Description:Title of the Invention
“A Diagnostic Test Kit for Bilirubin”

Field of the Invention
The present disclosure relates generally to field of medical devices. More specifically the present invention relates to test kit works on photometric method to determine bilirubin from serum / plasma with in vitro diagnostic test kit for laboratories and medical professionals.
Background of Invention
Bilirubin is a naturally occurring water-soluble, tetrapyrrolic yellow pigment that occurs in the normal catabolic pathway. Bilirubin is divided in two types, i.e., conjugated and unconjugated bilirubin. It is formed due to breakdown of heme from hemoglobin, myoglobin, peroxidases, and cytochromes in vertebrates. Normally, it is a waste product which is cleared by certain enzymes like glucuronosyl transferase in the liver. However, when the liver is improperly functioning, the bilirubin gets accumulated in the body due to improper breakdown of erythrocytes, liver failure, etc. This elevated Bilirubin can cause various diseases, and if not treated, can also lead to death. Elevated bilirubin may be due to genetic diseases, anaemia, liver issues, etc. The structure of bilirubin contains four pyrolle rings. It is of yellow – orange color in appearance. The chemical formula of Bilirubin is C 33 H 36 N 4 O 6. Bilirubin is also called as Neonatal Bilirubin, Direct Bilirubin, Conjugated Bilirubin, Indirect Bilirubin and Unconjugated Bilirubin. Normal values of direct bilirubin range from 0 to 0.4 mg/dL High bilirubin levels in a newborn means that the neonate is not processing, red cell breakdown effectively or an underlying cause is responsible High levels of Bilirubin can cause hepatitis, cirrhosis, gallstones. Symptoms of high bilirubinlevels in newborns are skin and/or jaundice. High bilirubin levels in a newborn means that the neonate is not processing red cell breakdown effectively or an underlying cause is responsible. Thus, there is a need to detect the bilirubin as early as possible.
Neonatal hyperbilirubinemia is one of the most frequent problems encountered in newborns. Highly elevated levels of bilirubin (degraded product of erythrocytes) can lead to kernicterus, jaundice, liver failure etc. and if delayed can also lead to death. The excessive amount of bilirubin within the body may be due to excessive hemolysis or liver failure.
Yet another approach is the Diazo method, which is based on different solubility properties of conjugated and unconjugated bilirubin. As also disclosed in US4030885A, bilirubin is reacted with diazo reagent in the presence of caffeine, benzoate, and acetate as accelerators to form azobilirubin (coloured compound) which acts as the indicator. However,this method may underestimate a low level of bilirubin in a given sample, requires multiple chemicals, reagents, 2accelerators and it is time consuming.
To overcome the complex process disclosed above, one may consider High Pressure Liquid chromatography, which is a rapid and precise method to determine the total bilirubin. However the drawback here is the high cost of operation. Non-invasive methods also involve the use of image capturing, electronic device or gadgets, etc.
Therefore, there is a need for a device that overcomes at least the above mentioned problems. There is a need for a rapid, precise, non-invasive and cost-effective device and method for the measurement of total bilirubin concentration. Moreover, there is a need for quantitatively measuring bilirubin concentration using whole blood.
The present disclosure relates generally to field of medical devices. More specifically the present invention relates to test kit works on photometric method to determine bilirubin from serum / plasma with in vitro diagnostic test kit for laboratories and medical professionals.
Object of the Invention
The primary object of the invention is to develop a test kit to determine the bilirubin from blood serum / plasma useful to detect the bilirubin effectively.
The secondary objective of the invention is that the test kit for bilirubin utilizes photometric method herein the kit configured with suitable reagents like Sulphanilic acid, Nitrite and Detergent solution 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 total bilirubin and direct bilirubin 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 bilirubin 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 bilirubin content.
A test kit for Bilirubin comprising a test kit characterized in that 100 ml size kit of three compartment cells reagents preferably consist of effective concentration of 5 mmol/l of Sulphanilic acid, 5 mmol/l of Detergent solution and 144 mmol/l Nitrite solution determines total bilirubin and direct bilirubin in vitro from blood serum / plasma sample effectively, wherein the three cell reagents composed of reagent I cell of 5 mmol/l of Sulphanilic acid and 5 mmol/l of Detergent solution; reagent II cell of 5 mmol/l of Sulphanilic acid; and reagent III cell of 144 mmol/l Nitrite solution, determines total bilirubin and direct bilirubin by taking the blood serum/ plasma sample into two clean test tube for test and blank sample; pipette out the reagent I and III from the kit separately to determine total bilirubin and the reagent II and III from the kit separately to determine direct bilirubin; 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 546 nm; and finally calculating the absorbance using factor to determine total bilirubin and direct bilirubin 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 three reagent cells ( I, II & III), which prefilled with the preferred reagent like Sulphanilic acid, Detergent solution and Nitrite solution reacts with serum/ plasma bilirubin evaluated through the absorbance on photometer.

One of the preferred embodiments of the present invention is that to determine total bilirubin and direct bilirubin 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 total bilirubin and direct bilirubin is estimated in vitro through the 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 to operate total bilirubin
Figure 2: Flow chart of method to operate direct bilirubin

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
Bilirubin is a naturally occurring water-soluble, tetrapyrrolic yellow pigment that occurs in the normal catabolic pathway. Bilirubin is divided in two types, i.e., conjugated and unconjugated bilirubin. It is formed due to breakdown of heme from hemoglobin, myoglobin, peroxidases, and cytochromes in vertebrates. Normally, it is a waste product which is cleared by certain enzymes like glucuronosyl transferase in the liver. However, when the liver is improperly functioning, the bilirubin gets accumulated in the body due to improper breakdown of erythrocytes, liver failure, etc. This elevated Bilirubin can cause various diseases, and if not treated, can also lead to death. Elevated bilirubin may be due to genetic diseases, anaemia, liver issues, etc.
A test kit for Bilirubin comprising a test kit characterized in that 100 ml size kit of three compartment cells reagents preferably consist of effective concentration of 5 mmol/l of Sulphanilic acid, 5 mmol/l of Detergent solution and 144 mmol/l Nitrite solution determines total bilirubin and direct bilirubin in vitro from blood serum / plasma sample effectively, wherein the three cell reagents composed of reagent I cell of 5 mmol/l of Sulphanilic acid and 5 mmol/l of Detergent solution; reagent II cell of 5 mmol/l of Sulphanilic acid; and reagent III cell of 144 mmol/l Nitrite solution, determines total bilirubin and direct bilirubin by taking the blood serum/ plasma sample into two clean test tube for test and blank sample; pipette out the reagent I and III from the kit separately to determine total bilirubin and the reagent II and III from the kit separately to determine direct bilirubin; 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 546 nm; and finally calculating the absorbance using factor to determine total bilirubin and direct bilirubin from the test serum / plasma sample effectively.

The present disclosure relates a diagnostic kit specifically to determine the bilirubin 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 bilirubin content.
Preferred embodiments of diagnostic kit for Bilirubin
i) Diagnostic Kit : The bilirubin kit comprises of 100 ml size wherein three compartment cells consist prefilled reagent for the determination of serum/ plasma bilirubin herein the key reagent Sulphanilic acid, Detergent solution and Nitrite solution used in different concentration, effectively reacts with serum / plasma bilirubin measures the absorbance using photometer of desired wavelength.
ii) Reagents: Blood serum /plasma, Sulphanilic acid, Detergent solution and Nitrite solution.
iii) Apparatus / Instrument: Test tubes, Photometer, Incubator etc.
Table no. 01: Preferred embodiments of the kit with concentrations

Particulars Name of ingredient with concentration
Reagent I 5 mmol/l Sulphanilic acid and 5 mmol/l Detergent solution
Reagent II 5 mmol/l Sulphanilic acid
Reagent III 144 mmol/l Nitrite solution
Shelf Life 24 months

Table no. 02: Method to operate Total Bilirubin

Particulars Blank Sample
Reagent I 1.0 ml 1.0 ml
Reagent III -- 0.050 ml
Serum/plasma test sample 0.050 ml 0.050 ml
Table no. 03: Method to operate Direct Bilirubin

Particulars Blank Sample
Reagent II 1.0 ml 1.0 ml
Reagent III -- 0.050 ml
Serum/plasma test sample 0.050 ml 0.050 ml

General Method to operate the device
Taking the 0.050 ml blood serum/ plasma sample into two clean test tubes for test and blank sample separately;
pipette out the reagent I (5 mmol/l of Sulphanilic acid, 5 mmol/l of Detergent solution) and III (144 mmol/l Nitrite solution ) from the kit separately to determine total bilirubin and the reagent II (5 mmol/l of Sulphanilic acid ) and III (144 mmol/l Nitrite solution ) from the kit separately to determine direct bilirubin; 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 546 nm; and finally calculating the absorbance using factor to determine total bilirubin and direct bilirubin from the test serum / plasma sample effectively.
The absorbance is calculated using below formula:
Total Bilirubin: 23 x Absorbance
Direct Bilirubin: 17 x Absorbance (Table No. 1, 2, 3 and Figure No. 1 and 2 )
Example 1
Taking the 0.050 ml blood serum/ plasma sample into two clean test tubes for test and blank sample separately;
pipette out the reagent I (5 mmol/l of Sulphanilic acid, 5 mmol/l of Detergent solution) and III (144 mmol/l Nitrite solution ) from the kit separately; 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 546 nm; and finally calculating the absorbance using factor to determine total bilirubin from the test serum / plasma sample effectively.
The absorbance is calculated using below formula:
Total Bilirubin: 23 x Absorbance
Example 2
Taking the 0.050 ml blood serum/ plasma sample into two clean test tubes for test and blank sample separately;
pipette out the reagent II (5 mmol/l of Sulphanilic acid ) and III (144 mmol/l Nitrite solution ) from the kit separately; 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 546 nm; and finally calculating the absorbance using factor to determine direct bilirubin from the test serum / plasma sample effectively.
The absorbance is calculated using below formula:
Direct Bilirubin: 17 x Absorbance

Scope of the Invention

A test kit for Bilirubin principally design to develop a simple method to analyse the serum / plasma bilirubin level using photometric method having size of 100 ml size kit of three compartment cells reagents preferably consist of effective concentration of 5 mmol/l of Sulphanilic acid, 5 mmol/l of Detergent solution and 144 mmol/l Nitrite solution determines total bilirubin and direct bilirubin 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 bilirubin comprising a test kit characterized in that 100 ml size kit of three compartment cells reagents preferably consist of effective concentration of 5 mmol/l of Sulphanilic acid, 5 mmol/l of Detergent solution and 144 mmol/l Nitrite solution determines total bilirubin and direct bilirubin in vitro from blood serum / plasma sample effectively.
2- The diagnostic test kit for bilirubin as claimed in claim 1, wherein the three cell reagents composed of reagent I cell of 5 mmol/l of Sulphanilic acid and 5 mmol/l of Detergent solution; reagent II cell of 5 mmol/l of Sulphanilic acid; and reagent III cell of 144 mmol/l Nitrite solution.

3- The diagnostic test kit for bilirubin as claimed in claim 1 and reagent cells in claim 2, operates through in vitro diagnostic method to determine total bilirubin comprises the following steps:

taking the blood serum/ plasma sample into two clean test tube for test and blank sample;

pipette out the reagent Sulphanilic acid and Detergent solution (I) and Nitrite solution (III) separately, transferring it into the test sample tube (excluding blank sample test tube) 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 546 nm; and

calculating the absorbance using factor to determine total bilirubin from the test serum / plasma sample effectively.

4- The diagnostic test kit for bilirubin as claimed in claim 1 and reagent cells in claim 2, operates through in vitro diagnostic method to determine direct bilirubin comprises the following steps:

taking the blood serum/ plasma sample into two clean test tube for test and blank sample;

pipette out the reagent Sulphanilic acid (II) and Nitrite solution (III) separately, transferring it into the test sample tube (excluding blank sample test tube) 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 546 nm; and

calculating the absorbance using factor to determine direct bilirubin from the test serum / plasma sample effectively.