT 197 0 (39 of 1970)
&
The Patents Rules, 2003 COMPLETE SPECIFICATION (See Section 10, and rule 13)
1. TITLE OF INVENTION
REAGENT KIT FOR DIRECT DETERMINATION OF HDL CHOLESTEROL;


2. APPLICANT(S)
a) Name : TRANSASIA BIO-MEDICALS LTD.
b) Nationality : INDIAN Company cj Address : TRANSASIA HOUSE,
8, CHANDIVALI STUDIO ROAD, MUMBAI - 400 072 MAHARASHTRA

3. PREAMBLE TO THE DESCRIPTION

The following specification particularly describes the invention and; the manner in which it is to be performed


Reagent kit for direct determination of HDL cholesterol
Field of invention :
The present invention provides a reagent for quantifying cholesterol in high density lipoprotein (HDL). More particularly the invention relates to a reagent for assaying HDL cholesterol, which comprises of a two reagent system involving a surfactant. The invention also provides a method for assaying cholesterol in HDL fraction in serum or plasma requiring no treatment for separation, such as centrifugation or electrophoresis and a reagent composition there for.
Background Art:
It is known that HDL is useful indicator for the risk of onset of arterial sclerosis. Methods for estimating cholesterol in HDL include ultra-centrifugation and electrophoresis, which are complex and are not commonly used. In routine clinical estimations, a precipitating agent is added to the sample to aggregate the lipoproteins other than HDL and then removing the precipitated lipoproteins by centrifugation. Cholesterol in the remaining supernatant containing HDL is measured. Although the method is simple, it involves separation, requires higher sample volumes and cannot be used on automated analyzers.
Methods employing cholesterol assaying reagent system comprising of antibodies against specific lipoprotein fraction or the use of poly-anion to aggregate lipoproteins other than HDL and enzymatically reacting the cholesterol in HDL or LDL alone have been proposed.

Other method includes the use of reagents having modified cholesterol esterase and oxidase in the presence of cyclodextrin for entrapment of HDL cholesterol. Results obtained with this method were not specific and abnormal values were obtained in samples having high triglyceride and bilirubin values.

Object of the present invention :


Therefore, in one aspect of the present invention, the object present invention is to provide a reagent system for the direct determination of cholesterol in HDL by the use of surfactants, which provides a preferential reactivity of lipoprotein fractions with the enzymatic cholesterol reagent system.
In preferred aspect of the invention, another object of the invention is to provide a reagent for assaying HDL cholesterol comprising a single reagent containing a surfactant.
In another aspect of the invention, the object of the present invention is to provide a method for the direct determination of cholesterol in HDL fraction in serum or plasma that does not require treatment for separation, such as centrifugation or electrophoresis and a reagent composition there for.
Statement of the invention :
For specific determination of HDL cholesterol, the cholesterol fraction in lipoproteins other than HDL is retarded in the first incubation phase and HDL is quantified in the second step by incubation with a cholesterol detection reagent system, which comprises cholesterol esterase and oxidase in a buffered aqueous medium comprising surfactants that specifically activates cholesterol in HDL fraction and measuring the product of the oxidation reaction (hydrogen peroxide) generated.
Description of the invention :
In one aspect of the present invention, a process for the direct determination of cholesterol in high density lipoprotein (HDL-C) comprises of elimination of cholesterol in lipoprotein fraction with cholesterol esterase and oxidase in the presence of a surfactant that entraps LDL, VLDL & chylomicrons in the first step;


adding a surfactant specific to HDL to reactivate the HDL cholesterol fraction for reacting with the cholesterol assaying reagent system in the second step. In the first pre-treatment step of the sample with the reagent system in the presence of a surfactant, the cholesterol in HDL is not substantially reacted while other lipoproteins such as LDL, VLDL and CM are reacted and eliminated. In the subsequent second reaction phase, the cholesterol in HDL is selectively measured. The entrapment of lipoprotein fractions for rendering it non-reactive to cholesterol reagent (other than HDL) is achieved through the use a combination of surfactants in the reagent system. Using the process of the invention, the maximum detectable range is 200 mg/dl of HDL-C.
In one aspect of the present invention, a reagent system for the direct determination of cholesterol in high density lipoprotein (HDL-C) comprises first reagent comprising a buffer with a pH in the range of 6 to 8, a cholesterol oxidase, a cholesterol esterase, a phenol based hydrogen donor compound and second reagent comprising a buffer with a pH in the range of 6 to 8 with the same molarity range as the first reagent and a surfactant for activating the cholesterol fraction in HDL.
The said buffer is selected from a phosphate, a Tris and a Good's buffer (MOPSO, BES, HEPES PIPES), which is preferably Good's buffer having a concentration in range from 25-75 mM. The said first reagent comprises the cholesterol oxidase in the concentration of 0.2 to 0.6 U/ml, the cholesterol esterase in the concentration of 0.5 to 1.0 U/ml, a phenol based hydrogen donor compound in the concentration of 0.3 to 1.0 mM and sutfactant in the concentration of 0.01%. The said surfactant -a poly hydroxy ethylene deivative has hydrophilicity lipophilicity balance (HLB) value between 13.0 to 14.0 and has the concentration in the range between 0.5 to 1.5%.
The reagent system further comprises a peroxidase in the concentration of about 1.0 to 4.0 U/l, divalent metal ions for reacting lipoprotein sub-fractions with the cholesterol reagent system, a phenolic substitute as a chromogen having high signal ratio for better sensitivity for detectinglower levels of concentration in the clinically


significant range. The reagent system further comprises a peroxidase enzyme stabilizer that contributes to the reagent stability.
In preferred aspect of the present invention, the reagent system is formulated as a two-reagent system in a 4:1 ratio and may be converted into a Mono-reagent pack. The said first reagent preferably comprises PIPES buffer pH 7.0 in the concentration of 50 mmol/I; the cholesterol esterase in the concentration of 0.8 u/ml; the cholesterol oxidase in the concentration of 0.4 u/ml; surfactant 0.01% and phenolic chromogen in the concentration of 0.7 mmol/l. The said second reagent preferably comprises PIPES buffer pH 7.0 in the concentration of 50 mmol/l; 4-aminoantipyrine in the concentration of 4 mmol/l; peroxidase in the concentration of 4.0 u/ml; and a polyoxyethylene derivative detergent in the concentration of 1.3%.
In preferred embodiment of the process of the present invention, 5 μl of sample is added 400 μl of first reagent and incubated for five minutes at 37°C and 100 μl of the second reagent is added and the mixture is allowed to react at 37°C for five minutes, followed by measurement of absorbance at 546 nm of the reaction mixture.
Detailed embodiment of the invention :
In one embodiment of the present invention, for HDL estimation the reagent and process comprises eliminating cholesterol in lipoprotein fraction with cholesterol esterase and oxidase in the presence of a surfactant that entraps LDL, VLDL & chylomicrons In further embodiment of the invention, adding a surfactant specific to HDL for reactivating the HDL cholesterol fraction to react with the cholesterol assaying reagent system in the second step.
In preferred embodiment of the invention, in the first pre-treatment step of the sample with the reagent system in the presence of a surfactant, the cholesterol in HDL is not substantially reacted while other lipoproteins such as LDL, VLDL and

CM are reacted and eliminated. Subsequent in second reaction phase, the cholesterol in HDL is selectively measured.
In another preferred embodiment of the invention, the reagent system for HDL cholesterol comprises first reagent comprising a buffer with a pH range of 6 to 8. The buffer is selected from a phosphate, Tris and Good's buffer (MOPSO, BES, HEPES PIPES). Under normal circumstances, Good's buffer with a concentration ranging from 25-75mM is preferred.
In further preferred embodiment, the first reagent comprises cholesterol oxidase in the concentration of 0.2- 0.6U/ml; cholesterol esterase in the concentration of 0.5-l.OU/ml; a surfactant that entraps LDL, VDLD & chylomicrons and a phenol based hydrogen donor compound in the concentration of 0.3-1.0mM.
In next preferred embodiment of the invention, the second reagent is also buffered between 6 to 8 with the same molarity range as the first reagent and comprises a surfactant, which activates the cholesterol fraction in HDL to react with the cholesterol assaying reagent system. The hydrophilicity lipophilicity balance (HLB) value of the surfactant used is between 13.0 to 14.0 and a concentration ranging between 0.5 to 1.5%. The concentration of peroxidase may preferable be about 1.0 to 4.0 U/l.
In another embodiment of the invention, the reagent system for the direct determination of cholesterol fraction of HDL is based on the use of surfactants (HLB value 12.8-13.2) and divalent metal ions in the reagent system, which renders the lipoprotein sub-fractions to preferentially react with the cholesterol reagent.
In one preferred embodiment, the present method of the invention is different in the sense that it does not involve the agglutination of lipoproteins other than HDL, either by the use of specific antibody or use of polyanions, ionic strength alterations,



or use of chemically modified enzymes for preferential entrapment of lipoprotein moieties for specific determination of HDL-C as documented in the prior art.
In another preferred embodiment, the reagent of the present invention does not include catalase enzyme as a scavenger for hydrogen peroxide produced in the pre-treatment step by the action of cholesterol esterase and oxidase on lipoprotein sub-fractions other than HDL-C, thus lowering the reagent cost.
In still another preferred embodiment of the invention, entrapment of lipoprotein fractions to render it non-reactive to cholesterol reagent (other than HDL) is achieved through the use a combination of surfactants in the reagent system.
In further preferred embodiment, the cholesterol reagent system of the invention is based on a modified Trinder reaction with a superior chelating agent, which improves the reagent stability at elevated temperatures.
In still further preferred embodiment of the invention, the reagent system comprises a phenolic substitute as a chromogen having a high signal ratio that provides a better sensitivity to the reagent to detect lower levels of concentration in the clinically significant range. The chromogen system is found to be very stable, unlike other peroxidase substrates.
In different preferred embodiment of the invention, low color development in blank renders the reagent to have at least 14 days calibration stability on automated analyzers.
In still different embodiment of the invention, the maximum detectable range is 200 mg/dl of HDL- C, which is at par with other reagent brands available in the market.
In separate preferred embodiment, the reagent is found to be stable for a period of 14 days at 37°C or 6 days at 45°C, which for a liquid reagent system extrapolates to 15 months of shelf life at 2-8°C

In more preferred embodiment, the reagent of the present invention is formulated as a two-reagent system in a 4:1 ratio and there is a good possibility of it being converted to a Mono-reagent pack.
In another more preferred embodiment, the reagent comprises a peroxidase enzyme stabilizer, which contributes to the reagent stability.
The formulation of the reagents system for the direct determination of cholesterol in HDL stated above will now be described by way of example :
Example 1 - Formulation of the first reagent:
The first reagent for the direct determination of cholesterol in HDL comprises PIPES buffer pH 7.0, 50 mmol/1; Cholesterol esterase, 0.8 u/ml; Cholesterol oxidase, 0.4 u/ml; surfactant 0.01% and phenolic chromogen, 0.7 mmol/1.
Example 2- Formulation of the first reagent:
The first reagent for the direct determination of cholesterol in HDL comprises PIPES buffer pH 7.0, 25 mmol/1; Cholesterol esterase, 1.0 u/ml; Cholesterol oxidase, 0.6 u/ml; surfactant 0.001 %and phenolic chromogen, 0.7 mmol/1.
Example 3 - Formulation of the second reagent:
The second reagent for the direct determination of cholesterol in HDL and LDL comprises PIPES buffer pH 7.0, 50 mmol/1; 4- aminoantipyrine, 4 mmol/1; Peroxidase, 4.0 u/ml; and Detergent A, 1.3%.
Example 4- Formulation of the second reagent:


The second reagent for the direct determination of cholesterol in HDL and LDL comprises PIPES buffer pH 7.0, 50 mmol/1; 4- aminoantipyrine, 4 mmol/l; Peroxidase, 4.0 u/ml; Surfactant A, 1.2%; and surfactant - B 0.1%.
Example 5- Direct determination of cholesterol in HDL :
5μl of sample was added 400 μl of Reagent-1 and incubated for five minutes at 37°C. Then 100 μl of the second reagent was added and the mixture was allowed to react at 37°C for five minutes, followed by measurement of absorbance at 546 nm of the reaction mixture.
Example 6- Calculations of HDL concentration :
Calculations are based on using a serum standard with a known value of HDL concentration, the serum standard being used in test batch in the same way as the sample.
Example 7 - Comparison of HDL determinations :

A comparison of HDL determinations according to examples stated above with a method using a specific antibody or polyanion as a reference gave the following statistical correlations for HDL as depicted in Table 1.

Table 1:

HDL Test Method
Slope 0.9900
y-intercept 0.6744
S 2.3412
r 0.9786
Bias 0.2265
SDdiff 2.3032
t-value 0.5386


WE CLAIM:
1. A reagent system for the direct determination of cholesterol in high density lipoprotein (HDL-C) comprises first reagent comprising a buffer with a pH in the range of 6 to 8, a cholesterol oxidase, a cholesterol esterase, selective surfactant and a phenol based hydrogen donor compound; and second reagent comprising a buffer with a pH in the range of 6 to 8 with the same molarity range as the first reagent, peroxidase and a surfactant for activating the cholesterol fraction in HDL.
2. The reagent system as claimed in claim 1, wherein said buffer is selected from a phosphate, a Tris and a Good's buffer (MOPSO, BES, HEPES PIPES).
3. The reagent system as claimed in claim 2, wherein said buffer is Good's buffer having a concentration in range from 25-75 mM.
4. The reagent system as claimed in claim 1, wherein said first reagent comprises the buffer with a pH of 6 to 8, the cholesterol oxidase in the concentration of 0.2 to 0.6 U/ml, the cholesterol esterase in the concentration of 0.5 to 1.0 U/ml, selective surfactant in the concentration of 0.01% - 0.1% and a phenol based hydrogen donor compound in the concentration of 0.3 to 1.0 mM.
5. The reagent system as claimed in claim 1, wherein said second reagent comprises the buffer with a pH of 6 to8, 4-aminoantipyrine in the concentration of 1 to 4mmol/l, the peroxidase in the concentration of 1 to 4 U/ml, and detergent in the concentration of 0.5 to 1.3%,
6. The reagent system as claimed in claim 1, wherein said surfactant has hydrophilicity Jipophilicity balance (HLB) value between 13.0 to 14.0 and has the concentration in the range between 0.5-1.5%.


7. The reagent system as claimed in claim 1, further comprises divalent metal ions for reacting lipoprotein sub-fractions with the cholesterol reagent system.
8. The reagent system as claimed in claim 1, further comprises a chromogen such as phenolic substitute having high signal ratio for better sensitivity for detecting lower levels of concentration in the clinically significant range.
9. The reagent system as claimed in claim 1, further comprises a peroxidase enzyme stabilizer that contributes to the reagent stability.
10. The reagent system as claimed in claim 1, wherein said reagent system is formulated as a two-reagent system in a 4:1 ratio and may be converted into a Mono-reagent pack.
11. The reagent system as claimed in claim 1, wherein said first reagent comprises PIPES buffer pH 7.0 in the concentration of 50 mmol/l; the cholesterol esterase in the concentration of 0.8 u/ml; the cholesterol oxidase in the concentration of 0.4 u/ml; selective surfactant in the concentration of 0.01 - 0.1%; and phenolic chromogen in the concentration of 0.7 mmol/l.
12. The reagent system as claimed in claim 1, wherein said second reagent comprises PIPES buffer pH 7.0 in the concentration of 50 mmol/l; 4-aminoantipyrine in the concentration of 4 mmol/l; peroxidase in the concentration of 4.0 u/ml; and detergent in the concentration of 1.3%,
13. A process for the direct determination of cholesterol in high density lipoprotein (HDL-C) comprises eliminating the cholesterol in lipoprotein fraction with cholesterol esterase and oxidase in the presence of a selective surfactant and removing the product of oxidation reaction in the first step;


adding a surfactant specific to HDL to reactivate the HDL cholesterol fraction for reacting with the cholesterol assaying reagent system in the second step.
14. The process as claimed in claimed in claim 13, wherein in the first pre-treatment step of the sample with the reagent system in the presence of a surfactant, the cholesterol in HDL is not substantially reacted while other lipoproteins such as LDL, VLDL and CM are reacted and eliminated.
15. The process as claimed in claimed in claim 13, wherein in the subsequent second reaction phase, the cholesterol in HDL is selectively measured.
16. The process as claimed in claimed in claim 13, wherein the entrapment of lipoprotein fractions for rendering it non-reactive to cholesterol reagent (other than HDL) is achieved through the use a combination of surfactants in the reagent system.
17. The process as claimed in claimed in claim 13, wherein the maximum detectable range is 200 mg/dl of HDL-C.
18. The process as claimed in claimed in claim 13, wherein 5 pal of sample is added 400 ui of first reagent and incubated for five minutes at 37°C and 100 fil of the second reagent is added and the mixture is allowed to react at 37°C for five minutes, followed by measurement of absorbance at 546 nm of the reaction mixture.
Dated this 2nd day of Feb. 2009.