FORM 2
THE PATENT ACT 1970 (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 LDL CHOLESTEROL;

2. APPLICANT(S)
a) Name
b) Nationality
c) Address

TRANSASIA BIO-MEDICALS LTD
INDIAN Company
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 : -


199
MUM2009 FEB 2009

Reagent kit for direct determination of LDL cholesterol
Field of invention :
The present invention provides an enzyme reagent system for the determination of cholesterol in low density lipoprotein (LDL). More particularly the invention relates to the reagent system for the direct determination of LDL cholesterol that comprises a single reagent comprising a surfactant. The present invention also relates a process for the direct determination of cholesterol in LDL fraction in serum or plasma without separation techniques, such as centrifugation, electrophoresis or a reagent composition.
Background Art:
In the prior art, it is well documented that HDL & LDL is useful indicator for the risk of onset of arterial sclerosis. Methods for estimating cholesterol in HDL & LDL 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^p^
*

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, an object present invention is to provide an enzyme reagent system for the direct determination of cholesterol in LDL by the use of surfactants, which provides a preferential reactivity of lipoprotein fractions with the enzymatic cholesterol reagent system.
Another object of the invention is to provide an enzyme reagent for assaying LDL cholesterol comprising a single reagent containing a surfactant.
Still another object of the present invention is to provide a process for the direct determination of cholesterol in LDL fraction in serum or plasma, which 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 LDL, the invention is characterized by the use of surfactants, which preferentially blocks very low density lipoproteins (VLDL) and HDL cholesterol fractions and subsequently determining the amount of LDL cholesterol using the cholesterol assaying enzyme reagent system of the present invention.
Description of the invention :
In one aspect of the present invention, an enzyme reagent system for the direct determination of cholesterol in low density lipoprotein (LDL-C) comprises a buffer with a pH in the range of 6 to 8, a cholesterol esterase, a cholesterol oxidase, a


peroxidase, 4-aminoantipyrine, pHBA and a polyhydroxy ethylene derivative detergent.
In preferred aspect of the invention, the said buffer of the enzyme reagent is selected from a phosphate buffer, Tris buffer and a Good's buffer (MOPSO, BES, HEPES PIPES) that is more preferably Good's buffer having a concentration in range from 25 to 75 mM. The said surfactant of the enzyme reagent system 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%.
In another preferred aspect of the invention, the enzyme reagent system further comprises the cholesterol esterase enzyme in the concentration range from 0.5 to 1.0 U/ml, the cholesterol oxidase enzyme in the concentration range from 0.5 to 1.0 U/l, the peroxidase enzyme in the concentration range from 1.0 to 3.0 U/ml, 4-aminoantipyrine in the concentration range from 1.0 to 3.0 mmol/1, pHBA in the concentration range from 7.5 to 12.5 mmol/1 and detergent in the concentration in range from 0.05 to 0.15%
In still another preferred aspect of the invention, the enzyme reagent system further comprises a superior chelating agent, which contributes for lowering reagent blanks, thereby extending on-board calibration stability of the enzyme reagent system.
In yet another preferred aspect of the invention, the enzyme reagent system further comprises a phenolic substitute, chromogen that ensures higher signal ratio at 505 nm, thereby detecting lower levels of concentration in the clinically significant range.

In further preferred aspect of the invention, the enzyme reagent system further comprises, the peroxidase enzyme stabilizer, which contributes for increasing stability of the enzyme reagent system.
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In another aspect of the present invention, a process for the direct determination of cholesterol in low density lipoprotein (LDL-C) comprises (i) removing the cholesterol in lipoprotein fraction with the enzymes such as cholesterol esterase and oxidase in the absence of a surfactant and further removing the product of oxidation reaction; and (ii) incorporating the surfactant specific to LDL for reactivating LDL cholesterol fraction to react with the cholesterol assaying enzyme reagent system.
In preferred embodiment of the process of present invention, in the step (i) a pre-treatment of the sample with the enzyme reagent system in the absence of a surfactant is performed, in which the cholesterol in LDL is not substantially reacted, whereas other lipoproteins, such as HDL, VLDL and CM are reacted and subsequently removed.
In another preferred embodiment of the process of present invention, in the subsequent step (ii), the cholesterol in LDL is selectively determined using chromogen reaction. An immobilization of lipoprotein fractions for making it non-reactive to cholesterol reagent (other than LDL) is achieved by using a single enzyme reagent system comprising a surfactant, in which the maximum detectable range is 600 mg/dl of LDL-C. In yet another preferred embodiment of the process of present invention, 5ul of the sample is added to the above reagent and incubated for 5 minutes at 37°C followed by measurement of absorbance at 505nm of the reaction mixture.
Detailed embodiment of the invention :

In one embodiment of the invention, determination of LDL comprises charging the sample to said enzyme reagent system comprising a surfactant, which preferentially blocks cholesterols in high density and very low density lipoproteins among lipoproteins and subsequently estimating the concentration of cholesterol that reacts there after.

In preferred embodiment of the invention, the enzyme reagent system of the present invention is formulated as a single reagent system for specific quantization of LDL cholesterol levels.
In another preferred embodiment of the invention, the enzyme reagent system for LDL cholesterol comprises a buffer with a pH range of 6 to 8. The said 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 25-75 mM is preferred.
In still preferred embodiment of the present invention, the enzyme reagent system is formulated as a single reagent system, which preferably comprises PIPES buffer pH 7.0 in the concentration of 50 mmol/I, the cholesterol esterase enzyme in the concentration of 0.75 U/ml, the cholesterol oxidase enzyme in the concentration of 0.75 U/l, the peroxidase enzyme in the concentration of 2.0 U/ml, 4-aminoantipyrine in the concentration of 2.0 mmol/1, pHBA in the concentration of 10.0 mmol/1 and detergent in the concentration of 0.1% The hydrophilicity lipophilicity balance (HLB) value of the surfactant used is between 13.0-14.0 and a concentration ranging between 0.5-1.5%.
In further embodiment of the invention, the enzyme reagent system for thedirect determination of cholesterol fraction of LDL is based on using surfactants (HLB value 12.8-13.2) that causes the lipoprotein sub-fractions to preferentially react with the said enzyme reagent system.
In yet further embodiment, the process of present invention is significantly different in the sense that it does not comprise agglutination of lipoproteins other than LDL, either by using specific antibody or using poly-anions, ionic strength alterations, or using chemically modified enzymes for preferentially immobilizing lipoprotein


In still further embodiment of the invention, the process is based on using specific surfactants, which have higher affinity for VLDL & HDL-C sub fractions, thereby causing them non-reactive to the said cholesterol enzyme reagent system and only LDL is be reacted.
In separate embodiment of the invention, the enzyme reagent system is formulated as a single reagent and does not require the pretreatment step as reported in the prior art published so far.
In next embodiment, an assay process of the present invention has a shortened incubation time of five minutes, thereby completing the reaction well within the specified time interval. The maximum detectable limit of LDL-C using the reagent system of the invention is 600 mg/dl and is well adaptable on a fully automated analyzer.
In different embodiment, the enzyme reagent system of the invention also comprises the superior chelating agent, which contributes for lowering the reagent blanks and thereby extending on board calibration stability of the enzyme reagent system.
In separate embodiment of the invention, the reagent system also comprises the peroxidase enzyme stabilizer, which increases the reagent system stability.
In distinct embodiment, the reagent system also comprises chromogenic detection system, which comprises a phenolic substitute solution that is very stable and produces higher signal ratio at 505 nm, and thus results show good correlation with other commercially available brands in the market.

In another embodiment of the invention, the enzyme reagent system is stable for a period of about three months at room temperature (20 to 28°C) that equivalent storage times for liquid reagents extrapolates to 2 years at 2 to 8°C.

In particular embodiment of the invention, the typical formulation of the above said enzyme reagents system for the direct determination of cholesterol in LDL is now described by way of following Examples 1 to 3.
Example 1 :
The enzyme reagent system for the determination of LDL-C comprises PIPES buffer pH 7.0, 50 mmol/1; the cholesterol esterase, 0.75 U/ml; the cholesterol oxidase, 0.75 U/l; the peroxidase enzyme, 2.0 U/ml; 4-aminoantipyrine, 2 mmol/1; pHBA, 10 mmol/1; and detergent, 0.1%
Example 2:
The enzyme reagent system for the determination of LDL-C comprises BES buffer pH 7.0, 25 mmol/1; the cholesterol esterase, 0.5 U/ml; the cholesterol oxidase, 0.5 U/l; the peroxidase enzyme, 1.0 U/ml; 4-aminoantipyrine, 1 mmol/1; pHBA, 75 mmol/1; and detergent, 0.05%
Example 3 :
The enzyme reagent system for the determination of LDL-C comprises HEPES buffer pH 7.0, 75 mmol/1; the cholesterol esterase, 1.0 U/ml; the cholesterol oxidase, 1.0 U/l; the peroxidase enzyme, 3.0 U/ml; 4-aminoantipyrine, 3 mmol/1; pHBA, 12.5 mmol/1; and detergent, 0.15%
Example 4 :

Determination of cholesterol in LDL - 10 ul of the sample is contacted with the reagent formulation of Example 1 and incubated for 5 minutes at 37°C followed by measurement of absorbance at 505 nm of the reaction mixture.
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Example 5 :
Determination of cholesterol in LDL - 5 ul of the sample is contacted with the reagent formulation of Example 2 and incubated for 5 minutes at 37°C followed by measurement of absorbance at 505 nm of the reaction mixture.
Example 6:
Determination of cholesterol in LDL - 5 ul of the sample is contacted with the reagent formulation oi Example 3 and incubated for 5 minutes at 37°C followed by msasurement of absorbance at 505 nm of the reaction mixture.
Example 7 :
Calculations of LDL concentrations - Calculations are based on using a serum calibrator with a known value of LDL concentration, the serum standard being used in test batch in the same way as the sample.
Example 8 :
Comparison of LDL determinations - A comparison of LDL determinations according to examples stated above with a method using polyanion as a reference gave the following statistical correlations for LDL as depicted in following Table 1.

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Table 1:

LDL Test Method
Slope 1.0092
y-intercept -0.8093
S 1.5154
R 0.9978
Bias 0.2300
S 1.5038
t-value 0.8377

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WE CLAIM :
1. An enzyme reagent system for the direct determination of cholesterol in low density lipoprotein (LDL-C) comprises a buffer with a pH in the range of 6 to 8r enzymes such as cholesterol esterase, cholesterol oxidase and peroxidase, 4-aminoantipyrine, pHBA and a detergent.
2. The reagent system as claimed in claim 1, wherein the buffer is selected from a phosphate buffer, Tris buffer or a Good's buffer (MOPSO, BES, HEPESPIPES), preferably Good's buffer with a concentration in range from 25 to 75 mM is selected.
3. The reagent system as claimed in claim 1, wherein the surfactant 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%.
4. The reagent system as claimed in claim 1, wherein the reagent system preferably comprises the cholesterol esterase enzyme in the concentration range from 0.5 to 1.0 U/ml, the cholesterol oxidase enzyme in the concentration range from 0.5 to 1.0 U/l, the peroxidase enzyme in the concentration range from 1.0 to 3.0 U/ml, 4-arninoantipyrine in the concentration range from 1.0 to 3.0 mmol/1, pHBA in the concentration range from 7.5 to 12.5 mmol/1 and detergent in the concentration in range from 0.05 to 0.15%
5. The reagent system as claimed in claim 1, wherein the reagent system further comprises a superior chelating agent, which contributes for lowering reagent blanks, thereby extending on-board calibration stability.
6. The reagent system as claimed in claim 1, wherein the reagent system further comprises a phenolic substitute, chromogen, which ensures higher

signal ratio at 505 nm, thereby detecting lower levels of concentration in the clinically significant range.
7. The reagent system as claimed in claim 1, wherein the reagent system further comprises the peroxidase enzyme stabilizer, which contributes for increasing stability of the said reagent system.
8. The reagent system as claimed in claim 1, wherein the reagent system more preferably is formulated as a single reagent system, which most preferably comprises PIPES buffer pH 7.0 in the concentration of 50 mmol/1, the cholesterol esterase in the concentration of 0.75 U/ml, the cholesterol oxidase in the concentration of 0.75 U/l, the peroxidase in the concentration of 2.0 U/ml, 4-aminoantipyrine in the concentration of 2.0 mmol/1, pHBA in the concentration of 10.0 mmol/1 and detergent in the concentration of 0.1%.

9. A process for the direct determination of cholesterol in low density lipoprotein (LDL-C) comprises (i) removing a cholesterol in lipoprotein fraction with enzymes such as cholesterol esterase and cholesterol oxidase in the absence of a surfactant and further removing a product of oxidation reaction; and (ii) incorporating then a surfactant specific to LDL for reactivating LDL cholesterol fraction to react with the cholesterol determining enzyme reagent system.
10. The process as claimed in claim 9, wherein in the step (i) a pre-treatment of the sample with the enzyme reagent system is performed in the absence of a surfactant in which the cholesterol in LDL is not substantially reacted, whereas other lipoproteins, such as HDL, VLDL and CM are reacted and subsequently removed.

11. The process as claimed in claim 9, wherein in the step (ii), the cholesterol in LDL is selectively determined by incorporating a surfactant specific to LDL and by using chromogenic phenolic substitute.
12. The process as claimed in claim 9, wherein an immobilization of lipoprotein fractions for making it non-reactive to the reagent system (other than LDL) is achieved by using a single enzyme reagent system comprising a surfactant, wherein maximum detectable range of LDL-C is 600 mg/dl.
13. The process as claimed in claim 9, wherein 5(il of the sample is added to the reagent system and incubated for 5 minutes at 37°C followed by measurement of absorbance at 505 nm of the reaction mixture.
2nd Februvary
Dated this , day of ' 2009.
ForTRANSASIA BIO-MEDICALS LTD.
RAVINDRAYEVLEVICE PRESIDENT - LEGAL, IP AND COMPANY SECRETARY
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