WO2019239430A1 - Biomonitoring device for detection and monitoring of cancer and a method thereof - Google Patents
Biomonitoring device for detection and monitoring of cancer and a method thereof Download PDFInfo
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- WO2019239430A1 WO2019239430A1 PCT/IN2019/050449 IN2019050449W WO2019239430A1 WO 2019239430 A1 WO2019239430 A1 WO 2019239430A1 IN 2019050449 W IN2019050449 W IN 2019050449W WO 2019239430 A1 WO2019239430 A1 WO 2019239430A1
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- Prior art keywords
- test strip
- holder
- test
- cancer
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/48—Other medical applications
- A61B5/4842—Monitoring progression or stage of a disease
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/145—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue
- A61B5/14546—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue for measuring analytes not otherwise provided for, e.g. ions, cytochromes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/48—Other medical applications
- A61B5/4848—Monitoring or testing the effects of treatment, e.g. of medication
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/43—Detecting, measuring or recording for evaluating the reproductive systems
- A61B5/4306—Detecting, measuring or recording for evaluating the reproductive systems for evaluating the female reproductive systems, e.g. gynaecological evaluations
- A61B5/4312—Breast evaluation or disorder diagnosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/43—Detecting, measuring or recording for evaluating the reproductive systems
- A61B5/4306—Detecting, measuring or recording for evaluating the reproductive systems for evaluating the female reproductive systems, e.g. gynaecological evaluations
- A61B5/4318—Evaluation of the lower reproductive system
- A61B5/4325—Evaluation of the lower reproductive system of the uterine cavities, e.g. uterus, fallopian tubes, ovaries
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/43—Detecting, measuring or recording for evaluating the reproductive systems
- A61B5/4306—Detecting, measuring or recording for evaluating the reproductive systems for evaluating the female reproductive systems, e.g. gynaecological evaluations
- A61B5/4318—Evaluation of the lower reproductive system
- A61B5/4331—Evaluation of the lower reproductive system of the cervix
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2440/00—Post-translational modifications [PTMs] in chemical analysis of biological material
- G01N2440/40—Post-translational modifications [PTMs] in chemical analysis of biological material addition of nucleotides or derivatives, e.g. adenylation, flavin attachment
Definitions
- the present invention relates to biomonitoring device for detection and monitoring of cancer. More particularly, the current invention relates to a biomonitoring device for rapid and semi quantitative/quantitative detection of the ADP ribose polymer adduct (ARP A) molecular marker which is correlated to cancer progression and regression.
- ARP A ADP ribose polymer adduct
- Cancer is recognized as a serious health care concern globally. While its treatment through chemotherapy, radiotherapy, immunotherapy, etc. gives varying degrees of success and cure, early detection and measuring the response of treatment continues to remain a challenge for the medical profession.
- the ELISA based Slot and Western blot assay described in all the above publications suffer from several drawbacks. For instance, it requires expensive and sophisticated equipments for performing the assay.
- the assay comprises several steps including protein estimation of the sample followed by Slot and Western blotting of the proteins.
- Western blotting includes subjecting the sample to 12% SDS-PAGE and transferring the resolved protein to an activated Nitrocellulose membrane (NCM) and immunoprobing. After blocking and exposing the NCM to primary and secondary antibodies as well as to washing steps followed by colour development step, the bands become visible. The bands obtained are quantified by a Densitometer and analysed using Electrophoresis Documentation and Analysis Software.
- the assay is time consuming, complicated, not economical and requires technical experts to perform it.
- WO 2008/012393 discloses an instrumental and medical kit for performing a home test for detection of precursor lesions in cervical cancer.
- the kit can be used to collect cells from the vagina and to store the same in a liquid medium for subsequent cytological analysis in a laboratory.
- the device is configured as a set or box which contains a bottle and a brush which in turn is housed in an outer sheath. The brush needs to be inserted into the vagina and rotated in order to collect vaginal cells, thus making it inconvenient for use. Also, there is no certainty that sufficient number of vaginal cells will be collected so as to perform the analysis in the laboratory.
- Another drawback of the device is that it detects only precursor lesions in cervical cancer. No other cell abnormalities/cancers can be detected using this device.
- biomonitoring devices known in prior art do not detect all types of cancers and also are not equipped to measure the efficacy of the ongoing treatment to cure cancer.
- the present inventors felt a need to develop a biomonitoring device in a kit form which is simple, quick, economical and sensitive, and which can detect many types of human cancers in the initiation, promotion and progression stages as well as measure response, regression or change in the status of cancer while the process of cancer therapy of the subject is in progress.
- the present invention provides a novel biomonitoring device for rapid and semi quantitative/quantitative detection of the ADP-ribose polymer adduct (ARP A) molecular marker which is correlated to cancer progression and regression.
- ARP A ADP-ribose polymer adduct
- the present invention discloses a biomonitoring device which uses ARPA as the molecular marker to detect many types of human cancers in different stages as well as measures response of the ongoing treatment such as chemotherapy, radiotherapy, immunotherapy, combined modalities etc. used to cure cancer.
- the invention discloses a method of identifying initiation/promotion/progression stages of a cancer in a subject and/or biomonitoring regression of cancer or change in the status of cancer during and while in the process of cancer therapy as being applied to a subject.
- Fig. 1 depicts the main components of the Biomonitoring device according to one of the embodiments.
- Figs. 2A and 2B depict the main steps of the assembly of the Biomonitoring device and its operational outline.
- Fig. 3 depicts the working illustration of the Biomonitoring device.
- Fig. 4 demonstrates the results of ARPA levels tested in 11 patients of H&N cancer undergoing chemotherapy.
- Fig. 5 demonstrates ARPA levels in 2 patients of breast cancer undergoing chemotherapy.
- Fig. 6 demonstrates ARPA levels in 2 patients of cervical cancer undergoing chemotherapy. Detailed Description of Drawings:
- the biomonitoring device of the invention comprises of three main components, namely, a test strip (11), a test reagent strip (12) and a test strip holder (13).
- test strip (11) is a single use component containing Nylon supported Nitrocellulose Membrane Filter (NCM) embedded in a bio-inert long plastic holder.
- NCM Nylon supported Nitrocellulose Membrane Filter
- test reagent strip (12) is a single use bio-inert embodiment containing fixed volumes of different reagents, arranged serially in the required fashion and desired volumes, in the in-built, breakable bubble-like contraptions (121).
- test reagent strip (12) may contain the following in breakable bubble like contraptions:
- the volumes of each of these components are fixed and predetermined.
- the anti-ARPA conjugate (1214) is a chemiluminescent or florescent dye(s), in which case, the colour developer (1216) shall be excluded as the same is not required.
- the anti-ARPA conjugate (1214) is an enzyme, such as horseradish peroxidase (HRP) or any other appropriate enzyme, respective colour developer (1216), shall be provided.
- HRP horseradish peroxidase
- the Test Strip Holder (13) is a reusable components made of bio-inert material such as plastic or the like.
- the Test Strip Holder (13) is equipped with features to hold the Test Strip (11) and the Test Reagent Strip (12) appropriately.
- the Test Strip (11) sits at the bottom of the Test Strip Holder (13) with Test Reagent Strip (12) above it in a way that when the in-built turn-key or knob (122) of the Test Reagent Strip (12) is rotated clockwise, the content of each of the bubble-like contraptions (121) is dispensed into the Test Strip Holder (13) sequentially so that the NCM side of the Test Strip (11) is fully dipped in the respective reagent.
- the assembly can be tilted to pour off and remove the reagent dipping the NCM side of the Test Strip (11) when desired at appropriate and predetermined time intervals.
- appropriate electronic device is used to measure chemiluminescence or fluorescence or colour, depending on whichever tag is used with the anti-APRA as a conjugate.
- Fig. 3 illustrates the working location of the breakable bubble like contraptions.
- the various components are detailed below.
- the Reagent Strip (12) comprises, in sequence, all the required reagents in appropriate volumes to perform the test, such as Activation cum Lysis Buffer (1211), Wash Buffer (1213), Anti- ARPA conjugate solution (1214), Wash Buffer (1215), optionally, a Colour Development Solution (1216) and Sterile Water (1217). It also has a hole (1212) for application of lysed blood sample onto the Test Strip with Nitro cellulose membrane (11).
- the colour development solution (1216) is required only when the Anti-APRP antibody is conjugated with an enzymatic tag. In case where the Anti-ARPA antibody is conjugated to a chemiluminescent/fluorescent tag, the (1216) the same may be eliminated.
- a separate pouch with appropriate volume of Activation cum Lysis Buffer (LB) (1218) is also provided, which shall be detached to add to the blood sample for testing before the Reagent Strip (12) is fitted onto the Test Strip Holder (13) (see illustrative Figures 2 & 3).
- the Reagent Strip (12) has a knob (122) so that it may be appropriately fitted into the slot provided in the Test Strip Holder (13) and turned clockwise. This turning of the knob will dispense the reagents packaged in the Test Reagent Strip (12) in a sequence into the bottom of Test Strip Holder (13) such that the nitrocellulose membrane portion of the Test Strip (11) is fully submerged in the reagent(s).
- the Test Strip Holder (13) has provision to drain off the reagent by decantation.
- the first bubble like contraption (1211) comprises of activation cum lysis buffer.
- the lysis buffer comprises of Ammonium chloride (MLCl), Potassium bicarbonate (KHCCh) and Ethylenediamine tetra acetic acid (EDTA).
- MLCl Ammonium chloride
- KHCCh Potassium bicarbonate
- EDTA Ethylenediamine tetra acetic acid
- the blood sample is placed at (1212).
- a wash buffer is contained in next bubble contraption (1213).
- Bubble contraption (1214) contains antibody conjugated with chemiluminescent or florescent dye on one hand or appropriate enzyme, such as horse-radish Peroxidase (HRP) on the other, which is an air & light-tight container with sufficient volume of instant unique polyclonal antibody against heterogeneous ADP-ribose polymer adduct (ARP A), raised in appropriate small or large animal, such as goat, horse, etc. shall be used after it has been chemically conjugated with appropriate tag which may be selected from chemiluminescent or florescent dye or an enzyme (e.g., HRP).
- HRP horse-radish Peroxidase
- the bubble contraption (1216) would contain a colour development solution.
- the (1215) bubble contraption contains phosphate buffered saline as wash buffer.
- Said wash buffer contains Sodium chloride (NaCl); Potassium chloride (KC1); Disodium hydrogen phosphate (Na2HP0 4 ) and Potassium dihydrogen phosphate (KH2PO4).
- the (1216) bubble contraption shall contain colour development (CD) solution.
- CD solution shall be obtained from commercial sources as a ready-made preparation. This preparation, supplied in an air & light-tight dark container as a lOx solution essentially contains the following 3,3’,5,5’-Tetramethylbenzidine (TMB) and Hydrogen peroxide (H2O2).
- the colour developer shall comprise a TMB/ H2O2 stock solution and used by diluting the supplied stock solution to lx or 2x working solution of CD, wherein lx working solution of CD containing 1 : 10 dilution of CD in distilled water, and 2x containing 2: 10 dilution of CD in sterile and distilled water.
- HRP horse-radish Peroxidase
- Figs. 4, 5 and 6 illustrate the results of clinical validation study conducted over 15 patients suffering from different cancers and are presently undergoing treatment.
- the clinical sample consisted of 11 patients suffering from head and neck cancer, 2 suffering from breast cancer and 2 suffering from cervical cancer.
- the blood samples of these patients were testing using the technology described in the invention.
- the Fig. 4 illustrates a plot of the ARPA levels in blood samples of patients who were treated for head and neck cancer using either chemotherapy or radiotherapy or combination thereof.
- the ARPA concentration in blood sample of patients was assayed before the treatment was started to determine the initial (basal) concentration of ARPA. Subsequent samples were tested after first, second or third rounds of treatment in this ongoing study.
- the testing of the ARPA levels with the technology of the present invention substantially reduces the treatment burden on the patient (which is not really helping the patient) and provides a quantitative measure to clinicians/oncologist for mid-term course correction of therapy for the benefit of the patient(s).
- Fig. 5 presents the pre-treatment and post-treatment ARPA concentrations in 2 patients suffering from breast cancer
- Fig. 6 indicates plot of 2 patients suffering from cervical cancer. In all the four cases mentioned hereinbefore the ARPA concentration demonstrated a significant improvement, establishing the fact that the treatment they were undergoing was effective.
- Described herein is a biomonitoring device for detection and monitoring of cancer progression as well as regression.
- the device is configured for rapid and semi quantitative/quantitative detection of the ADP Ribose Polymer Adduct (ARP A) molecular marker which is correlated to cancer progression and regression.
- ARP A ADP Ribose Polymer Adduct
- the present invention relates to a novel biomonitoring device which is simple, quick, economical, sensitive and helpful to medical practitioners to detect various types of cancers during its development in human body and also to measure the efficacy of the on-going treatment for cancer including chemotherapy, radiotherapy, immunotherapy, combined modalities etc. given to the subject by using it as a biomarker.
- the types of cancer include, but are not limited to, gastric cancer, large bowel cancer, breast cancer, lung cancer, pancreatic cancer, liver cancer, ovarian cancer, prostate cancer, cancer of head and neck region, malignant lymphoma, leukaemia, brain tumour, uterine cancer and bladder cancer.
- the biomonitoring device of the invention consists of the following components:
- the Test Strip (11) is a single use strip of Nylon supported Nitrocellulose Membrane Filter (NCM) embedded in a bio-inert long plastic holder.
- NCM Nitrocellulose Membrane Filter
- the Test Reagent Strip (13) is a single use strip made of a bio-inert material containing fixed volumes of different reagents (1211 - 1217, optionally 1218), arranged serially in the required fashion and desired volumes, in the in-built, breakable bubble-like contraptions.
- the Test Reagent Strip (12) may contain: (i) a Lysis cum Activation buffer (1211), (ii) Washing buffer (1213), (iii) an Anti- ARPA conjugated with chemiluminescent/fluorescent or enzymatic tag (1214), (iv) washing buffer (1215), (v) optionally, a colour developer, preferably, when enzyme is conjugated with anti-ARPA (1216), (vi) Distilled water (1217).
- the volumes of each of the components are fixed and predetermined.
- the test reagent strip (13) may contain additional reagents, if required by the testing procedure, similarly one or more of the mentioned reagents may be eliminated, if not required by the testing procedure.
- the test strip (11) further comprises a hole for application of lysed blood sample onto the Test Strip with NCM.
- a separate pouch (1218) with appropriate volume of Activation cum Lysis Buffer (LB) is also provided, which shall be detached to add to the blood sample for testing before the test Reagent Strip (12) is fitted onto the Test Strip Holder (13) (see illustrative Figures 2 & 3).
- the Reagent Strip has a knob (122) so that it may be appropriately fitted into the slot provided in the Test Strip Holder and turned clockwise. This turning of the knob will dispense the reagents packaged in the Reagent Strip in a sequence into the bottom of Test Strip Holder such that the NCM portion of the Test Strip is fully submerged in the reagent.
- the Test Strip Holder has provision to drain off the reagent by decantation.
- the Test Strip Holder (13) is a reusable bio-inert embodiment (e.g., plastic, etc.) with features to hold the Test Strip (11) and the Test Reagent Strip (12).
- the Test Strip (11) sits at the bottom of the Test Strip Holder (13) with Test Reagent Strip (12) above such that when the in-built knob (122) of the Test Reagent Strip (12) is rotated clockwise, the content of each of the bubble-like contraption (1211 - 1218) dispenses its content into the Test Strip Holder sequentially (13) so that the Nitrocellulose Membrane (NCM) side of the Test Strip (11) is fully dipped in the respective reagent.
- the assembly can be tilted to pour off and remove the reagent dipping the NCM side of the Test Strip when desired.
- the invention provides a biomonitoring kit for detection and monitoring of progression or regression of cancer, whereby the kit comprises of:
- NCM Nitrocellulose Membrane
- NCM Nitrocellulose Membrane
- NCM is a commercially available nitrocellulose transfer membrane, manufactured by Whatman GmbH, Germany under trade name‘Protran’ or equivalent.
- a sterile, thin, inert, plastic Test Strip of appropriate dimension may also be used.
- the Test Strip would have a NCM well of a specified dimension (circular, rectangular or square), such that when the plastic Test Strip is fixed in its appropriate Test Strip Holder and filled with a solution dispensed by the Reagent Strip, the NCM portion of the Test Strip would be fully dipped in the solution as shown in illustrative Figure 2.
- These sterile Test Strips are to be packaged in an air & light-tight contraption.
- Test Reagent Strip (12) as shown in illustrative Figures 2, 3 & 4.
- Said test reagent strip (12) comprises, in sequence, all the required reagents in appropriate volumes to perform the test, such as Activation cum Lysis Buffer, Wash Buffer, Anti-ARPA Ab-HRP Conjugate Solution, Wash Buffer, Colour Development (CD) Solution, if required, and Distilled Water, etc. It also has a hole for application of lysed blood sample onto the Test Strip with NCM.
- a separate pouch with appropriate volume of Activation cum Lysis Buffer is also provided in the kit, which shall be detached to add to the blood sample for testing before the Reagent Strip is fitted onto the Test Strip Holder (see illustrative Figures 2 & 3).
- LB Activation cum Lysis Buffer
- the Reagent Strip (12) is provided with a knob (122) so that it may be appropriately fitted into the slot provided in the Test Strip Holder (13) and turned clockwise.
- the turning of the knob (122) dispenses the reagents packaged in the test Reagent Strip (12) in a sequence into the bottom well of Test Strip Holder (13) such that the NCM portion of the Test Strip (11) is fully submerged in the reagent.
- the Test Strip Holder has provision to drain off the reagent by decantation.
- An air & light-tight bubble-type embodiment with sufficient volume of polyclonal antibody against heterogeneous ADP-ribose polymer adduct (ARP A), raised in rabbit or any other appropriate small or large animal, such as goat, horse, etc. shall be used after it has been chemically conjugated with appropriate tag (e.g., chemiluminescent or florescent dye, or enzyme, such as Horse-radish Peroxidase (HRP)).
- appropriate tag e.g., chemiluminescent or florescent dye, or enzyme, such as Horse-radish Peroxidase (HRP)
- HRP Horse-radish Peroxidase
- the Test Strip Holder (13) is a reusable bio-inert embodiment (e.g., plastic, etc.) with features to hold the Test Strip (11) and the Test Reagent Strip (12).
- the invention provides a process for conducting the biomonitoring analysis using the biomonitoring device of the invention as follows:
- the process for conducting the biomonitoring analysis may involve additional steps (after step 8 above) depending on the nature of‘tag’ conjugated with the anti-ARPA antibody.
- the assay process continues into the following steps:
- Test Strip (11) inserting the Test Strip (11) into an electronic gadget configured to read Chemiluminescence or Fluorescence, to read the chemiluminescence or fluorescence value of bound dye, as the case maybe.
- the assay process continues into the following steps:
- step (vi) inserting the developed Test Strip (11) of step (vi) into a Densitometer to read the colour intensity value of developed colour.
- an appropriate reusable Electronic or Digital Denstitometric device, Densitometer or equivalent, operated by chargeable battery or normal dry cells shall be part of the biomonitoring kit/biomonitoriong device.
- a filter strip where the intensity graded violet/purple colour strip is used to interpret the results of the bioassay.
- the intensity graded violet/purple colour strip is graduated to show a ‘Green’ or safe-zone (higher range of violet/purple colour intensity), ‘Yellow’ or caution-zone (mid-range of violet/purple colour intensity) and ‘Red’ or dangerous-zone (lower range of violet/purple colour intensity).
- the inverse correlation (down-regulation) of ARPA with all stages of carcinogenesis essentially means that as carcinogenesis progresses, the level of ARPA goes down or is down-regulated. Contrastingly, the reverse happens during cancer regression. That is, when cancer regresses after therapeutic intervention, the ARPA levels start going upwards (up-regulation).
- the technology of the biomonitoring kit is capable of measuring the level of ARPA thereby facilitating in quantitative assessment of the progression of disease and effectiveness of therapy by monitoring cancer regression.
- the present biomonitoring kit/biomolecular device and the process for the bioassay of the instant invention is also simple, economical and requires only about 1 hour to complete the process.
- the competitive advantages of the Cancer Biomonitoring Kit/Biomolecular Device of the instant invention are described below:
- Non-invasive nature can even use blood sample drawn even from finger-tip or from a blood storage tube with appropriate anti-coagulant
- All components of the kit have a high shelf-life at room temperature.
- the Fig. 4 illustrates a plot of the ARPA levels in blood samples of patients who were treated for head and neck cancer using either chemotherapy or radiotherapy or combination thereof.
- the ARPA concentration in blood sample of patients was assayed before the treatment was started to determine the initial (basal) concentration of ARPA. Subsequent samples were tested after first, second or third rounds of treatment in this ongoing study.
- the Fig. 4 illustrates a plot of the ARPA levels in blood samples of patients who were treated for head and neck cancer using either chemotherapy or radiotherapy or combination thereof.
- the ARPA concentration in blood sample of patients was assayed before the treatment was started to determine the initial (basal) concentration of ARPA. Subsequent samples were tested after first, second or third rounds of treatment in this ongoing study.
- patients 2, 3, 6, 7, 8, 9, 10 and 11 showed a variable higher levels of ARPA in post therapy (first, second or third rounds of treatments) blood samples.
- the up-regulation of ARPA concentrations in post-therapy cycles were low, medium of high in different patients.
- the up-regulation or increase in the ARPA concentration, as compared to patient’s pre-therapy level, is a direct indicator that the treatment is effective in regressing the cancer.
- a non-significant change in ARPA concentration, as compared to patient’s pre-therapy level indicates that the treatment which they were undergoing was not effective despite 3 rd cycle of Chemotherapy in regressing the cancer and hence a change in dose or treatment could be warranted.
- a small volume or a drop of blood sample to be tested shall be put on the Test Strip and immediately mixed with the same volume of 1% (or, approximately 26 mM) aqueous solution of Ethylene diamine tetra acetic acid (EDTA) at pH 8 and mixed by gentle tapping and is left to stand at room temperature for lysis (say for 5 min. or as required).
- EDTA Ethylene diamine tetra acetic acid
- the WB may consist of the following components:
- the PBS can also be obtained from commercial sources and used as WB.
- the Test Strip Holder is filled with WB to submerge the plastic Test Strip with NCM and left to stand at room temperature for 2 minutes or as required. Development of the NCM strip:
- the assay process continues into the following steps:
- the content (Wash buffer) is poured off completely from the Test Strip Holder by tilting the holder.
- the content (Colour Developer) is poured off completely from the Test Strip Holder by tilting the holder.
- the knob of the Test Reagent Strip is rotated again till the next index to release its content (Distilled Water) into the well of the Test Strip Holder submerging the NCM area of the Test Strip.
- Test Strip is dislodged from the Test Strip Holder.
- the developed Test Strip is then inserted into the electronic gadget, the Densitometer, to read the colour intensity value of developed colour.
- the quantitative value of the colour intensity shall be the indicator of the level of ARPA and, therefore, an indicator of the status of cancer progression or regression after therapeutic intervention.
- ARPA in blood samples was assayed (a) prior to start of the therapeutics and (b) followed the same after end of I st and subsequent cycles of therapeutics.
- the results are shown in Figures 4, 5 and 6.
- the plot of Fig. 4 indicates the amount of ARPA, as determined by the biomonitoring kit of the invention, in 11 patients who were treated for head and neck cancer.
- the plot of Fig. 5 indicates the amount of ARPA, as determined by the biomonitoring kit of the invention, in 2 patients who were treated for breast cancer.
- the plot of Fig. 6 indicates the amount of ARP A, as determined by the biomonitoring kit of the invention, in 2 patients who were treated for cervical cancer.
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Abstract
The present invention describes to a biomonitoring device and kit for monitoring progression and regression of cancer by quantitative detection of the ADP ribose polymer adduct (ARPA) molecular marker. The invention further describes a method for monitoring progression and regression of cancer by quantitative detection of the ADP ribose polymer adduct (ARPA) molecular marker.
Description
“BIOMONITORING DEVICE FOR DETECTION AND MONITORING OF CANCER AND A METHOD THEREOF”
Field of Invention:
The present invention relates to biomonitoring device for detection and monitoring of cancer. More particularly, the current invention relates to a biomonitoring device for rapid and semi quantitative/quantitative detection of the ADP ribose polymer adduct (ARP A) molecular marker which is correlated to cancer progression and regression.
Background of Invention:
Cancer is recognized as a serious health care concern globally. While its treatment through chemotherapy, radiotherapy, immunotherapy, etc. gives varying degrees of success and cure, early detection and measuring the response of treatment continues to remain a challenge for the medical profession.
While it is admitted that cancer can be managed, treated or cured much better if detected early, the cost/inconvenience in diagnosis as well as reliability of available diagnostic tests are poor leading to progression of the disease before initiation of medical intervention. Even after progression, when the treatment for cancer is in progress, the oncologist often faces the challenge of measuring the biological response, except through symptomatic relief and degree of pain reported by the patient. If the oncologist is equipped to measure the physiological or biological response during the treatment effectively and reliably, the direction of the treatment could be continued or altered, including the dose and frequency of therapeutics during the course of the on-going treatment. This will facilitate higher clinical efficacy of cancer treatment even with the existing therapeutic modalities.
An article titled‘ Negative correlation between poly-ADP -ribosylation of spleen cell histone proteins and initiation duration of dimethyl nitrosamine exposure to mice in vivo measured by Western blot immunoprobe assay: A possible biomarker for
cancer detection’ by Devi et al. published in Cancer Detection and Prevention Vol. 29 in 2005 discloses Poly-ADP-Ribosylation (PAR) of spleen cell histone proteins as a possible biomarker for cancer detection. The ELISA based Slot and Western Blot assay is used to study the relationship between PAR and initiation of cancer.
An article titled‘ Detection and quantification of poly-ADP-ribosylated cellular proteins of spleen and liver tissues of mice in vivo by slot and Western blot immunoprobing using polyclonal antibody against mouse ADP-ribose polymer’ by R.N. Sharan et al. published in Molecular and Cellular Biochemistry Vol. 278 in 2005 discloses the assay for detecting and quantifying Poly-ADP-ribosylated cellular proteins of spleen and liver tissues of mice using the ELISA based Slot and Western Blot immunoprobe assays.
A review article titled‘ Poly-ADP-ribosylation in cancer’ by Sharan, published in Cancer Epigenetics in 2009 discusses, in detail, the process of Poly-ADP- Ribosylation and the influence of ADP-Ribose polymers on the chromatin organization and cellular physiology. It also throws light on the involvement of ADP-Ribose polymers in carcinogenesis.
The ELISA based Slot and Western blot assay described in all the above publications suffer from several drawbacks. For instance, it requires expensive and sophisticated equipments for performing the assay. The assay comprises several steps including protein estimation of the sample followed by Slot and Western blotting of the proteins. Western blotting includes subjecting the sample to 12% SDS-PAGE and transferring the resolved protein to an activated Nitrocellulose membrane (NCM) and immunoprobing. After blocking and exposing the NCM to primary and secondary antibodies as well as to washing steps followed by colour development step, the bands become visible. The bands obtained are quantified by a Densitometer and analysed using Electrophoresis Documentation and Analysis Software. The assay is time consuming, complicated, not economical and requires technical experts to perform it.
WO 2008/012393 discloses an instrumental and medical kit for performing a home test for detection of precursor lesions in cervical cancer. According to the disclosure of WO‘393, the kit can be used to collect cells from the vagina and to store the same in a liquid medium for subsequent cytological analysis in a laboratory. The device is configured as a set or box which contains a bottle and a brush which in turn is housed in an outer sheath. The brush needs to be inserted into the vagina and rotated in order to collect vaginal cells, thus making it inconvenient for use. Also, there is no certainty that sufficient number of vaginal cells will be collected so as to perform the analysis in the laboratory. Another drawback of the device is that it detects only precursor lesions in cervical cancer. No other cell abnormalities/cancers can be detected using this device.
Moreover, the biomonitoring devices known in prior art do not detect all types of cancers and also are not equipped to measure the efficacy of the ongoing treatment to cure cancer.
In view of the shortcomings of the prior arts, the present inventors felt a need to develop a biomonitoring device in a kit form which is simple, quick, economical and sensitive, and which can detect many types of human cancers in the initiation, promotion and progression stages as well as measure response, regression or change in the status of cancer while the process of cancer therapy of the subject is in progress.
Therefore, the present inventor has investigated the problem and has sought to find a solution which has not been described in earlier publications and hereby provide a novel biomonitoring device which can detect and monitor cancer progression and regression.
Summary of Invention:
The present invention provides a novel biomonitoring device for rapid and semi quantitative/quantitative detection of the ADP-ribose polymer adduct (ARP A) molecular marker which is correlated to cancer progression and regression.
In an aspect, the present invention discloses a biomonitoring device which uses ARPA as the molecular marker to detect many types of human cancers in different stages as well as measures response of the ongoing treatment such as chemotherapy, radiotherapy, immunotherapy, combined modalities etc. used to cure cancer.
In another aspect, the invention discloses a method of identifying initiation/promotion/progression stages of a cancer in a subject and/or biomonitoring regression of cancer or change in the status of cancer during and while in the process of cancer therapy as being applied to a subject.
Brief Description of Drawings:
Fig. 1 depicts the main components of the Biomonitoring device according to one of the embodiments.
Figs. 2A and 2B depict the main steps of the assembly of the Biomonitoring device and its operational outline.
Fig. 3 depicts the working illustration of the Biomonitoring device.
Fig. 4 demonstrates the results of ARPA levels tested in 11 patients of H&N cancer undergoing chemotherapy.
Fig. 5 demonstrates ARPA levels in 2 patients of breast cancer undergoing chemotherapy.
Fig. 6 demonstrates ARPA levels in 2 patients of cervical cancer undergoing chemotherapy.
Detailed Description of Drawings:
As illustrated in Fig. 1, the biomonitoring device of the invention comprises of three main components, namely, a test strip (11), a test reagent strip (12) and a test strip holder (13).
In a typical example of the invention, the test strip (11) is a single use component containing Nylon supported Nitrocellulose Membrane Filter (NCM) embedded in a bio-inert long plastic holder.
The test reagent strip (12) is a single use bio-inert embodiment containing fixed volumes of different reagents, arranged serially in the required fashion and desired volumes, in the in-built, breakable bubble-like contraptions (121).
The test reagent strip (12) may contain the following in breakable bubble like contraptions:
(i) Lysis cum Activation buffer (1211),
(ii) Hole or empty slot (1212),
(iii) Washing buffer (1213),
(iv) Anti-ARPA conjugated with a tag (1214),
(v) Washing buffer (1215),
(vi) Colour developer, if required (1216),
(vii) Distilled water (1217).
The volumes of each of these components are fixed and predetermined.
In an optional embodiment, it may be possible to include or exclude reagents as required by the subject test, which may be selected based on the anti- ARP A conjugate used. The anti-ARPA conjugate (1214) is a chemiluminescent or florescent dye(s), in which case, the colour developer (1216) shall be excluded as the same is not required. Alternatively, if the anti-ARPA conjugate (1214) is an
enzyme, such as horseradish peroxidase (HRP) or any other appropriate enzyme, respective colour developer (1216), shall be provided.
The Test Strip Holder (13) is a reusable components made of bio-inert material such as plastic or the like. The Test Strip Holder (13) is equipped with features to hold the Test Strip (11) and the Test Reagent Strip (12) appropriately.
With reference to Figs. 2A and 2B, when the device is assembled, the Test Strip (11) sits at the bottom of the Test Strip Holder (13) with Test Reagent Strip (12) above it in a way that when the in-built turn-key or knob (122) of the Test Reagent Strip (12) is rotated clockwise, the content of each of the bubble-like contraptions (121) is dispensed into the Test Strip Holder (13) sequentially so that the NCM side of the Test Strip (11) is fully dipped in the respective reagent. The assembly can be tilted to pour off and remove the reagent dipping the NCM side of the Test Strip (11) when desired at appropriate and predetermined time intervals. Further, appropriate electronic device is used to measure chemiluminescence or fluorescence or colour, depending on whichever tag is used with the anti-APRA as a conjugate.
Fig. 3 illustrates the working location of the breakable bubble like contraptions. The various components are detailed below.
In an embodiment, the Reagent Strip (12), as shown in illustrative Figs 1, 2 & 3, comprises, in sequence, all the required reagents in appropriate volumes to perform the test, such as Activation cum Lysis Buffer (1211), Wash Buffer (1213), Anti- ARPA conjugate solution (1214), Wash Buffer (1215), optionally, a Colour Development Solution (1216) and Sterile Water (1217). It also has a hole (1212) for application of lysed blood sample onto the Test Strip with Nitro cellulose membrane (11).
In accordance with above embodiment, the colour development solution (1216) is required only when the Anti-APRP antibody is conjugated with an enzymatic tag. In case where the Anti-ARPA antibody is conjugated to a chemiluminescent/fluorescent tag, the (1216) the same may be eliminated.
In another embodiment, for lysis of blood, a separate pouch with appropriate volume of Activation cum Lysis Buffer (LB) (1218) is also provided, which shall be detached to add to the blood sample for testing before the Reagent Strip (12) is fitted onto the Test Strip Holder (13) (see illustrative Figures 2 & 3). The Reagent Strip (12) has a knob (122) so that it may be appropriately fitted into the slot provided in the Test Strip Holder (13) and turned clockwise. This turning of the knob will dispense the reagents packaged in the Test Reagent Strip (12) in a sequence into the bottom of Test Strip Holder (13) such that the nitrocellulose membrane portion of the Test Strip (11) is fully submerged in the reagent(s). The Test Strip Holder (13) has provision to drain off the reagent by decantation.
In an embodiment, the first bubble like contraption (1211) comprises of activation cum lysis buffer. The lysis buffer comprises of Ammonium chloride (MLCl), Potassium bicarbonate (KHCCh) and Ethylenediamine tetra acetic acid (EDTA). The blood sample is placed at (1212).
A wash buffer is contained in next bubble contraption (1213).
Bubble contraption (1214) contains antibody conjugated with chemiluminescent or florescent dye on one hand or appropriate enzyme, such as horse-radish Peroxidase (HRP) on the other, which is an air & light-tight container with sufficient volume of instant unique polyclonal antibody against heterogeneous ADP-ribose polymer adduct (ARP A), raised in appropriate small or large animal, such as goat, horse, etc. shall be used after it has been chemically conjugated with appropriate tag which may be selected from chemiluminescent or florescent dye or an enzyme (e.g., HRP). In accordance with this embodiment, if the Anti-ARPA antibody is conjugated with
the enzyme, the bubble contraption (1216) would contain a colour development solution.
The (1215) bubble contraption contains phosphate buffered saline as wash buffer. Said wash buffer contains Sodium chloride (NaCl); Potassium chloride (KC1); Disodium hydrogen phosphate (Na2HP04) and Potassium dihydrogen phosphate (KH2PO4).
If present, the (1216) bubble contraption shall contain colour development (CD) solution. CD solution shall be obtained from commercial sources as a ready-made preparation. This preparation, supplied in an air & light-tight dark container as a lOx solution essentially contains the following 3,3’,5,5’-Tetramethylbenzidine (TMB) and Hydrogen peroxide (H2O2).
If the enzyme tag is a horse-radish Peroxidase (HRP) tag conjugated to anti-ARPA, the colour developer shall comprise a TMB/ H2O2 stock solution and used by diluting the supplied stock solution to lx or 2x working solution of CD, wherein lx working solution of CD containing 1 : 10 dilution of CD in distilled water, and 2x containing 2: 10 dilution of CD in sterile and distilled water.
Figs. 4, 5 and 6 illustrate the results of clinical validation study conducted over 15 patients suffering from different cancers and are presently undergoing treatment. The clinical sample consisted of 11 patients suffering from head and neck cancer, 2 suffering from breast cancer and 2 suffering from cervical cancer. The blood samples of these patients were testing using the technology described in the invention.
The Fig. 4 illustrates a plot of the ARPA levels in blood samples of patients who were treated for head and neck cancer using either chemotherapy or radiotherapy or combination thereof. The ARPA concentration in blood sample of patients was assayed before the treatment was started to determine the initial (basal)
concentration of ARPA. Subsequent samples were tested after first, second or third rounds of treatment in this ongoing study.
As can be seen in the plot of Fig. 4, patients 2, 3, 6, 7, 8, 9, 10 and 11 showed a variable higher levels of ARPA in post therapy (first, second or third rounds of treatments) blood samples. As can be noticed, the up-regulation of ARPA concentrations in post-therapy cycles were low, medium of high in different patients. The up-regulation or increase in the ARPA concentration, as compared to patient’s pre-therapy level, is a direct indicator that the treatment is effective in regressing the cancer. In case of patients 1, 4 and 5 a non-significant change in ARPA concentration, as compared to patient’s pre-therapy level, indicates that the treatment which they were undergoing was not effective despite 3rd cycle of Chemotherapy in regressing the cancer and hence a change in dose or treatment could be warranted.
In the light of the above outcome especially in the case of patient numbers 1, 4 and 5, the testing of the ARPA levels with the technology of the present invention substantially reduces the treatment burden on the patient (which is not really helping the patient) and provides a quantitative measure to clinicians/oncologist for mid-term course correction of therapy for the benefit of the patient(s).
Similar observations could be made based on the plots illustrated in Figs. 5 and 6. While Fig. 5 presents the pre-treatment and post-treatment ARPA concentrations in 2 patients suffering from breast cancer, Fig. 6 indicates plot of 2 patients suffering from cervical cancer. In all the four cases mentioned hereinbefore the ARPA concentration demonstrated a significant improvement, establishing the fact that the treatment they were undergoing was effective.
Detailed Description of the Invention:
Described herein is a biomonitoring device for detection and monitoring of cancer progression as well as regression. The device is configured for rapid and semi
quantitative/quantitative detection of the ADP Ribose Polymer Adduct (ARP A) molecular marker which is correlated to cancer progression and regression.
The present invention relates to a novel biomonitoring device which is simple, quick, economical, sensitive and helpful to medical practitioners to detect various types of cancers during its development in human body and also to measure the efficacy of the on-going treatment for cancer including chemotherapy, radiotherapy, immunotherapy, combined modalities etc. given to the subject by using it as a biomarker. The types of cancer include, but are not limited to, gastric cancer, large bowel cancer, breast cancer, lung cancer, pancreatic cancer, liver cancer, ovarian cancer, prostate cancer, cancer of head and neck region, malignant lymphoma, leukaemia, brain tumour, uterine cancer and bladder cancer.
In an embodiment, the biomonitoring device of the invention consists of the following components:
1. a Test Strip (11);
2. a Test Reagent Strip (12),
3. a Reusable Test Strip Holder (13),
The Test Strip (11) is a single use strip of Nylon supported Nitrocellulose Membrane Filter (NCM) embedded in a bio-inert long plastic holder.
The Test Reagent Strip (13) is a single use strip made of a bio-inert material containing fixed volumes of different reagents (1211 - 1217, optionally 1218), arranged serially in the required fashion and desired volumes, in the in-built, breakable bubble-like contraptions.
In accordance with above embodiment, the Test Reagent Strip (12) may contain: (i) a Lysis cum Activation buffer (1211), (ii) Washing buffer (1213), (iii) an Anti- ARPA conjugated with chemiluminescent/fluorescent or enzymatic tag (1214), (iv) washing buffer (1215), (v) optionally, a colour developer, preferably, when enzyme
is conjugated with anti-ARPA (1216), (vi) Distilled water (1217). The volumes of each of the components are fixed and predetermined. The test reagent strip (13) may contain additional reagents, if required by the testing procedure, similarly one or more of the mentioned reagents may be eliminated, if not required by the testing procedure.
The test strip (11) further comprises a hole for application of lysed blood sample onto the Test Strip with NCM. For lysis of blood, a separate pouch (1218) with appropriate volume of Activation cum Lysis Buffer (LB) is also provided, which shall be detached to add to the blood sample for testing before the test Reagent Strip (12) is fitted onto the Test Strip Holder (13) (see illustrative Figures 2 & 3). The Reagent Strip has a knob (122) so that it may be appropriately fitted into the slot provided in the Test Strip Holder and turned clockwise. This turning of the knob will dispense the reagents packaged in the Reagent Strip in a sequence into the bottom of Test Strip Holder such that the NCM portion of the Test Strip is fully submerged in the reagent. The Test Strip Holder has provision to drain off the reagent by decantation.
The Test Strip Holder (13) is a reusable bio-inert embodiment (e.g., plastic, etc.) with features to hold the Test Strip (11) and the Test Reagent Strip (12).
In accordance with above embodiment, when the device is assembled, the Test Strip (11) sits at the bottom of the Test Strip Holder (13) with Test Reagent Strip (12) above such that when the in-built knob (122) of the Test Reagent Strip (12) is rotated clockwise, the content of each of the bubble-like contraption (1211 - 1218) dispenses its content into the Test Strip Holder sequentially (13) so that the Nitrocellulose Membrane (NCM) side of the Test Strip (11) is fully dipped in the respective reagent. The assembly can be tilted to pour off and remove the reagent dipping the NCM side of the Test Strip when desired.
In an embodiment, the invention provides a biomonitoring kit for detection and monitoring of progression or regression of cancer, whereby the kit comprises of:
1. A set of sterile rectangular Nylon supported Nitrocellulose Membrane (NCM) filter strips (11);
2. A Test Reagent Strip (12);
3. A Test Strip holder (13), and
4. An instruction manual.
The set of sterile rectangular Nylon supported Nitrocellulose Membrane (NCM) filter strips (11) of appropriate dimensions are packaged in an air & light-tight container wherein, one active end of the nitrocellulose membrane contains a fixed area for blood spotting and the other passive end for holding the strip. The blood taken in the capillary tube is placed on the active end of the Nitrocellulose Membrane (NCM) where it gets absorbed by capillary action and electro- static interaction.
NCM is a commercially available nitrocellulose transfer membrane, manufactured by Whatman GmbH, Germany under trade name‘Protran’ or equivalent.
In addition to this, a sterile, thin, inert, plastic Test Strip of appropriate dimension, specially designed for this test, as shown in an illustrative Figures 1, 2, 3 & 4, may also be used. In this, the Test Strip would have a NCM well of a specified dimension (circular, rectangular or square), such that when the plastic Test Strip is fixed in its appropriate Test Strip Holder and filled with a solution dispensed by the Reagent Strip, the NCM portion of the Test Strip would be fully dipped in the solution as shown in illustrative Figure 2. These sterile Test Strips are to be packaged in an air & light-tight contraption.
The Test Reagent Strip (12), as shown in illustrative Figures 2, 3 & 4. Said test reagent strip (12) comprises, in sequence, all the required reagents in appropriate volumes to perform the test, such as Activation cum Lysis Buffer, Wash Buffer,
Anti-ARPA Ab-HRP Conjugate Solution, Wash Buffer, Colour Development (CD) Solution, if required, and Distilled Water, etc. It also has a hole for application of lysed blood sample onto the Test Strip with NCM. For lysis of blood, a separate pouch with appropriate volume of Activation cum Lysis Buffer (LB) is also provided in the kit, which shall be detached to add to the blood sample for testing before the Reagent Strip is fitted onto the Test Strip Holder (see illustrative Figures 2 & 3).
The Reagent Strip (12) is provided with a knob (122) so that it may be appropriately fitted into the slot provided in the Test Strip Holder (13) and turned clockwise. The turning of the knob (122) dispenses the reagents packaged in the test Reagent Strip (12) in a sequence into the bottom well of Test Strip Holder (13) such that the NCM portion of the Test Strip (11) is fully submerged in the reagent. The Test Strip Holder has provision to drain off the reagent by decantation.
An air & light-tight bubble-type embodiment with sufficient volume of polyclonal antibody against heterogeneous ADP-ribose polymer adduct (ARP A), raised in rabbit or any other appropriate small or large animal, such as goat, horse, etc. shall be used after it has been chemically conjugated with appropriate tag (e.g., chemiluminescent or florescent dye, or enzyme, such as Horse-radish Peroxidase (HRP)). For conjugation, either standard chemical methods or appropriate ready- to-use, commercially available, kit may be used. The polyclonal antibody conjugated with appropriate tag as above is supplied in the test kit in an appropriate buffer fortified with protein stabilizer.
The Test Strip Holder (13) is a reusable bio-inert embodiment (e.g., plastic, etc.) with features to hold the Test Strip (11) and the Test Reagent Strip (12).
Furthermore, an appropriate hand-held Electronic or Digital Densitometric device (not shown), Densitometer or equivalent, operated by chargeable battery or normal dry cells may also be part of the biomonitoring kit.
In one more embodiment, the invention provides a process for conducting the biomonitoring analysis using the biomonitoring device of the invention as follows:
1. Placing a small volume or a drop of blood sample to be tested, say‘x’ pl (microlitre), on the Test Strip (11) and immediately mixed with the same volume of 1% (or, approximately 26 mM) aqueous solution of Ethylene diamine tetra acetic acid (EDTA) at pH 8 contained in bubble contraption (1218) and mixed by gentle tapping and is left to stand at room temperature for 5 min;
2. fixing the Test Strip (11) in the well of the Test Strip Holder (13) and placing the Test Reagent Strip (12) appropriately;
3. releasing the content of the of the bubble contraption (1211), that is, Lysis cum Activation buffer till the reaction is complete by manually rotating the knob clock- wise till the Lysis cum Activation buffer contained in (1211) is discharged in the well of the Test Strip Holder (13) submerging the NCM area of the Test Strip (11);
4. incubating the test strip holder (13) till the reaction is complete then the content is decanted by tilting the Test Strip Holder (13);
5. rotating the knob (122) till the Wash buffer contained in (1213) is discharged in the well of the Test Strip Holder (13) submerging the NCM area of the Test Strip (11);
6. incubating the test strip holder (13) till the washing step is complete; then the content is decanted by tilting the Test Strip Holder (13);
7. rotating the knob (122) again till the Anti-ARPA antibody conjugated with appropriate tag is released into the well of the Test Strip Holder (13) submerging the NCM area of the Test Strip (11).
8. incubating the test strip holder (13) till the reaction is complete, then the content is decanted by tilting the Test Strip Holder (13); and
9. rotating the knob (122) again till the wash buffer contented in (1214) is released into the well of the Test Strip Holder (13) submerging the NCM area of the Test Strip (11). The content is decanted by tilting the Test Strip Holder (13).
In accordance with above embodiment, the process for conducting the biomonitoring analysis may involve additional steps (after step 8 above) depending on the nature of‘tag’ conjugated with the anti-ARPA antibody.
In accordance with above embodiment, where the tag is a chemiluminescent/fluorescent dye, the assay process continues into the following steps:
i. Pouring of the Wash buffer from the Test Strip Holder (13) by tilting the test strip holder (13);
ii. dislodging the Test Strip (11) from the Test Strip Holder (13);
iii. inserting the Test Strip (11) into an electronic gadget configured to read Chemiluminescence or Fluorescence, to read the chemiluminescence or fluorescence value of bound dye, as the case maybe.
In accordance with above embodiment, where the tag is an enzyme, e.g., HRP, the assay process continues into the following steps:
i. Pouring of the Wash buffer from the Test Strip Holder (13) by tilting the holder. ii. rotating the knob (122) again till the colour developer contained in (1216) is released into the well of the Test Strip Holder (13) submerging the NCM area of the Test Strip;
iii. incubating the test strip holder (13) till the reaction is complete;
iv. rotating the knob (122) till the distilled water contained in (1218) is released into the well of the Test Strip Holder (13) submerging the NCM area of the Test Strip
(ii);
v. incubating the test strip holder (13) till the reaction is complete;
vi. dislodging the Test Strip (11) from the Test Strip Holder, and
vii. inserting the developed Test Strip (11) of step (vi) into a Densitometer to read the colour intensity value of developed colour.
In accordance with above embodiment, an appropriate reusable Electronic or Digital Denstitometric device, Densitometer or equivalent, operated by chargeable
battery or normal dry cells shall be part of the biomonitoring kit/biomonitoriong device. The rectangular strip of NCM or the sterile plastic Test Strip with NCM, as the case may be, shall be inserted into the slot to quantitatively read the pixel density of the colour developed on the sample spot.
Alternatively, a filter strip where the intensity graded violet/purple colour strip is used to interpret the results of the bioassay. The intensity graded violet/purple colour strip is graduated to show a ‘Green’ or safe-zone (higher range of violet/purple colour intensity), ‘Yellow’ or caution-zone (mid-range of violet/purple colour intensity) and ‘Red’ or dangerous-zone (lower range of violet/purple colour intensity).
In accordance with above embodiment, the inverse correlation (down-regulation) of ARPA with all stages of carcinogenesis (initiation to promotion to progression stages) essentially means that as carcinogenesis progresses, the level of ARPA goes down or is down-regulated. Contrastingly, the reverse happens during cancer regression. That is, when cancer regresses after therapeutic intervention, the ARPA levels start going upwards (up-regulation). The technology of the biomonitoring kit is capable of measuring the level of ARPA thereby facilitating in quantitative assessment of the progression of disease and effectiveness of therapy by monitoring cancer regression.
While the novel and inventive features of the present invention is embodied in the detection and monitoring of cancer at and during all the three stages of carcinogenesis, the present biomonitoring kit/biomolecular device and the process for the bioassay of the instant invention is also simple, economical and requires only about 1 hour to complete the process. The competitive advantages of the Cancer Biomonitoring Kit/Biomolecular Device of the instant invention are described below:
1. Detection and monitoring of cancer at and any time during initiation, promotion and progression stages.
2. Simplicity, specificity and sensitivity employing direct ELISA principles;
3. Non-invasive nature (can even use blood sample drawn even from finger-tip or from a blood storage tube with appropriate anti-coagulant);
4. Quick semi-quantitative or quantitative results;
5. No hospitalization;
6. Non-radioactive nature;
7. Relatively inexpensive and easy to perform/apply.
All components of the kit have a high shelf-life at room temperature.
The clinical validation study of the technology, which is currently ongoing. We report here preliminary results of 15 patients at this instance who are suffering from cancer and are undergoing chemotherapy treatment. The results have been plotted as illustrated in Figs. 4, 5 and 6. The sample consisted of 11 patients suffering from head and neck cancer, 2 suffering from breast cancer and 2 suffering from cervical cancer. The blood samples of these patients were assayed using the technology described in the invention.
The Fig. 4 illustrates a plot of the ARPA levels in blood samples of patients who were treated for head and neck cancer using either chemotherapy or radiotherapy or combination thereof. The ARPA concentration in blood sample of patients was assayed before the treatment was started to determine the initial (basal) concentration of ARPA. Subsequent samples were tested after first, second or third rounds of treatment in this ongoing study.
The Fig. 4 illustrates a plot of the ARPA levels in blood samples of patients who were treated for head and neck cancer using either chemotherapy or radiotherapy or combination thereof. The ARPA concentration in blood sample of patients was assayed before the treatment was started to determine the initial (basal) concentration of ARPA. Subsequent samples were tested after first, second or third rounds of treatment in this ongoing study.
As can be seen in the plot of Fig. 4, patients 2, 3, 6, 7, 8, 9, 10 and 11 showed a variable higher levels of ARPA in post therapy (first, second or third rounds of treatments) blood samples. As can be noticed, the up-regulation of ARPA concentrations in post-therapy cycles were low, medium of high in different patients. The up-regulation or increase in the ARPA concentration, as compared to patient’s pre-therapy level, is a direct indicator that the treatment is effective in regressing the cancer. In case of patients 1, 4 and 5 a non-significant change in ARPA concentration, as compared to patient’s pre-therapy level, indicates that the treatment which they were undergoing was not effective despite 3rd cycle of Chemotherapy in regressing the cancer and hence a change in dose or treatment could be warranted.
EXAMPLES:
The invention will now be illustrated with the help of examples. The aforementioned embodiments and below mentioned examples are for illustrative purpose and are not meant to limit the scope of the invention. Various modifications of aforementioned embodiments and below mentioned examples are readily apparent to a person skilled in the art. All such modifications may be construed to fall within the scope and limit of this invention as defined by the appended claims. Example 1:
1. Prepping blood sample using Ethylene diamine tetra acetic acid (EDTA) at pH
8,
A small volume or a drop of blood sample to be tested, say‘x’ pl (microlitre), shall be put on the Test Strip and immediately mixed with the same volume of 1% (or, approximately 26 mM) aqueous solution of Ethylene diamine tetra acetic acid (EDTA) at pH 8 and mixed by gentle tapping and is left to stand at room temperature for lysis (say for 5 min. or as required).
2. The embodiment is assembled and next steps are carried out as per the assemblage.
Antibody Conjugated with appropriate tag:
This describes the case in which HRP conjugated polyclonal antibody are to be used as tag.
After discharge of the HRP conjugated polyclonal antibody solution onto the Test Strip fixed in the Test Stip Holder, it is left to stand at room temperature for 15 minutes or as required. After the required time, the container is decanted from the Test Strip Holder in which the plastic Test Strip was fitted. Phosphate buffered saline (PBS) as Washing Buffer (WB). pH 7 4:
The WB may consist of the following components:
i. Sodium chloride (NaCl) : l37 mM
ii. Potassium chloride (KC1) :2.7 mM
iii. Di sodium hydrogen phosphate (Na2HP04) : 10 mM
iv. Potassium dihydrogen phosphate (KH2PO4) :2 mM
The PBS can also be obtained from commercial sources and used as WB.
The Test Strip Holder is filled with WB to submerge the plastic Test Strip with NCM and left to stand at room temperature for 2 minutes or as required. Development of the NCM strip:
The assay process continues into the following steps:
i. The content (Wash buffer) is poured off completely from the Test Strip Holder by tilting the holder.
ii. The knob of the Test Reagent Strip is rotated again till the next index to release its content (Colour Developer. In case of HRP) into the well of the Test Strip Holder submerging the NCM area of the Test Strip.
iii. This is left to stand for appropriate time period at room temperature.
iv. The content (Colour Developer) is poured off completely from the Test Strip Holder by tilting the holder.
v. The knob of the Test Reagent Strip is rotated again till the next index to release its content (Distilled Water) into the well of the Test Strip Holder submerging the NCM area of the Test Strip.
vi. This is left to stand for 5 min at room temperature.
vii. The Test Strip is dislodged from the Test Strip Holder.
The developed Test Strip is then inserted into the electronic gadget, the Densitometer, to read the colour intensity value of developed colour.
5. Interpretation of Result:
The quantitative value of the colour intensity shall be the indicator of the level of ARPA and, therefore, an indicator of the status of cancer progression or regression after therapeutic intervention.
Example 2:
Over 60 patients have been recruited for the study so far, which is ongoing. We are presenting preliminary results of 15 cases (11 patients with head and neck cancer, 2 with breast cancer and 2 with cervical cancer) available till date. The details of the 15 patients whose results have been presented in Figs. 4, 5 & 6, are given in Table 1 below.
Table: 1
For these 15 patients, ARPA in blood samples was assayed (a) prior to start of the therapeutics and (b) followed the same after end of Ist and subsequent cycles of therapeutics. The results are shown in Figures 4, 5 and 6.
The quantitative results of testing the sample using the biometric device of the invention are plotted in Figs. 4, 5 and 6.
As can be seen from the above mentioned plots, the ARPA concertation has effectively increased post-treatment of cancer patients either with chemotherapy or radiotherapy or combination thereof. The plot of Fig. 4 indicates the amount of ARPA, as determined by the biomonitoring kit of the invention, in 11 patients who were treated for head and neck cancer. The plot of Fig. 5 indicates the amount of ARPA, as determined by the biomonitoring kit of the invention, in 2 patients who
were treated for breast cancer. The plot of Fig. 6 indicates the amount of ARP A, as determined by the biomonitoring kit of the invention, in 2 patients who were treated for cervical cancer.
Form the Figures 4, 5 and 6, following could be concluded:
1. The status of ARPA level at pot-therapy cycles, compared with pre-therapy level, gives quantitative measure of patient response to the ongoing therapeutic intervention. Up-regulation of ARPA levels at post-therapy cycles, as compared to the pre-treatment ARPA level, indicates cancer regression and‘positive’ patient response.
2. Different cancer patients show different extents of up-regulation of ARPA levels after therapeutic intervention exhibiting differential individualistic response to therapeutics. This could, in principle, lay foundation of‘personalized and precise therapy’, thereby enhancing the clinical efficacy of ongoing therapeutics.
3. Some patients do not show any significant differences in the ARPA levels after therapeutic intervention as compared to the level prior to start of the therapy. This set of patients are not responding to the ongoing therapeutics, thereby meaning that either a higher dose of the chemo is required or another chemo-therapeutic agent might be required for cancer regression in these patients.
Claims
1. A biomonitoring device for monitoring progression and regression of cancer by semi-quantitative/quantitative detection of the ADP ribose polymer adduct (ARP A) molecular marker, wherein said device comprises of:
a. a Test Strip (11) made of Nylon supported or plastic based Nitrocellulose Membrane Filter (NCM) embedded in a bio-inert long plastic holder; b. a Test Reagent Strip (12) containing fixed volumes of different reagents, arranged serially in the required fashion and desired volumes contained in in-built, breakable bubble-like contraptions (121), made of bio-inert material, wherein the test reagent strip (12) comprises of:
i. a lysis cum activation buffer (1211) containing Ammonium chloride (NH4CI), Potassium bicarbonate (KHCO3) and Ethylene diamine tetra acetic acid (EDTA);
ii. a washing solution (1213) containing phosphate buffered saline
(PBS);
iii. an anti-ARPA conjugated with chemiluminescent/fluorescent or enzymatic tag (1214);
iv. a washing buffer (1215) containing Sodium chloride (NaCl),
Potassium chloride (KC1), Disodium hydrogen phosphate (Na2HP04) and Potassium dihydrogen phosphate (KH2PO4), and
v. Distilled water (1217). c. a Test Strip Holder (13) is a reusable component made of bio-inert
material with features to hold the Test Strip and the Test Reagent Strip appropriately.
2. The biomonitoring device as claimed in Claim 1, wherein the enzymatic tag may be appropriate chemiluminescent dye or a fluorescent dye.
3. The biomonitoring device as claimed in Claim 2, wherein the enzymatic tag is Horse-radish Peroxidase (HRP) or the like.
4. The biomonitoring device as claimed in Claims 1 to 3, wherein the test reagent strip optionally comprises a colour developer (1216) containing 3, 3’, 5,5’- Tetramethylbenzidine (TMB) and Hydrogen peroxide (H2O2), when the anti- ARPA antibody is conjugated with an enzymatic tag
5. A kit for monitoring progression and regression of cancer by quantitative detection of the ADP ribose polymer adduct (ARP A) molecular marker, wherein said device comprises of:
a. a Test Strip (11) made of Nylon supported or plastic based Nitrocellulose Membrane Filter (NCM) embedded in a bio-inert long plastic holder; b. a Test Reagent Strip (12) containing fixed volumes of different reagents, arranged serially in the required fashion and desired volumes contained in in-built, air- and light-tight, breakable bubble-like contraptions (121), made of bio-inert material;
c. a reusable Test Strip Holder (13) made of bio-inert material with features to hold the Test Strip and the Test Reagent Strip appropriately, and d. instruction manual.
6. The kit as claimed in Claim 5, wherein the kit may further comprises:
a. an appropriate battery-operated reusable Electronic or Digital
Denstitometric device to read the value of APRA level depending on the type of conjugate used with the anit-ARPA antibody
7. A method for monitoring progression and regression of cancer by quantitative detection of the ADP ribose polymer adduct (ARP A) molecular marker using a device as claimed in Claim 1, wherein the method comprises:
a. placing the Test Strip (11) with blood sample to be tested mixed with the same volume of 1% aqueous solution of Ethylene diamine tetra acetic acid (EDTA) contained in in-built, breakable bubble-like contraptions (1218), put on one end of the strip, in the well of the Test Strip Holder (13);
b. placing the Test Reagent Strip (12) above the test strip, such that knob (122) of the test reagent strip (12) fits is the slot (131) provided in the test strip holder (13), and
c. reading the results through chemiluminescent or fluorescent or
colorimetric means.
8. A method as claimed in Claim 7, wherein the method comprises :
a. placing a small volume or a drop of blood sample to be tested, say‘x’ pl (microlitre), on the Test Strip (11) and immediately mixed with the same volume of 1% (or, approximately 26 mM) aqueous solution of Ethylene diamine tetra acetic acid (EDTA) at pH 8 contained in bubble contraption (1218) and mixed by gentle tapping and is left to stand at room temperature for 5 min;
b. fixing the Test Strip (11) in the well of the Test Strip Holder (13) and placing the Test Reagent Strip (12) appropriately;
c. releasing the content of the of the bubble contraption (1211), that is, Lysis cum Activation buffer by manually rotating the knob clock-wise till the Lysis cum Activation buffer contained in (1211) is discharged in the well of the Test Strip Holder (13) submerging the NCM area of the Test Strip
(ii);
d. incubating the test strip holder (13) till the reaction is complete;
e. rotating the knob (122) till the Wash buffer contained in (1213) is discharged in the well of the Test Strip Holder (13) submerging the NCM area of the Test Strip (11);
f. incubating the test strip holder (13) till the reaction is complete;
g. rotating the knob (122) again till the Anti-ARPA antibody conjugated with appropriate tag is released into the well of the Test Strip Holder (13) submerging the NCM area of the Test Strip (11);
h. incubating the test strip holder (13) till the reaction is complete, and i. rotating the knob (122) again till the wash buffer contented in (1214) is released into the well of the Test Strip Holder (13) submerging the NCM area of the Test Strip (11).
9. The method as claimed in Claim 8, wherein the method further comprises of : a. pouring of the Wash buffer from the Test Strip Holder (13) by tilting the test strip holder (13);
b. dislodging the Test Strip (11) from the Test Strip Holder (13);
c. inserting the Test Strip (11) into an electronic gadget configured to read Chemiluminescence or Fluorescence, to read the chemiluminescence or fluorescence value of bound dye.
10. The method as claimed in Claim 8, wherein the method further comprises of : a. pouring of the Wash buffer from the Test Strip Holder (13) by tilting the holder;
b. rotating the knob (122) again till the colour developer contained in (1216) is released into the well of the Test Strip Holder (13) submerging the NCM area of the Test Strip;
c. incubating the test strip holder (13) till the reaction is complete;
d. rotating the knob (122) till the distilled water contained in (1218) is released into the well of the Test Strip Holder (13) submerging the NCM area of the Test Strip (11);
e. incubating the test strip holder (13) till the reaction is complete;
f. dislodging the Test Strip (11) from the Test Strip Holder, and
g. inserting the developed Test Strip (11) of step (vi) into a Densitometer to read the colour intensity value of developed colour.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| IN201831022176 | 2018-06-13 | ||
| IN201831022176 | 2018-06-13 |
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| WO2019239430A1 true WO2019239430A1 (en) | 2019-12-19 |
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ID=68842033
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/IN2019/050449 WO2019239430A1 (en) | 2018-06-13 | 2019-06-12 | Biomonitoring device for detection and monitoring of cancer and a method thereof |
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| WO (1) | WO2019239430A1 (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100196909A1 (en) * | 2009-01-29 | 2010-08-05 | Mathias Ziegler | Parp-based cytochemical and histochemical detection methods and kits therefore |
| CN104614524A (en) * | 2015-03-01 | 2015-05-13 | 河南省科隆医疗器械有限公司 | Test strip for rapid cervical cancer HPV detection device |
-
2019
- 2019-06-12 WO PCT/IN2019/050449 patent/WO2019239430A1/en active Application Filing
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100196909A1 (en) * | 2009-01-29 | 2010-08-05 | Mathias Ziegler | Parp-based cytochemical and histochemical detection methods and kits therefore |
| CN104614524A (en) * | 2015-03-01 | 2015-05-13 | 河南省科隆医疗器械有限公司 | Test strip for rapid cervical cancer HPV detection device |
Non-Patent Citations (1)
| Title |
|---|
| "ADP-ribose polymer - a novel and general biomarker of human cancers of head & neck, breast, and cervix", MOLECULAR CANCER, October 2010 (2010-10-01), XP021078024 * |
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