Description:Title of the Invention
“A Diagnostic Test Kit for Alkaline Phosphatase (ALP)”

Field of the Invention
The present disclosure relates generally to field of medical devices. More specifically the present invention relates to diagnostic test kit works on photometric method to determine Alkaline Phosphatase (ALP) from blood serum / plasma in vitro for laboratories and medical professionals.
Background of Invention
ALP is found in the cytosol of liver cells and the canalicular membrane of hepatocytes.ALP is found in decreasing concentrations in various organs such as the placenta, ileal mucosa, kidney, bone, and liver. Over 80% of the ALP in serum originates from the liver and bone, with minor contributions from the intestine. Although ALPs are present in various tissues throughout the body, their precise physiological function remains elusive. An alkaline phosphatase (ALP) test measures the amount of ALP in your blood. ALP is an enzyme found in many parts of your body. Each part of your body produces a different type of ALP. Most ALP is found in your liver, bones, kidneys, and digestive system. Abnormal levels of ALP in your blood may be a sign of a wide range of health conditions, including liver disease, bone disorders, and chronic kidney disease. An alkaline phosphatase test is often used to screen for or help diagnose diseases of the liver or bones. The test may also help diagnose or monitor other health conditions. Many conditions may affect ALP levels, so the test is often done with other blood tests. These other tests include a comprehensive metabolic panel (CMP) or liver function tests that check how well your liver is working. An alkaline phosphatase test may also be ordered if you have symptoms of liver damage or a bone disorder. Symptoms of liver disease include: Nausea and vomiting, Lack of appetite, Fatigue, Weakness, Jaundice, a condition that causes your skin and eyes to turn yellow, Swelling and/or pain in your abdomen (belly), Swelling in your ankles and legs, Dark-colored urine (pee) and/or light-colored stool (poop), Frequent itching while symptoms of bone disorders include:Pain in your bones, Inflammation and arthritis in your joints, Bones that are unusually shaped and/or too large, Broken bones.
An alkaline phosphatase test is a type of blood test. Alkaline phosphatases (ALPs) are a group of isoenzymes located on the outer layer of the cell membrane. They catalyze the hydrolysis of organic phosphate esters found in the extracellular space. Zinc and magnesium are essential cofactors of this enzyme. Despite the diverse tissue distribution and varying physiochemical properties of ALPs, they are classified as true isoenzymes due to their shared ability to catalyze the same reaction.
Various clinical methods are available to assess serum ALP levels, distinguished by the substrate used, alkaline pH buffer, and established normal reference values. These tests, in principle, rely on the enzyme's ability to hydrolyze phosphate esters. In the most widely used international method, p-nitrophenol phosphate serves as the substrate, while amino alcohol is used as the buffering agent. The rate at which p-nitrophenol phosphate is released from the substrate serves as a measurable marker of ALP activity, and the outcomes are typically expressed in international units per liter (IU/L).Various methods appear equally effective in identifying abnormal values associated with different clinical conditions. Comparing results obtained from different tests can be straightforward by using multiples of the upper limit of the established normal range.
The ALP test is performed on semiautomatic or fully automated analyzers that operate on the principle of photometry. Photometry entails the quantification of light absorption within the ultraviolet (UV), visible (VIS), and infrared (IR) spectrum range. This measurement determines the amount of a particular analyte in a solution or liquid. Photometers utilize a specific light source and detectors capable of converting the light transmitted through a sample solution into an electrical signal proportionate to the light's intensity. These detectors can include photodiodes, photoresistors, or photomultipliers

Therefore, there is a need for a device that overcomes at least the above mentioned problems. There is a need for a rapid, precise, and cost-effective diagnostic kit and method for the measurement of Alkaline Phosphatase (ALP)
The present disclosure relates generally to field of medical devices. More specifically the present invention relates to diagnostic test kit works on photometric method to determine Alkaline Phosphatase (ALP) from serum / plasma, in vitro for laboratories and medical professionals.
Object of the Invention
The primary object of the invention is to develop a diagnostic test kit to determine the Alkaline Phosphatase (ALP) from blood serum / plasma useful effectively.
The secondary objective of the invention is that the diagnostic test kit for Alkaline Phosphatase (ALP) utilizes photometric method herein the kit configured with suitable reagents like AMP buffer and Substrate reagent taken in effective concentration of blood serum /plasma.
Another objective of the invention is that the prepared blood serum/plasma samples with reagents estimated for absorbance in photometer at desired wavelength and room temperature.
Further objective of the invention is that Alkaline Phosphatase (ALP) is determined through calculation by multiplying the absorbance with factor.
Summary of the Invention

The present disclosure relates a diagnostic kit specifically to determine the Alkaline Phosphatase (ALP) concentration in the blood serum/plasma wherein the desired size kit is developed, essentially consist of reagents for reaction with blood serum/plasma samples, detected on photometer at desired wavelength to measure the absorbance and finally calculated to determine the Alkaline Phosphatase (ALP) concentration.
A diagnostic test kit for Alkaline Phosphatase (ALP) comprising a test kit characterized in that 25 ml and 75 ml size kit of two compartment cell reagents preferably consist of effective concentration of 0.35 mol/l of AMP buffer and 10 mmol/l of Substrate reagent determines Alkaline Phosphatase (ALP) in vitro from blood serum / plasma sample effectively, wherein the two cell reagent composed of reagent I cell of 0.35 mol/l of AMP buffer; reagent II cell of 10 mmol/l of Substrate reagent determines Alkaline Phosphatase (ALP) by taking blood serum/ plasma sample 0.015 ml to 0.020 ml into two clean test tube for test and blank sample; pipette out the reagent I and II from the kit separately in effective ratio of 4:1 respectively to determine Alkaline Phosphatase (ALP ); transferring it into the test tubes respectively at room temperature (37oC); incubating the reaction mixture for 5 minutes; determining the absorbance of said test sample and blank sample on photometer at a wavelength of 405 nm for 60-180 seconds; and finally calculating the absorbance using factor to determine Alkaline Phosphatase (ALP ) from the test serum / plasma sample effectively.

One of the preferred embodiments of the present invention is the unique design of the kit consist of two reagent cell ( I & II ), which prefilled with the preferred reagent like AMP buffer and Substrate reagent reacts with serum/ plasma Alkaline Phosphatase (ALP ) evaluated through the absorbance on photometer.

One of the preferred embodiments of the present invention is that to determine Alkaline Phosphatase (ALP ) from the varied concentration more preferably making the test sample and blank sample to infer the clinical findings effectively.

One of the preferred embodiments of that Alkaline Phosphatase (ALP) is estimated in vitro through all in one reagent kit.

Brief Description of Drawings

The following thorough explanation of the various aspects of the invention, taken in conjunction with the corresponding drawing that represents various aspects and other features of the disclosure invention.

Figure 1: Flow chart of method of estimation of Alkaline Phosphatase (ALP)

Detailed Description of the Invention

The following description is of exemplary embodiments only and is not intended to limit the scope, applicability or configuration of the invention in any way. Rather, the following description provides a convenient illustration through explanation and figures for implementing exemplary embodiments of the invention. Various changes to the described embodiments may be made in the function and arrangement of the ingredients described without departing from the scope of the invention.
The use of “including”, “comprising” or “having” variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. The terms “a” and “an” herein do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced items. Further, the use of terms “first”, “second”, and “third”, and the like, herein do not denote any order, quantity, or importance, but rather are used to distinguish one element from another.
The terms used in the invention
mmol: milimole
mmol/l: millimole per liter
ALP is found in the cytosol of liver cells and the canalicular membrane of hepatocytes.ALP is found in decreasing concentrations in various organs such as the placenta, ileal mucosa, kidney, bone, and liver. Over 80% of the ALP in serum originates from the liver and bone, with minor contributions from the intestine. Although ALPs are present in various tissues throughout the body, their precise physiological function remains elusive. An alkaline phosphatase (ALP) test measures the amount of ALP in your blood. ALP is an enzyme found in many parts of your body. Each part of your body produces a different type of ALP. Most ALP is found in your liver, bones, kidneys, and digestive system. Abnormal levels of ALP in your blood may be a sign of a wide range of health conditions, including liver disease, bone disorders, and chronic kidney disease. An alkaline phosphatase test is often used to screen for or help diagnose diseases of the liver or bones. The test may also help diagnose or monitor other health condition
A diagnostic test kit for Alkaline Phosphatase (ALP) comprising a test kit characterized in that 25 ml and 75 ml size kit of two compartment cell reagents preferably consist of effective concentration of 0.35 mol/l of AMP buffer and 10 mmol/l of Substrate reagent determines Alkaline Phosphatase (ALP) in vitro from blood serum / plasma sample effectively, wherein the two cell reagent composed of reagent I cell of 0.35 mol/l of AMP buffer; reagent II cell of 10 mmol/l of Substrate reagent determines Alkaline Phosphatase (ALP) by taking blood serum/ plasma sample from 0.015 ml to 0.020 ml into two clean test tube for test and blank sample; pipette out the reagent I and II from the kit separately in effective ratio of 4:1 respectively to determine Alkaline Phosphatase (ALP ); transferring it into the test tubes respectively at room temperature (37oC); incubating the reaction mixture for 5 minutes; determining the absorbance of said test sample and blank sample on photometer at a wavelength of 405 nm for 60-180 seconds; and finally calculating the absorbance using factor to determine Alkaline Phosphatase (ALP ) from the test serum / plasma sample effectively.

The present disclosure relates a dognostic kit specifically to determine the Alkaline Phosphatase (ALP) concentration in the blood serum/plasma wherein the desired size kit is developed, essentially consist of reagents for reaction with blood serum/plasma samples, detected on photometer at desired wavelength to measure the absorbance and finally calculated to determine Alkaline Phosphatase (ALP ) concentration.
Preferred embodiments of Diagnostic Test Kit for Alkaline Phosphatase
i) Diagnostic Kit : The bilirubin kit comprises of 25 ml and 75 ml size wherein two compartment cell consist prefilled reagent for the determination of blood serum/ plasma Alkaline Phosphatase (ALP ), herein the key reagent AMP buffer and Substrate reagent used in different concentration, effectively reacts with serum / plasma Alkaline Phosphatase (ALP ) measures the absorbance using photometer of desired wavelength.
ii) Reagents: Blood serum /plasma, AMP buffer and Substrate reagent.
iii) Apparatus / Instrument: Test tubes, Photometer, Incubator etc.
Table no. 01: Preferred embodiments and prameters of the kit with concentrations

Particulars Name of ingredient with concentration
Serum/ plasma sample 0.015 ml to 0.020 ml
Reagent I 0.35 mol/l of AMP buffer
Reagent II 10 mmol/l of Substrate reagent
Effective Ratio of reagent I : reagent II 4:1
Shelf Life 24 months
Kit Size 25 ml & 75 ml
Wavelength 405 nm
Temperature 37oC
Time of Estimation 60 to 180 seconds
Factor 2720

Table no. 02: Method to operate Alkaline Phosphatase

Particulars Blank Sample
Reagent I 4.0 ml 4.0 ml
Reagent II -- 1.0 ml
Serum/plasma test sample 0.020 ml 0.020 ml

General Method to operate the device
Taking the 0.015 ml to 0.020 ml blood serum/ plasma sample into two clean test tubes for test and blank sample separately;
pipette out the reagent I (0.35 mol/l of AMP buffer) and II (10 mmol/l of Substrate reagent ) in 4:1 ratio from the kit separately to prepare test sample and only reagent I (0.35 mol/l of AMP buffer) used to prepare blank sample; transferring it into the serum/ plasma sample respectively at room temperature (37oC) and uniformly mixed; incubating the reaction mixture for 5 minutes; determining the absorbance of said test sample and blank sample on photometer at a wavelength of 405 nm for 60-180 seconds; and finally calculating the absorbance using factor to determine Alkaline Phosphatase (ALP ) from the test serum / plasma sample effectively.

The absorbance is calculated using below formula:
Alkaline Phosphatase (ALP ): 2720 x Absorbance
(Table No. 1, 2 and Figure No. 1 )
Example 1
Taking the 0.015 ml blood serum/ plasma sample into two clean test tubes for test and blank sample separately;
pipette out the reagent I (0.35 mol/l of AMP buffer) and II (10 mmol/l of Substrate reagent ) in 4:1 ratio from the kit separately to prepare test sample and only reagent I (0.35 mol/l of AMP buffer) used to prepare blank sample; transferring it into the serum/ plasma sample respectively at room temperature (37oC) and uniformly mixed; incubating the reaction mixture for 5 minutes; determining the absorbance of said test sample and blank sample on photometer at a wavelength of 405 nm for 60-180 seconds; and finally calculating the absorbance using factor to determine Alkaline Phosphatase (ALP ) from the test serum / plasma sample effectively.

The absorbance is calculated using below formula:
Alkaline Phosphatase (ALP): 2720 x Absorbance
Example 2
Taking the 0.020 ml blood serum/ plasma sample into two clean test tubes for test and blank sample separately;
pipette out the reagent I (0.35 mol/l of AMP buffer) and II (10 mmol/l of Substrate reagent ) in 4:1 ratio from the kit separately to prepare test sample and only reagent I (0.35 mol/l of AMP buffer) used to prepare blank sample; transferring it into the serum/ plasma sample respectively at room temperature (37oC) and uniformly mixed; incubating the reaction mixture for 5 minutes; determining the absorbance of said test sample and blank sample on photometer at a wavelength of 405 nm for 60-180 seconds; and finally calculating the absorbance using factor to determine Alkaline Phosphatase (ALP ) from the test serum / plasma sample effectively.

The absorbance is calculated using below formula:
Alkaline Phosphatase (ALP): 2720 x Absorbance

Scope of the Invention

A test kit for Alkaline Phosphatase (ALP ) principally design to develop a simple method to analyse the serum / plasma Alkaline Phosphatase (ALP ) level using photometric method having size of 25 ml and 75 ml size kit of two compartment cell reagent preferably consist of effective concentration of 0.35 mol/l of AMP buffer and 10 mmol/l of Substrate reagent in effective ratio of 4:1 determines Alkaline Phosphatase (ALP )in vitro from blood serum / plasma sample effectively, could be potential diagnostic and economical method, compare to expensive conventional methods in future.

It is to be understood that the present invention is not limited to the embodiments described above, it should be clear that various modifications and alterations can be made along with various features of one embodiment included in other embodiments, within the scope of the present invention.
, Claims:We claim,
1- A diagnostic test kit for Alkaline Phosphatase (ALP) comprising a test kit characterized in that 25 ml and 75 ml size kit of two compartment cell reagent preferably consist of effective concentration of 0.35 mol/l of AMP buffer (Reagent I) and 10 mmol/l of Substrate reagent (Reagent II) in effective ratio of 4:1 respectively, estimates Alkaline Phosphatase (ALP) in vitro from blood serum / plasma sample taken from 0.015 ml to 0.020 ml, using photometer effectively.

2- The diagnostic test kit for Alkaline Phosphatase (ALP) as claimed in claim 1, operates through in vitro diagnostic method to determine Alkaline Phosphatase (ALP) comprises the following steps:
taking the 0.015 ml to 0.020 ml blood serum/ plasma sample into two clean test tubes for test and blank sample separately;

pipette out the reagent I (0.35 mol/l of AMP buffer) and II (10 mmol/l of Substrate reagent ) in 4:1 ratio from the kit separately to prepare test sample and only reagent I (0.35 mol/l of AMP buffer) used to prepare blank sample;

transferring it into the serum/ plasma sample respectively at room temperature (37oC) and uniformly mixed;

incubating the reaction mixture for 5 minutes;

determining the absorbance of said test sample and blank sample on photometer at a wavelength of 405 nm for 60-180 seconds; and

calculating the absorbance using factor to determine Alkaline Phosphatase (ALP ) from the test serum / plasma sample effectively.