[0001] The present disclosure relates generally to the field of biochemical kits. More
5 specifically, the disclosure is directed to a diagnostic kit for the determination of PD-1
immunological complex in a sample.
BACKGROUND OF THE INVENTION
[0002] Background description includes information that may be useful in
10 understanding the present invention. It is not an admission that any of the information
provided herein is prior art or relevant to the presently claimed invention, or that any
publication specifically or implicitly referenced is prior art.
[0003] Cancer is a disease infecting about 10 million people in the world each year. It
involves rapid abnormal multiplication of cells that may also have the potential to invade
15 other parts of the body apart from the primary site. Early detection of cancer, to enable
therapeutic treatment from the onset of the disease, has been inefficient due to lack of precise
methods and detection kits. Early detection may result in successful treatment in many cases.
Tumor cells express certain proteins which are liable to be used as tumor markers. Such
protein molecules do exist in normal cells, however in cancer cells these protein markers are
20 expressed at a certain threshold which circulates in the body and is detectable. P53 is one
such marker which is present in 80% of the cancers, but the focus has now shifted to more
precise protein markers which can detect cancer at early stages.
[0004] All types of cancers release cytokines and inflammatory markers which trigger
protein gene expressions. In the markers that are used in clinical practice now, the substance
25 is expressed when the tumor is well established; as a result it is not possible to detect it at the
early stages of cancer onset, i.e. when the patient is asymptomatic. Hence, there is a need to
explore early detectors and over-expressed markers, based on which treatments can be
directed.
[0005] Programmed Death or Programmed Cell Death Protein-1, more familiarly
30 known as PD-1 or PDCD-1, is a protein encoded by the PDCD 1 gene and is generally found
on the surface of the cells of the adaptive immune system, specifically the T-cells. It acts as a
checkpoint molecule that regulates the immune response of the system, for example it keeps
the immune system in check developing tolerance and also avoiding autoimmune diseases.
3
On the other hand, it may also lead to tumor cells evading the immune system completely
allowing them to multiply.
[0006] As such, PD-1 is one of the pivotal and least used clinically relevant markers
for physiologic regulation of the immune system. Akt, Zap70 and Rap1 pathways get
5 inhibited by the PD1 pathway which in turn inhibits production of cytokines, proliferation
and adhesion. In the tumor microenvironment, PD-1 binds the ligand PD-L1 found on
antigen-presenting cells or tumor cells leading to an over expression of PD-1 that exhibits an
inhibitory effect on the immune system’s defense. Therefore, when PD-1 is bound to PD-L1
the tumor cells evade the T-cells or immune checkpoints (down-regulated response) leading
10 to an immunosuppressant activity. This knowledge has backed several immunotherapies for
cancer treatment.
[0007] Various cancers express early markers which are triggered by immune
response against the emergence of tumor cells putting the body in action for a defense. Even
though the patients do not show symptoms, the body narrates and prepares both for a
15 defensive as well as a tumor-conducive environment. Therefore, the patient expresses both
types of markers. In conventional kits, based on previous evidences of marker expression in
cancer, PD-1 has been least studied. However, it expresses at a certain threshold to be
detectable. In 60% of the cancers it gets expressed at a very early stage of cancer onset.
[0008] Hence, the inventors of the present invention have overcome the drawbacks of
20 conventional techniques. They have arrived at a diagnostic kit that detects the presence of
protein (PD-1) immunological complex leading to early diagnosis of cancer.
OBJECTS OF THE INVENTION
[0009] An object of the present disclosure is to provide a kit for detecting, monitoring
25 or analyzing the presence of PD-1 immunological complex in a sample.
[0010] An object of the present disclosure is to provide a kit for detecting, monitoring
or analyzing the presence of PD-1 immunological complex that helps in early detection and
treatment of cancer.
[0011] An object of the present disclosure is to provide a kit for detecting, monitoring
30 or analyzing the presence of PD-1 immunological complex that has high specificity.
[0012] An object of the present disclosure is to provide a kit for detecting, monitoring
or analyzing the presence of PD-1 immunological complex that is easy to use.
4
[0013] An object of the present disclosure is to provide a kit for detecting, monitoring
or analyzing the presence of PD-1 immunological complex that does not use invasive tests
like biopsy.
5 SUMMARY OF THE INVENTION
[0014] This summary is provided to introduce a selection of concepts in a simplified
form that are further described below in Detailed Description section. This summary is not
intended to identify key features or essential features of the claimed subject matter, nor is it
intended to be used as an aid in determining the scope of the claimed subject matter.
10 [0015] The present disclosure is directed to a diagnostic kit for the determination of
PD-1 immunological complex in a sample, which is a promising biomarker.
[0016] In an aspect, the present disclosure relates to a diagnostic kit for detecting,
monitoring or analyzing PD-1 immunological complex in a sample. PD-1 forms an
immunological complex with auto-antibodies present in circulation of a cancer patient.
15 [0017] In an aspect, the present disclosure relates to a diagnostic kit for detecting,
monitoring or analyzing PD-1 immunological complex in a sample, the kit comprising:
a) a dissolution solution A comprising ammonium sulfate, Ethylenediaminetetraacetic
acid (EDTA), and trifluoroethanol;
b) a reaction medium B comprising a copper solution and a base; and
20 c) a denaturation solution C comprising an acid.
[0018] In an embodiment, the dissolution solution A further comprises water.
[0019] In an embodiment, the copper solution may be selected from cupric chloride,
cupric acetate, and cupric sulfate.
[0020] In an embodiment, the base may be selected from those well known in the art,
25 including sodium hydroxide, potassium hydroxide, barium hydroxide, strontium hydroxide
and calcium hydroxide.
[0021] In an embodiment, the acid present in the denaturation solution C may be
selected from any mineral acid, including but not limited to hydrochloric acid, acetic acid and
sulfuric acid. The denaturation solution C exposes a specific amino acid sequence of the
30 bound PD-1 protein that gives pink coloration to the solution thereby detecting the presence
or absence of the PD-1 immunological complex.
[0022] In an embodiment, the specific amino acid sequence of the PD-1
immunological complex is serine-threonine-tyrosine i.e. SER-THR-TYR. A western blot may
be used for confirming the presence of the protein sequence.
5
[0023] In an embodiment, the sample may preferably be selected from blood and/or
urine.
[0024] In an aspect, the present disclosure relates to a method of detecting PD-1
immunological complex in a sample.
5 [0025] In an embodiment, the method of detecting, monitoring or analyzing PD-1
immunological complex in a sample comprises the following steps:
(a) dissolving the sample in a dissolution solution A followed by incubation;
(b) optionally vortexing the solution from step (a) and reacting with a reaction
medium B; and
10 (c) mixing the product of step (b) with a denaturation solution C to give a coloration
to the solution.
[0026] In an embodiment, the present disclosure relates to an apparatus or diagnostic
equipment comprising the diagnostic kit for detecting, monitoring or analyzing PD-1
immunological complex in a sample.
15 [0027] These and other features, aspects, and advantages of the present subject matter
will be better understood with reference to the following description and appended claims.
This summary is provided to introduce a selection of concepts in a simplified form. This
summary is not intended to identify key features or essential features of the claimed subject
matter, nor is it intended to be used to limit the scope of the claimed subject matter.
20 [0028] Figure 1: Colour formation of samples C, T1, T2 and T3 using the claimed kit
[0029] Figure 2: Western blot analysis of samples C, T1, T2 and T3
DETAILED DESCRIPTION OF THE INVENTION
[0030] The following is a detailed description of embodiments of the disclosure. The
25 embodiments are in such detail as to clearly communicate the disclosure. However, the
amount of detail offered is not intended to limit the anticipated variations of embodiments; on
the contrary, the intention is to cover all modifications, equivalents, and alternatives falling
within the spirit and scope of the present disclosure as defined by the appended claims.
[0031] All publications herein are incorporated by reference to the same extent as if
30 each individual publication or patent application were specifically and individually indicated
to be incorporated by reference. Where a definition or use of a term in an incorporated
reference is inconsistent or contrary to the definition of that term provided herein, the
definition of that term provided herein applies and the definition of that term in the reference
does not apply.
6
[0032] Reference throughout this specification to “one embodiment” or “an
embodiment” means that a particular feature, structure or characteristic described in
connection with the embodiment is included in at least one embodiment. Thus, the
appearances of the phrases “in one embodiment” or “in an embodiment” in various places
5 throughout this specification are not necessarily all referring to the same embodiment.
Furthermore, the particular features, structures, or characteristics may be combined in any
suitable manner in one or more embodiments.
[0033] In some embodiments, numbers have been used for quantifying volumes,
percentages, concentrations, and so forth, to describe and claim certain embodiments of the
10 invention and are to be understood as being modified in some instances by the term “about.”
Accordingly, in some embodiments, the numerical parameters set forth in the written
description and attached claims are approximations that can vary depending upon the desired
properties sought to be obtained by a particular embodiment. In some embodiments, the
numerical parameters should be construed in light of the number of reported significant digits
15 and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges
and parameters setting forth the broad scope of some embodiments of the invention are
approximations, the numerical values set forth in the specific examples are reported as
precisely as practicable. The numerical values presented in some embodiments of the
invention may contain certain errors necessarily resulting from the standard deviation found
20 in their respective testing measurements.
[0034] Various terms as used herein are shown below. To the extent a term used in a
claim is not defined below, it should be given the broadest definition persons in the pertinent
art have given that term as reflected in printed publications and issued patents at the time of
filing.
25 [0035] As used in the description herein and throughout the claims that follow, the
meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates
otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on”
unless the context clearly dictates otherwise.
[0036] Unless the context requires otherwise, throughout the specification which
30 follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising”
are to be construed in an open, inclusive sense that is as “including, but not limited to.”
[0037] The recitation of ranges of values herein is merely intended to serve as a
shorthand method of referring individually to each separate value falling within the range.
7
Unless otherwise indicated herein, each individual value is incorporated into the specification
as if it were individually recited herein.
[0038] All methods described herein can be performed in any suitable order unless
otherwise indicated herein or otherwise clearly contradicted by context. The use of any and
5 all examples, or exemplary language (e.g. “such as”) provided with respect to certain
embodiments herein is intended merely to better illuminate the invention and does not pose a
limitation on the scope of the invention otherwise claimed. No language in the specification
should be construed as indicating any non-claimed element essential to the practice of the
invention.
10 [0039] Groupings of alternative elements or embodiments of the invention disclosed
herein are not to be construed as limitations. Each group member can be referred to and
claimed individually or in any combination with other members of the group or other
elements found herein. One or more members of a group can be included in, or deleted from,
a group for reasons of convenience and/or patentability. When any such inclusion or deletion
15 occurs, the specification is herein deemed to contain the group as modified.
[0040] The description that follows, and the embodiments described therein, is
provided by way of illustration of an example, or examples, of particular embodiments of the
principles and aspects of the present disclosure. These examples are provided for the
purposes of explanation, and not of limitation, of those principles and of the disclosure.
20 [0041] It should also be appreciated that the present disclosure can be implemented in
numerous ways, including as a system, a method or a device. In this specification, these
implementations, or any other form that the invention may take, may be referred to as
processes. In general, the order of the steps of the disclosed processes may be altered within
the scope of the invention.
25 [0042] The headings and abstract of the invention provided herein are for convenience
only and do not interpret the scope or meaning of the embodiments.
[0043] The following discussion provides many example embodiments of the
inventive subject matter. Although each embodiment represents a single combination of
inventive elements, the inventive subject matter is considered to include all possible
30 combinations of the disclosed elements. Thus if one embodiment comprises elements A, B,
and C, and a second embodiment comprises elements B and D, then the inventive subject
matter is also considered to include other remaining combinations of A, B, C, or D, even if
not explicitly disclosed.
8
[0044] The present disclosure relates to biochemical kits for detection. Specifically,
the disclosure is directed to a diagnostic kit for the determination of PD-1 immunological
complex in a sample, which is a promising biomarker for early stages of cancer.
[0045] In an embodiment, the present disclosure relates to a diagnostic kit for
5 detecting, monitoring or analyzing PD-1 immunological complex in a sample with a
promising biomarker PD-1. PD-1 is a protein present in a biological sample that is capable of
forming a complex, PD-1 immunological complex, with auto-antibodies present in a tumor
environment. The formation of the PD-1 immunological complex is further enhanced in the
environment provided by the diagnostic kit of the present disclosure. The kit leads to the
10 isolation and consequent detection of the PD-1 immunological complex.
[0046] In an embodiment, the present disclosure relates to a diagnostic kit for
detecting, monitoring or analyzing PD-1 immunological complex in a sample, the kit
comprising:
a) a dissolution solution A comprising ammonium sulfate, Ethylenediaminetetraacetic
15 acid (EDTA), and trifluoroethanol;
b) a reaction medium B comprising a copper solution and a base; and
c) a denaturation solution C comprising an acid.
[0047] In an embodiment, the dissolution solution A dissolves free PD-1 protein, free
auto-antibodies and PD-1 immunological complex present in the sample. Said dissolution
20 solution A may further comprise water.
[0048] In an embodiment, the ammonium sulfate may be present in the concentration
selected from the range of about 0.1M to about 0.2M; preferably the concentration is about
0.15M.
[0049] In an embodiment, the EDTA may be present in the concentration selected
25 from the range of about 0.5M to about 1.5M; preferably the concentration is about 1M.
EDTA is used as an anticoagulant for the sample. Preferably for hematogolical studies
potassium EDTA solution is employed.
[0050] In an embodiment, the reaction medium B helps in further enhancing the
formation of the PD-1 immunological complex between the free PD-1 and the free auto
30 antibodies. This increases the amount of the immunological complex which consequently
improves the optical detection. In an embodiment, the copper solution may be selected from
cupric chloride, cupric acetate, and cupric sulfate. Preferably the copper solution is cupric
chloride, wherein the concentration of cupric chloride present in the solution may range from
about 0.1M to about 0.5M; preferably it is about 0.3M.
9
[0051] In an embodiment, the base may be selected from any base known to a person
of skill in the art. Preferably the base maybe selected from sodium hydroxide, potassium
hydroxide, barium hydroxide, strontium hydroxide and calcium hydroxide. Preferably the
base is sodium hydroxide, wherein the concentration of sodium hydroxide present in the
5 solution may range from about 0.5M to about 1.5M; preferably it is about 1M.
[0052] Without being bound to any theory, it is believed that conformational changes
occur in the protein PD-1 on binding with the auto-antibodies. These conformational changes
when denatured by the denaturation solution leads to exposure of specific amino acid
sequence of the PD-1 protein that reacts to give a pink coloration to the sample. This pink
10 coloration is not seen in non-tumor or very low risk samples. This difference is seen because
the specific amino acid sequence of the unbound protein is not exposed in the non-tumor or
least risk samples because of the conformational changes brought about in the PD-1 protein
in the PD-1 immunological complex.
[0053] In an embodiment, the acid in the denaturation solution C may be selected
15 from any mineral acid, including but not limited to hydrochloric acid, acetic acid and sulfuric
acid.
[0054] In an embodiment, the specific amino acid sequence of PD-1 in the PD-1
immunological complex that is exposed is serine-threonine-tyrosine i.e. SER-THR-TYR.
Said amino acid sequence may be detected via any analytical technique known in the art,
20 specifically it may be detected via western blotting.
[0055] In an embodiment, the method of analysis of the diagnostic kit is optical based
on the coloration of the solution. The color may be further analyzed using well known
analytical tools in the art, for example, spectrophotometry. These techniques may also be
used for the quantification of the PD-1 immunological complex or PD-1.
25 [0056] In an embodiment, the sample is a body fluid and may be selected from blood
serum, plasma, urine, saliva or feces; preferably the sample is a blood or urine sample. The
sample is collected from a subject based on methods known in the art. The samples are
collected into EDTA containers where they are stored before being used for testing. In the
case wherein the sample is a blood sample, plasma or serum of blood is obtained before
30 testing by spinning or spinning and clotting as the case may be.
[0057] In an embodiment, the kit may further comprise equipment that can collect a
sample, a vessel for the solutions, a spectrophotometer and a standard against which the
developed color is measured.
10
[0058] In an embodiment, the components of the kit may be added simultaneously or
subsequently in the sample. In another embodiment, the sample may be added to the
diagnostic kit components.
[0059] In an embodiment, the kit may be incorporated in the form of a testing strip or
5 chip. Addition of the solutions is made in a way such that a sample coming in contact with
the strip develops coloration in the presence of the PD-1 immunological complex.
[0060] In an embodiment, the present disclosure relates to a method of detecting PD-1
immunological complex in a sample.
[0061] In an embodiment, the method of detecting, monitoring or analyzing PD-1
10 immunological complex in a sample comprises the following steps:
(a) dissolving the sample in a dissolution solution A followed by incubation;
(b) optionally vortexing the solution from step (a) and reacting with a reaction
medium B;
(c) mixing the product of step (b) with a denaturation solution C to give a coloration
15 in the solution.
[0062] In an embodiment, the coloration is pink in the presence of a PD-1
immunological complex, thereby positively identifying its presence in the sample.
[0063] In an embodiment, the sample maybe selected from blood and/or urine. The
sample is collected from a subject based on methods known in the art. They are collected into
20 EDTA containers where they are stored before being used for testing. In the case wherein the
sample is a blood sample, plasma or serum of blood is obtained before testing by spinning or
spinning and clotting as the case may be.
[0064] In an embodiment, the dissolution solution A dissolves the free PD-1 protein,
free auto-antibodies and PD-1 immunological complex that may or may not be present in the
25 sample. After the addition of dissolution solution A, the obtained solution is left for
incubation. The incubation period ranges from about 2 hours to about 4 hours, preferably
about 3 hours. The incubation is preferably done at room temperature.
[0065] In an embodiment, before contacting the solution from step (a) with the
reaction medium B the solution from step (a) is preferably vortexed. Vortexing is done for a
30 very small time period from about 2 seconds to about 10 seconds, preferably 3 seconds. This
step of vortexing followed by reaction with the reaction medium B provides the perfect
environment for enhancing the reaction between the free PD-1 and the free auto-antibodies
present in the sample. Apart from the immunological complex of PD-1 already present in the
11
sample, this step enhances the amount of the complex. This leads to improved optical
detection of the immunological complex.
[0066] In an embodiment, subsequent to the formation of the PD-1 immunological
complex in step (b), the solution is treated to the denaturation solution C and thereafter rested
5 for 3-4 hours. This denaturation solution exposes the specific amino acid sequence of the
bound PD-1 protein to give a pink coloration that confirms the complex formation. The
coloration develops in about 20 minutes of the start of the reaction.
[0067] In an embodiment, the kit helps to detect the PD-1 immunological complex
even at micromolar levels.
10 [0068] In an embodiment, the presence of the PD-1 immunological complex may be
used as a diagnostic test for multiple forms of cancer. Specifically, the kit may be used to
detect early stages of all malignant tumors.
[0069] In an embodiment, the present disclosure relates to an apparatus or diagnostic
equipment comprising the diagnostic kit for detecting, monitoring or analyzing PD-1
15 immunological complex in a sample. The present disclosure is also related to a strip or chip
incorporating the kit for ease of detection in a pathological laboratory, home centers or other
medical centers. Said chip/strip may be disposed after use.
[0070] In an embodiment, the kit for detecting, monitoring or analyzing PD-1
immunological complex in a sample is easy to prepare and use.
20 [0071] In an embodiment, the kit for detecting, monitoring or analyzing PD-1
immunological complex in a sample can give results within 3-4 hours of testing.
[0072] In an embodiment, the kit for detecting, monitoring or analyzing PD-1
immunological complex in a sample can be used for early stage detection of a number of
cancers and thereby help in providing early treatment and higher recovery rates.
25 [0073] In an embodiment, the kit may detect the PD-1 immunological complex even
in patients placed at a high risk of developing cancer.
[0074] In an embodiment, the kit for detecting, monitoring or analyzing PD-1
immunological complex in a sample as in the present disclosure has a higher sensitivity
compared to traditionally known ELISA kits. They generate lesser negative results in the
30 early stages of the disease compared to ELISA.
[0075] In an embodiment, the kit for detecting, monitoring or analyzing PD-1
immunological complex in a sample can be used easily in a strip based method for ease of
detection.
12
[0076] In an embodiment, the kit for detecting, monitoring or analyzing PD-1
immunological complex in a sample uses a minimally invasive technique, unlike biopsy, for
testing.
[0077] In an embodiment, the results obtained from the kit can be further analyzed or
5 corresponded with clinical observations.
[0078] In an embodiment, owing to the ease of use the present kit can be used in
medical centers, home care, diagnostic centers, hospitals, military centers and the like.
[0079] The disclosure will now be illustrated with working examples, which is
intended to illustrate the working of disclosure and not intended to take restrictively to imply
10 any limitations on the scope of the present disclosure. Unless defined otherwise, all technical
and scientific terms used herein have the same meaning as commonly understood to one of
ordinary skill in the art to which this disclosure belongs. Although methods and materials
similar or equivalent to those described herein can be used in the practice of the disclosed
methods and compositions, the exemplary methods, devices and materials are described
15 herein. It is to be understood that this disclosure is not limited to particular methods, and
experimental conditions described, as such methods and conditions may vary.
Example 1: Compositions of the solutions for the kit
[0080] The diagnostic kit was prepared with the three component solutions as
provided in Table No. 1.
20 Table No. 1: Solutions of the kit
S. no Type of Solution Composition
1
Dissolution
solution A
Ammonium sulfate (0.15M)
EDTA (1M)
Trifluoroethanol
H2O
2
Reaction
medium B
Cupric chloride solution (0.3 M)
NaOH (1M)
3
Denaturation
solution C
HCl (0.01 mM)
H2O
Example 2: Testing the kit
13
[0081] Four blood samples were taken for testing the kit. The samples were named: C
for control from a patient with no tumor or not at risk of developing cancer, T1 was the
sample for a patient at risk; T2 from a patient who had cancer, T3 from a patient who is in the
chronic stages of cancer. All samples were treated with the claimed diagnostic kit to confirm
5 the presence of PD-1 immunological sample in the blood. The results have been provided in
the Figures 1 and 2. Once PD-1 was obtained in the bound stage, the PD-1 immunological
complex was denatured, exposing the unique sequence that gives the pink coloration.
[0082] Figure 1: C showed blue color, indicating absence/underexpression of PD-1
complex in non-cancer or least risk patients. T1 showed strong pink color indicating presence
10 of complex at early stages in cancer. T2 and T3 showed less color formation indicating its
presence however the color is not as intense as seen in the early stages of cancer onset. Figure
2 shows the western blot analysis in the three different types of samples. B-actin is a house
keeping gene. Control (non-tumor) sample showed no significant band.
[0083] While the foregoing describes various embodiments of the disclosure, other
15 and further embodiments of the disclosure may be devised without departing from the basic
scope thereof. The scope of the invention is determined by the claims that follow. The
invention is not limited to the described embodiments, versions or examples, which are
included to enable a person having ordinary skill in the art to make and use the invention
when combined with information and knowledge available to the person having ordinary skill
20 in the art.
ADVANTAGES OF THE PRESENT INVENTION
[0084] The present disclosure provides a kit for detecting, monitoring or analyzing
PD-1 immunological complex in a sample.
25 [0085] The present disclosure provides a kit that is easy to prepare and use.
[0086] The present disclosure provides a kit that provides a crucial biomarker that can
be used for early stage detection of a number of cancers resulting in early treatment and
consequent higher recovery rates.
[0087] The present disclosure provides a kit that has high sensitivity.
30 [0088] The present disclosure provides a kit that uses minimally invasive techniques
for testing with uncompromised analytical precision.

We Claim:

1. A diagnostic kit for detecting, monitoring or analyzing PD-1 immunological complex in a
5 sample, the kit comprising:
a) a dissolution solution A comprising ammonium sulfate, Ethylenediaminetetraacetic
acid (EDTA), and trifluoroethanol;
b) a reaction medium B comprising a copper solution and a base; and
c) a denaturation solution C comprising an acid.
10 2. The diagnostic kit as claimed in claim 1, wherein the copper solution is selected from
cupric chloride, cupric acetate, and cupric sulfate.
3. The diagnostic kit as claimed in claim 1, wherein the base is selected from sodium
hydroxide, potassium hydroxide, barium hydroxide, strontium hydroxide and calcium
hydroxide.
15 4. The diagnostic kit as claimed in claim 1, wherein the acid is selected from hydrochloric
acid, sulfuric acid, and acetic acid.
5. The diagnostic kit as claimed in claim 1, wherein the concentration of ammonium sulfate is
in the range of 0.1M to 0.2 M.
6. The diagnostic kit as claimed in claim 1, wherein the concentration of EDTA is in the
20 range of 0.5M to 1.5 M.
7. The diagnostic kit as claimed in claim 1, wherein the copper solution is cupric chloride
with concentration in the range of 0.1M to 0.5M.
8. The diagnostic kit as claimed in claim 1, wherein the base is sodium hydroxide with
concentration in the range of 0.5M to 1.5M.
25 9. The diagnostic kit as claimed in claim 1, wherein the sample is selected from blood serum,
plasma or urine.
10. The diagnostic kit as claimed in claim 1, wherein additionally water is present in the
dissolution solution A or reaction medium B or both.
11. The diagnostic kit as claimed in claim 1, wherein the kit is incorporated in the form of a
30 strip or chip for testing.
15
12. An apparatus comprising the diagnostic kit as claimed in claim 1.