APPARATUS AND METHOD FOR DETECTING CANINE CANCER

Abstract

It has been found that canine ECPKA protein secrets in a high level and an autoantibody against the canine ECPKA protein is formed in dogs with cancer. It is also found that human ECPKA does not selectively bind to a canine ECPKA autoantibody and cannot serve as a biomarker. In addition, canine ECPKA autoantibody detection can be used as a meaningful diagnosis tool for cancer in dogs only when quantitative measurement of such antibodies is adapted. When the measurement of canine ECPKA autoantibody is not conclusive, measuring CRP can provide supplemental data that can be used to improve the predictability of the canine ECPKA autoantibody measurement.

Claims

1. A lateral flow kit for quantitatively detecting an antibody of canine ECPKA in blood of a dog, comprising: a first solid phase having an immobilized purified recombinant canine PKA C protein wherein the recombinant canine PKA C protein has an amino acid sequence comprising SEQ ID NO. 005, a conjugate pad comprising a conjugated coloring agent with an optical density wherein the conjugated coloring agent is conjugated with a first binding protein, and a second solid phase comprising a second binding protein, wherein the conjugated coloring agent is configured to provide a first color intensity in the first solid phase and a second color intensity in the second solid phase, and wherein the first color intensity quantitatively depends on the concentration of the antibody in the blood of the dog and the second color intensity is independent of the concentration of the antibody

2. The lateral flow kit according to claim 1, wherein the optical density of the conjugated coloring agent is between 5-30.

3. The lateral flow kit according to claim 1, wherein the optical density of the conjugated coloring agent is between 10-20.

4. The lateral flow kit according to claim 1, wherein the lateral flow kit detects cancer in the dog with a specificity of at least about 80% and a sensitivity of at least about 80%.

5. The lateral flow kit according to claim 1, wherein the first color intensity provides the concentration of the antibody by comparing the first color intensity with a set of correlating data between the first color intensity and the concentration of the antibody.

6. The lateral flow kit according claim 5, wherein a portion of the set of correlating data can be expressed approximately by a linear equation, which allows determination of the concentration when the first color intensity does not exactly match with any of the set of correlating data.

7. The lateral flow kit according claim 1, wherein the first binding protein is selected from a group of streptavidin, biotin, protein A, anti-canine IgG Rat, anti-canine IgG Rabbit, anti-canine IgG goat, anti-canine IgG sheep and a protein capable of binding to the antibody.

8. The lateral flow kit according to claim 1, wherein the second binding protein is selected from a group of streptavidin, biotin, protein A, anti-rat IgG, anti-Rabbit IgG, anti-goat IgG, anti-sheep IgG and a protein capable of binding to IgG.

9. The lateral flow kit according to claim 1, wherein the coloring agent is selected from a group of gold, latex, gfp, fitc, and a UV active conjugating agent.

10. The lateral flow kit according to claim 1, further comprising a filter phase to filter blood cells.

11. A method of determining a concentration of an antibody of canine ECPKA in blood of a dog, comprising: (a) preparing a test sample from the blood of the dog for a lateral flow kit, (b) applying the test sample to the lateral flow kit, (c) allowing the lateral flow kit to develop, (d) obtaining a digital information by taking a digital picture of the developed lateral flow kit including the test line, and (e) obtaining the concentration of the antibody wherein the concentration is determined by comparing the digital information with a set of data correlating a digital value with a concentration of the antibody wherein the lateral flow kit comprises a first solid phase having an immobilized purified recombinant canine PKA C protein wherein the recombinant canine PKA C protein has an amino acid sequence comprising SEQ ID NO. 5, a conjugate pad comprising a conjugated coloring agent with an optical density wherein the conjugated coloring agent is conjugated with a first binding protein, and a second solid phase comprising a second binding protein, wherein the conjugated coloring agent is configured to provide a first color intensity in the first solid phase and a second color intensity in the second solid phase wherein the first color intensity quantitatively depends on the concentration of the antibody in the blood of the dog and the second color intensity is independent of the concentration of the antibody

12. The method according to claim 11, further comprising determining a likelihood that the dog has a cancer.

13. The method according to claim 11, further comprising sending the digital information is sent to an external server via a wireless communication.

14. The method according to claim 11, wherein the obtaining the digital information is conducted using a reader box comprising a wireless communication module, a camera module, a light source, and a slot designed to accommodate the lateral flow kit.

15. The method according to claim 14 wherein the wireless communication module operates based on a short range wireless communication protocol.

16. The method according to claim 11, wherein the optical density is higher than 5.

17. The method according to claim 11, wherein the optical density is higher than 10.

18. The method according to claim 11 wherein the method detects cancer in the dog with a specificity of at least about 80% and a sensitivity of at least about 80%.

19. The method according to claim 11, wherein the concentration of the antibody is extrapolated using a linear equation with an R.sup.2 value higher than 0.9.

20. A method of determining a concentration of an antibody of ECPKA in blood of a mammal, comprising: (a) preparing a test sample from the blood of the mammal for a lateral flow kit comprising; (b) applying the test sample to the lateral flow kit; (c) allowing the lateral flow kit to develop; (d) obtaining a digital information by taking a digital picture of the developed lateral flow kit; and (e) obtaining the concentration of the antibody wherein the concentration is determined by comparing the digital information of the test line with a set of data correlating a digital value obtained from the digital picture with a concentration of the antibody wherein the lateral flow kit comprises a first solid phase having an immobilized purified recombinant mammal PKA C protein wherein the recombinant mammal PKA C protein has an amino acid sequence, a conjugate pad comprising a conjugated coloring agent with an optical density of 5 or higher wherein the conjugated coloring agent is conjugated with a first binding protein, and a second solid phase comprising a second binding protein, wherein the conjugated coloring agent is configured to provide a first color intensity in the first solid phase and a second color intensity in the second solid phase, and wherein the first color intensity quantitatively depends on the concentration of the antibody in the blood of the dog and the second color intensity is independent of the concentration of the antibody.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0038] FIG. 1 shows comparison between amino acid sequences of a subunit of human ECPKA and a subunit of canine ECPKA.

[0039] FIG. 2 shows comparison between mRNA sequences of a subunit of human ECPKA and a subunit of canine ECPKA.

[0040] FIG. 3 illustrates canine ECPKA autoantibody measurements of normal dogs, dogs with benign tumors, dogs with tumors and dogs that have been surgically treated for cancer.

[0041] FIG. 4 shows a illustrative receiver operating characteristic graph where A is the area under the ROC curve.

[0042] FIG. 5 illustrates an ROC curve of a cancer detection method according to an embodiment of the invention.

[0043] FIG. 6 illustrates relationships between CRP level in a plasma and various tested subjects.

[0044] FIG. 7 illustrates relationships between CRP level in a plasma and various tested subjects.

[0045] FIG. 8 illustrates correlation between CRP measurements and canine ECPKA autoantibodies level.

[0046] FIG. 9 illustrates correlations between human ECPKA and canine ECPKA in detecting cancers of dogs.

[0047] FIG. 10 illustrates correlations between color intensity obtained using one embodiment lateral flow kit and the concentration of the antibody in a sample wherein the correlation is approximately leaner with R.sup.2 value higher than 0.9.

[0048] FIG. 11 illustrate the top view of a reader according to one embodiment.

[0049] FIG. 12 illustrate the bottom plate inside of a reader according to one embodiment.

[0050] FIG. 13 illustrate a side view of a reader according to one embodiment.

[0051] FIG. 14 illustrate a side view of a reader according to one embodiment.

[0052] FIG. 15 illustrate an see-through view of a reader according to one embodiment.

[0053] FIG. 16 illustrate an see-through view of a reader according to one embodiment.

[0054] FIG. 17 illustrates test results of a kit according to one embodiment.

[0055] FIG. 18 illustrates an ROC curve of test results of a kit according to one embodiment.

[0056] FIG. 19 summarizes test results of a kit according to one embodiment.

DETAILED DESCRIPTION OF THE INVENTION

[0057] Through extensive researches, it has been found that canine ECPKA protein secrets in a high level and an autoantibody against the canine ECPKA protein is formed in dogs with cancers. It is also found that human ECPKA does not selectively bind to a canine ECPKA autoantibody and cannot serve as a biomarker. In addition, canine ECPKA autoantibody detection can be used as a meaningful diagnosis tool for cancer in dogs only when quantitative measurement of such antibodies is adapted. When the measurement of canine ECPKA autoantibody is not conclusive, measuring CRP can provide supplemental data that can be used to improve the predictability of the canine ECPKA autoantibody measurement as described further below.

[0058] FIG. 1 is alignment of amino acid sequences of PKA C from human (NP_002721.1), dog (NP_001003032.1) and cat (XP_006928552.1). Dot boxes indicates amino acid residues showing difference between human and dog. While the amino acid sequences of human and dog share highly similarities but there are four different sequences are spread over the entire sequences including the c-terminus.

[0059] FIG. 2 compares mRNA sequences encoding PKA C from human (NP_002721.1), dog (NP_001003032.1) and cat (XP_006928552.1). There are a large number of differences in between mRNA sequences of human and dog.

[0060] FIG. 3 illustrates the test results of ECPKA autoantibody measurements in various test subjects: dogs (Cancers or Cancer) diagnosed as having cancer, dogs (Benign tumor) with benign tumor, dogs (Control or Non tumor disease) with no tumor disease, and dogs (Tx or Treatment). As shown in FIG. 3, the test subjects with cancers show much a higher level of ECPKA autoantibody measurement comparing to the other test subjects. Dogs that had tumor diseases but were treated through surgery show low levels of ECPKA autoantibody measurements. Table 1 summarizes total numbers of the test subjects and test results. For categorizing the test results, the positive results were counted when the ECPKA autoantibody is detected 41 unit and higher, and the negative results were determined when the level of the ECPKA autoantibody detection is less than 41 unit.

TABLE-US-00001 TABLE 1 Cancer Control Benign Tumor Treatment Positive 81 25 2 0 Negative 1 160 23 19 Total 93 185 25 19

[0061] Table 2 summarizes the sensitivities, specificity, positive predictive value, and negative predictive value for the canine ECPKA autoantibody detection as a cancer diagnosis method.

TABLE-US-00002 TABLE 2 Selectivity .sup.87% Positive Predictive Value .sup.75% Specificity 88.4% Negative Predictive Value 94.4%

[0062] There are many ways to verify or evaluate accuracy of a particular test method. Among those, sensitivity and specificity are commonly used. In general, the sensitivity and specificity means how good a method is in distinguishing between the targeted result and untargeted result. For example, in our case, the sensitivity means how well cancers in dogs can be found by using the detection of the autoantibody of canine ECPKA. On the other hand, the specificity relates to how well the method could distinguish dogs with cancer from dogs without cancer. In addition to the sensitivity and specificity, Receiver Operating Characteristics (ROC) curve is often used to determine usefulness and cut-off value of a method. The ROC curve is drawn using the rate of false positive in x axis value and the rate of true positive in y axis value. Whether a particular test method is accurate or not can be measured by using the ROC curve. In the below illustrative ROC curve, in which the positive proportion is plotted against the false positive proportion for various possible settings of the decision criterion, the area under the ROC curve (AUC) of 1 means that the test method is perfect and accurate but if the AUC is 0.5, the test method is useless and inaccurate. As the curve is closer to the upper left corner, the test method is more accurate and more useful. Typically, it is treated that a test method is not informative when the AUC is 0.5, is not so accurate when the AUC is between 0.5 and 0.7, is accurate when the AUC is between 0.7 and 0.9, and is very accurate when the AUC is 0.9 and 1. Accordingly, the ROC curve and its AUC value are a good tool to determine and evaluate a new test method. See Using the Receive Operating Characteristic (ROC) Curve to Measure Sensitivity and Specificity, Korean J. Fam. Med. Vol. 30, No. 11, November 2009, 30:841-842.

[0063] FIG. 5 shows a ROC curve of the canine ECKPA autoantibody measurement for detecting cancer of dogs. The AUC value is 0.9061, which indicates that the canine ECKPA autoantibody measurement of one embodiment of the present invention is a highly effective and accurate diagnostic tool.

[0064] FIG. 6 shows measurements of C-reactive protein (CRP) in various test subjects: dogs with cancer, dogs with benign tumors, dogs with no tumor diseases, dogs with non-cancer diseases. FIG. 7 shows measurements of CRP in dogs with cancer (Cancer), dogs with false native results in the canine ECKPA autoantibody measurement (FN), dogs with benign tumors (BT), dogs without cancer (Negative) and dogs with false positive results in the canine ECKPA autoantibody measurement (FP).

[0065] FIG. 8 plots the CRP measurement results against the canine ECKPA autoantibody measurement results of the various test subject groups. Table 3 show negative predictive values and positive predictive values when the CRP and the ECKPA autoantibody measurements are used together. The positive predictive values increase to 91% and 100% from the overall positive predictive value of 73.6% in the areas of C and E, and the negative predictive value improves to 97.6% in the area of C from the overall negative predictive value of 93.4%. Thus, measurement of CRP can improve the accuracy of the cancer diagnosis using the canine ECKPA autoantibody measurement.

TABLE-US-00003 TABLE 3 Benign Area NPV (%) PPV (%) Cancer Tumor Non-tumor A 62 38 8 4 9 B 97.6 2.4 4 19 138 C 9 91 20 0 2 D 50 50 25 2 23 E 0 100 13 0 0 F 11 89 16 0 2

[0066] FIG. 9 is a chart that the test results of dogs based on human ECPKA is plotted against the test results of dog based on canine ECPKA. When the r.sup.2 value is 1, the test results closely correlate to each other and human ECPKA or its autoantibodies can be used to detect cancer in dogs. However, as shown in Fig. x, the r.sup.2 value is 0.15, suggesting there is not much correlation between human ECPKA and Dog ECPKA and human ECPKA or its autoantibodies is not a good biomaker to detect cancers of dogs.

[0067] The table 4 summarizes various cancer detected in the test subjects. It is also found that the cancer detection method of one embodiment of the present invention can be used regardless of the cancer type, which makes the detection method unique and highly useful.

TABLE-US-00004 TABLE 4 Tumor type (Number) Diagnosis (Number) Carcinoma (57) Mammary gland Carcinoma (14) TCC (11) Hepatic carcinoma (9) Adenocarcinoma (5) SCC (5) Other carcinoma (13) Hematopoietic cancer (2) Lymphoma (14) Mast cell tumor (5) Leukemia (1) Sarcoma (16) Hemangiosarcoma (5) Melanoma (4) Soft tissue sarcoma (4) Other sarcoma (3) Benign (25) Adenoma (9) Lipoma (5) Histiocytoma (2) Benign mixed tumor (2) Other benign tumors (7)

[0068] The lateral flow kit structure may follow typical lateral flow kit structures. However, the coloring agent and proteins used are specially designed to detect the antibody of the canine ECPKA in a quantitative way. The kit has an application place where a test sample is applied. The test sample is typically prepared from blood of a test subject. Serum obtained from the blood is applied to the application place. The serum sample flows along the test strip and passes through a conjugate pad where a conjugated coloring agent is placed. The coloring agent is applied with pressure and baked in oven. Typical optical density used in lateral flow kits is around 2-3. However, a much higher optical density is used to obtain better correlation between the digitized test result of the expression and the concentration data. Various coloring agent can be used. Gold and latex are typical coloring agent. For UV active coloring agents include gfp, fitc, and UV active conjugating agents. The color agent exhibits different color intensity. The inherent intensity affects the digitization. FIG. 10 shows an example correlation between the color intensity received after testing using a lateral flow kit according to one embodiment and the concentration of the antibody.

[0069] The antibody of the canine ECPKA and other antibodies in the sample will bind to the conjugated first binding protein, effectively coating the antibodies with the coloring agent. The first binding protein may be selected from a group of streptavidin, biotin, protein A, anti-canine IgG Rat, anti-canine IgG Rabbit, anti-canine IgG goat, anti-canine IgG sheep and other protein capable of binding to the antibody. When the sample passes through the first solid phase of the kit, which contains an immobilized purified recombinant canine PKA C protein, the antibody of the canine ECPKA binds to the immobilized purified recombinant canine PKA C protein. After the complete development of the kit, only the antibody of the canine ECPKA stays in the first solid phase, exhibiting a color intensity, which can be used to find the corresponding concentration of the antibody. The expressed test result is digitized by take a digital camera and the digitized information is compared with the correlation data to determine the actual concentration. The digitized information uses color intensity to obtain a digital expression value. The embodiment shown in FIG. 10 provides a linear relationship between the digitized expression value and the concentration. Thus, it allows extrapolation of a concentration for which matching data does not exist in the data set. For easier extrapolation, the color intensity of the coloring agent for various concentrations of the antibody is desirable to provide a linear relationship.

[0070] Types of camera, color temperature setting, distance between the sample and camera, light source and exposure setting would affect the digitized value of the color intensity. Thus, it is desirable to have the digitization of the expressed color intensity is desirably done in a constant condition. FIGS. 11-16 shows a reader box 100, which has a housing 200. A camera module 300, monitoring and controlling unit 400 and bar code scanner 500 are provided on the top surface of the housing. Inside the reader, there is a slot 600 where the kit is placed through the opening 900. With the internal lighting system 1000 such as LED, the lighting inside of the reader is controlled to optimize to provide a constant condition for the digitization. When the digital camera module takes a picture of the kit on the slot, the digital image is transferred to a server via the wireless communication module 100. The wireless communication module uses a short distance communication protocol, allowing the reader to connect to a local internet portal such as WI-FI hot spot. The wavelength of the lighting source can be adjusted for various coloring agents such as UV active coloring agents. Because the reader has own communication module, it can operate independent of a mobile network.

[0071] FIGS. 17-19 shows test results of a kit according to an embodiment where the kit shows sensitivity of 84.7% and specificity of 84.4%.

EXAMPLES

Cloning of Canine Cyclic AMP-Dependent Protein Kinase Catalytic Subunit C (PKA C)

[0072] Total RNAs were isolated from canine adipose tissue homogenized in Trizol reagent (Invitrogen) using RNeasy columns (Qiagen). 1 g of total RNA was then reverse transcribed to cDNA with oligo (dT) primers using Improm-II Reverse Transcription System (Promega) according to manufacturer's instructions. The canine PKA C cDNA was amplified by polymerase chain reaction (PCR) using exTaq polymerase (Takara) and the canine PKA C primers containing restriction enzyme recognition sites, NcoI and XhoI. The sequences of primers are as follows: forward, AAT CCA TGG GCA ACG CCG CCG CCA AGA AGG GCA G and reverse, GCC GTC GAC GAA CTC ACA AAA CTC CTT GCC ACA CTT C.

[0073] The amplified cDNA fragments of canine PKA C was then digested with restriction enzymes, NcoI and XhoI (Takara), inserted into pET-22b(+) plasmid (Novagen), a bacterial expression vector and sequenced with T7 promoter and terminator primers (T7 promoter primer, AAT ACG ACT CAC TAT AGG and T7 terminator primer, GCT AGT TAT TGC TCA GCG G).

Purification of Recombinant Canine PKA C

[0074] pET-22b(+) plasmid encoding canine PKA C tagged with six histidine residues (6-His Epitope) at the C-terminus was introduced into Escherichia coli strain, BL21(DE3) and the expression of canine PKA C was induced with 1 mM Isopropyl -D-1-thiogalactopyranoside (IPTG) at room temperature for overnight. Cells were harvested, resuspended in 50 mM Tris.HCl (pH 7.4) containing 0.2M NaCl and sonicated. Recombinant canine PKA C was then purified with two sequential immobilized metal affinity chromatography using IDA Excellose resin (Bioprogen) followed by ion exchange chromatography using SP Sepharose resin (GE healthcare). The eluted recombinant protein was dialyzed and stored at a concentration of 1 mg/ml in 50 mM Tris.HCl (pH 7.4) supplemented with 0.15M NaCl and 1% sucrose at 80 C. until further use.

Western Blotting

[0075] 50 ng of purified recombinant canine PKA C and human PKA C, a positive control, proteins were separated on 10% SDS PAGE gel, transferred onto PVDF membrane and sequentially probed with rabbit polyclonal anti-PKA C antibody (Abcam) and goat anti-rabbit IgG antibody conjugated with horse radish peroxidase (HRP) (Bethyl Laboratories) followed by immunodetection with enhanced chemiluminescence (Pierce).

Enzyme-Linked Immunosorbent Assay (ELISA) Assay

[0076] The presence and level of extracellular PKA C in canine serum was assessed with anti-canine PKA C ELISA kit (Genorise) following the manufacturer's instructions. Briefly, 100 l of 4-fold diluted canine serum samples in reagent diluent was added to the 96 well ELISA plates precoated with anti-canine PKA C antibodies and incubated for 1 hr at room temperature. The plates were further incubated with canine PKA C detection antibodies for 1 hr at room temperature followed by incubation with HRP conjugate for 20 min at room temperature. The plates were then developed with substrate solution for 10 min at room temperature and the reaction was stopped with 50 l stop solution. The absorbance was determined at 450 nm with a scanning multi-well spectrophotometer (Molecular Device).

[0077] Autoantibodies against extracellular PKA C in canine serum were measured using solid phase ELISA method. Briefly, 96 well polystyrene ELISA strip plates (Santa Cruz) were coated with 100 l of recombinant canine PKA C diluted at 1 g/ml in carbonate coating buffer (pH 9.6) (Sigma) for overnight at room temperature, washed once with PBS containing 0.1% Tween 20 (pH 7.4), blocked with 1% bovine serum albumin (BSA) in PBS for 2 hrs at room temperature and washed twice with washing buffer (50 mM sodium citrate supplemented with 0.15M NaCl and 0.1% Tween 20 (pH 5.2)). The plates were then incubated with 100 l of canine serum samples diluted at 1:500 in sample dilution buffer (PBS containing 0.25% BSA and 0.05% Tween 20 (pH 7.4)) for 1 hr at room temperature, washed four times with washing buffer, further incubated with 100 l of goat anti-canine IgG antibody (Abcam) conjugated with HRP diluted at 1:20,000 in sample dilution buffer for 1 hr at room temperature, washed five times with washing buffer, and developed with 100 l of 3,3,5,5-Tetramethylbenzidine (TMB) Liquid Substrate solution (Thermo-Fisher) for 15 min at room temperature. The reaction was then stopped with 50 l of 2N H.sub.2SO.sub.4 solution and the absorbance was measured at 450 nm using a scanning multi-well spectrophotometer.

Detection of Extracellular PKA C in Sera from Dogs with Malignant Tumors

[0078] It has been shown that extracellular PKA produced by cancer cells is markedly increased in the sera of cancer patients and elicits the generation of autoantibodies against this it in those patients. In addition, it was reported that the titer of autoantibodies for PKA in serum is significantly correlated with the presence of cancers of various cell types. Hence, the autoantibody against PKA is considered as a novel potential biomarker for diagnosis of cancers in human. However, the presence of PKA autoantibody and its correlation with cancer have never been determined in mammals other than human.

Detection of Autoantibodies against Extracellular PKA C in Sera from Dogs with Malignant Tumors

[0079] To determine whether the titer of PKA C autoantibodies is positively correlated with cancer of various cell types in dogs as shown in humans, first, we cloned canine PKA C gene from canine adipose tissues, bacterially expressed and purified the recombinant protein tagged with 6-His Epitope (Figure). The presence and titer of PKA C autoantibodies were then assessed with ELISA assay using the purified canine PKA C protein as an antigen.

Preparation of a Lateral Flow Kit

[0080] ECPKA (0.5-4 mg/ml) and control protein (1-2 mg/ml) were diluted protein buffer was dispensed on nitrocellulose membrane by Biodot low volume precision dispensing equipment. The nitrocellulose membrane is dried overnight under 10% humidity. A sample pad is dipped into a sample pad butter and is dried at 37 C. for overnight under 15% humidity. A suspension of high density gold particle conjugated with a protein for the detection of canine IgG is sprayed with pressure into sliced conjugate pad at a room temperature to obtain a high optical density. The conjugation pad was then dried for overnight at 25 C. under 10% humidity. The kit was assembled by putting nitrocellulose membrane on a backing pad, putting the sliced conjugation pad filled with gold particle on backing pad which has to be overlapping with nitrocellulose membrane in front area, putting the sample pad over conjugation pad, putting an adsorption pad overlapping with nitrocellulose membrane in tail part, cutting the assembled backing pad by 4 mm wide and putting the cut assembled backing pad into a housing.

[0081] Since the above embodiments are described only for examples, the present invention is not limited to the above embodiments and various modifications or alterations can be easily made therefore by those skilled in the art without departing from the scope of the present invention.