Method of fibrin / fibrinogen degradation product detection in companion animals

Abstract

Two methods for quantifying fibrin / fibrinogen degradation products ("FDPs") in blood samples. The first method involves obtaining a blood sample from a non-human animal, centrifuging the blood sample to obtain a first supernatant and collecting the first supernatant, centrifuging the first supernatant to obtain a second supernatant and collecting the second supernatant, diluting the second supernatant, contacting the diluted second supernatant with a reagent containing an antibody specifically binding to FDP, and detecting the amount of the antibody specifically binding to FDP, thereby quantifying the FDP in the blood sample. The second method involves subjecting the diluted second supernatant to a quantitative immunoassay specifically detecting FDP, thereby quantifying the amount of FDP in the non-human blood sample.

Description

[Technical Field]

[0001] The present invention relates to a method for detecting biomarkers in companion animals. [Previous Technology]

[0002] Background of the Invention

[0003] Like humans, companion animals live longer due to advancements in pharmaceuticals, preventative care, and improved nutrition. The longer a pet lives, the more likely it is to develop age-related diseases, particularly cancer. An estimated 6 million dogs and nearly 6 million cats are diagnosed with cancer each year.

[0004] There are nearly 100 types of cancer in animals. Cancer can be found in the skin, bones, breasts, head and neck, lymphatic system, abdomen, and testes of pets. Leukemia is the most common type of cancer in cats, while lymphoma and mammary cancer are the most common types of cancer in dogs.

[0005] Compared to other companion animals, dogs are affected by more forms of cancer. According to the US Veterinary Cancer Society, cancer is the leading cause of death in 47% of dogs, especially those over ten years old.

[0006] Many biomarkers present in human serum are known to be associated with cancer, such as carcinoembryonic antigen (CEA) and fibrin / fibrinogen degradation products. Certain biomarkers have been identified as predicting the progression of human cancer. However, research on cancers in non-human animals remains sporadic.

[0007] There is a need to develop methods to detect biomarkers associated with cancer in animals so that appropriate treatment can be administered at an earlier stage of cancer, thereby improving survival rates. [Summary of the Invention]

[0008] Summary of the Invention

[0009] To meet this need, a method for quantifying fibrin / fibrinogen degradation products ("FDP") in a blood sample is provided. The method is performed by: obtaining a blood sample from a non-human animal; centrifuging the blood sample to obtain a first supernatant and collecting the supernatant; centrifuging the first supernatant to obtain a second supernatant and collecting the second supernatant; diluting the second supernatant; contacting the diluted second supernatant with a reagent containing an antibody specifically binding to FDP; removing antibodies not bound to FDP; and detecting the amount of antibodies specifically bound to FDP, thereby quantifying the FDP in the blood sample.

[0010] A second method for quantifying FDP in a blood sample is also provided. This method is achieved by obtaining a blood sample from a non-human animal, centrifuging the blood sample to obtain a first supernatant and collecting the supernatant, centrifuging the first supernatant to obtain a second supernatant and collecting the second supernatant, diluting the second supernatant, and subjecting the diluted second supernatant to a quantitative immunoassay specifically detecting FDP to quantify the FDP in the blood sample.

[0011] Details of several embodiments of the present invention are set forth in the following description. Other features, objectives, and advantages of the present invention will become apparent from the description, the drawings, and the appended claims.

Implementation Method

[0013] Detailed Description of Preferred Embodiments

[0014] As outlined above, a first method for quantifying FDP in blood samples is disclosed. This method is characterized by a step of centrifuging a non-human blood sample to obtain a first supernatant.

[0015] Preferably, the non-human blood sample is derived from a mammal, such as a dog, cat, rabbit, guinea pig, ferret, or pig. The blood can be obtained by any method known in this art.

[0016] In a particular first method, anticoagulated blood may be used. For example, fresh blood may be treated with ethylenediaminetetraacetic acid or sodium citrate to prevent clotting. The claimed invention does not require the non-human blood sample to be anticoagulated, as coagulated whole blood may also be used.

[0017] The non-human blood sample is centrifuged to remove blood cells, thereby obtaining a first supernatant. Centrifugation may be performed at 10×g to 2,000×g (e.g., 10×g, 50×g, 100×g, 250×g, 500×g, 1000×g, 1250×g, 1500×g and 2000×g) for 30 seconds to 30 minutes (e.g. 30 seconds, 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes and 30 minutes).

[0018] After collecting the first supernatant, for example by dispensing from the top of the hemoglobin aggregate, the first supernatant is centrifuged again to obtain the second supernatant. This second centrifugation step is performed at 1,000×g to 50,000×g (e.g., 1,000×g, 2,500×g, 5,000×g, 10,000×g, 11,000×g, 12,000×g, 13,000×g, 14,000×g, 15,000×g, 16,000×g, 17,000×g, 18,000×g, 19,000×g, 20,000×g, 25,000×g, 30,000×g, 35,000×g, 40,000×g, 45,000×g and 50,000×g) for 30 seconds to 30 minutes (e.g. 30 seconds, 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes and 30 minutes).

[0019] After collecting the second supernatant, it is prepared by diluting it with an appropriate diluent for immunoassay. For example, the second supernatant can be diluted with phosphate-buffered saline ("PBS").

[0020] The second supernatant can be diluted at a ratio of 1:5 to 1:10,000, more preferably 1:50 to 1:5,000, and even more preferably 1:100 to 1:1,000. Acceptable dilutions may be, for example, 1:5, 1:25, 1:50, 1:100, 1:200, 1:300, 1:400, 1:500, 1:750, 1:1,000, 1:1,500, 1:2,000, 1:2,500, 1:3,000, 1:3,500, 1:4,000, 1:4,500, 1:5,000, 1:7,500, and 1:10,000.

[0021] Next, the diluted second supernatant is placed in contact with a reagent containing an antibody that specifically binds to FDP.

[0022] Developing antibodies that specifically bind to FDP is within the skill of a person skilled in the art. For example, rabbits can be inoculated with FDP isolated from animals of interest, such as dogs, to obtain polyclonal sera containing antibodies that specifically bind to FDP. Mice can be inoculated with FDP and spleen cells can be isolated from positively reacting animals to obtain monoclonal antibodies against FDP. In a particular instance, some antibodies that bind to human FDP cross-react with FDP from other species. Thus, polyclonal or monoclonal antibodies against human FDP can be used as reagents.

[0023] The above method requires placing the diluted second supernatant in contact with the reagent. This can be achieved, for example, by adsorbing the diluted second supernatant onto the surface of a plate and exposing the plate surface to a reagent containing anti-FDP antibody. In another example, the diluted second supernatant and reagent are mixed in a test tube.

[0024] After allowing sufficient time for the anti-FDP antibody in the reagent to bind with the FDP in the diluted second supernatant, the unbound antibody is removed. For example, if the diluted second supernatant is adsorbed onto a plate, the unbound anti-FDP antibody can be removed by washing with a suitable cleaning solution (e.g., PBS / polysorbate-20). Alternatively, if the diluted second supernatant is mixed with the reagent in a test tube, the unbound antibody can be separated and removed from the bound antibody by adding a second antibody that also binds to FDP and capturing the second antibody.

[0025] As mentioned above, the method includes a step of detecting the amount of antibody specifically bound to FDP after the contact step. The anti-FDP antibody in the reagent may be fluorescently or enzyme-labeled, and the amount of anti-FDP antibody bound to FDP may be measured, for example, by fluorescence intensity (if using a fluorescently labeled antibody) or by colorimetry (if using an enzyme-labeled antibody). Any method known in this art for detecting specifically bound anti-FDP antibodies may be used.

[0026] The amount of FDP in the second supernatant can be quantified in various ways. In one particular example, the amount of anti-FDP antibody bound to a standard of purified FDP at different dilutions is compared with the amount detected in the second supernatant.

[0027] For comparative purposes, the above method can be performed on blood samples from several animals of the same species. For example, the method can be used to determine the FDP level in blood samples from dogs without cancer to establish a baseline. The same method can be used to determine the FDP level in blood samples from dogs with cancer to determine whether these levels differ from those in animals without cancer.

[0028] A second method for quantifying FDP in a blood sample is mentioned in the Summary of the Invention. This second method shares certain steps with the first method described in detail above. Similar to the first method, the second method requires the following steps: (i) obtaining a blood sample from a non-human animal, (ii) centrifuging the blood sample to obtain a first supernatant, (iii) collecting the first supernatant, (iv) centrifuging the first supernatant to obtain a second supernatant, (v) collecting the second supernatant, and (vi) diluting the second supernatant. The second method is characterized by subjecting the diluted second supernatant to a quantitative immunoassay specifically detecting FDP, thereby quantifying the amount of FDP in the blood sample.

[0029] Similar to the first method described above, the second method does not require anticoagulation of non-human blood samples. Clotted whole blood can be used.

[0030] The centrifugation and dilution steps in the second method are performed in the same manner as in the first method. To reiterate, non-human blood samples are centrifuged at 10×g to 2,000×g for 30 seconds to 30 minutes to obtain a first supernatant. The first supernatant is then centrifuged at 1,000×g to 50,000×g for 30 seconds to 30 minutes to obtain a second supernatant, which is then diluted at a ratio of 1:5 to 1:10,000 for quantitative immunoassay.

[0031] Quantitative immunoassay may include, but is not limited to, enzyme-linked immunosorbent assay ("ELISA"), enzyme multiplication immunoassay, quantitative immunohistochemistry, radioimmunoassay, immunofluorescence assay, flow cytometry, protein microarray assay, electrochemiluminescence assay, microbead assay, microfluidic immunoassay, or surface plasma resonance immunoassay.

[0032] In one specific example, the quantitative immunoassay is an ELISA using multiple rabbit serums containing antibodies that specifically bind to FDP. In another example, the quantitative immunoassay is an ELISA using a monoclonal antibody that specifically binds to FDP. The ELISA may be a sandwich ELISA using a second anti-FDP antibody to increase sensitivity.

[0033] Finally, in the second method, the non-human blood sample is from a mammal, such as a dog, cat, rabbit, guinea pig, ferret, or pig.

[0034] Without further detailed description, it is believed that those skilled in the art can make full use of the disclosure herein. Therefore, the following specific examples should be understood as descriptive only and in no way limit the remainder of this disclosure. Example 1: Preparation of blood samples

[0035] Blood samples were obtained from 262 dogs with cancer confirmed by cytology and / or histopathology. Blood samples were also obtained from 54 healthy animals. In all cases, informed consent was obtained from the pet owners for the use of clinical data for research purposes.

[0036] Whole blood was collected in test tubes treated with an anticoagulant (i.e., ethylenediaminetetraacetic acid). Immediately after collection, the tubes were centrifuged at 1500 RPM for 10 minutes, and then the supernatant was removed and centrifuged at 16,000 × g for 10 minutes. The resulting supernatant was diluted 1:200 in PBS for immunoassay. Example 2: Enzyme-linked immunosorbent assay ("ELISA")

[0037] Rabbit sera were generated against the DR-70 antigen (an antigen resulting from the combination of fibrin and FDP). FDP in the blood samples was quantitatively measured using an anti-DR-70 antigen antibody in a sandwich ELISA assay as described below.

[0038] Affinity-purified anti-DR-70 rabbit serum was spread onto the wells of a 96-well plate and then incubated for 30 minutes at room temperature with 100 µL of diluted blood sample. After washing the wells, horseradish peroxidase ("HRP")-bound anti-DR70 antibody was added to each well, incubated for 30 minutes, and washed. The HRP-bound anti-DR70 antibody was detected by adding tetramethylbenzidine receptor and stopping the reaction with 0.1 N HCl after sufficient color development. The color intensity was measured at 450 nm using a micro-quantitative disc reader. The DR-70 concentration was calculated by comparing it with a standard curve obtained from purified DR-70. The results are shown in Figure 1A.

[0039] The median DR-70 concentration in blood samples from healthy animals was 1.163 µg / mL (0.1010–1.775 µg / mL; n=54). The median DR-70 concentration in blood samples from dogs with cancer was 2.132 µg / mL (0.3495–4.355 µg / mL; n=262), which was significantly higher than that from healthy animals (p<0.0001; Kruskal-Wallis test). Example 3: Diagnostic Practicality

[0040] The utility of using the DR-70 criterion for diagnosing cancer in dogs was tested by preparing recipient operating characteristic curves and calculating the area under the curve ("AUC"). The results are shown in Figure 1B. The AUC for DR-70 was 0.9185 (95% confidence interval 0.8876 to 0.9494; p < 0.0001), indicating that the DR-70 criterion can be used to easily distinguish dogs with cancer from healthy animals.

[0041] The optimal cutoff value for DR-70 blood concentration was determined by calculating the sensitivity and specificity at different cutoff values. The data are shown in Table 1 below. Table 1. Sensitivity and specificity of DR-70 at selected cutoff values DR-70 (μg / mL) Sensitivity (%) 95% CI Sensitivity (%) 95% CI > 1.492 85.50 80.72% to 89.25% 79.63 67.10% to 88.23% > 1.494 85.11 80.30% to 88.92% 79.63 67.10% to 88.23% > 1.497 84.73 79.88% to 88.58% 79.63 67.10% to 88.23% > 1.503 84.73 79.88% to 88.58% 81.48 69.16% to 89.62% > 1.508 84.35 79.46% to 88.25% 81.48 69.16% to 89.62% > 1.513 84.35 79.46% to 88.25% 83.33 71.26% to 90.98% > 1.518 83.97 79.04% to 87.92% 83.33 71.26% to 90.98% > 1.523 83.59 78.62% to 87.58% 83.33 71.26% to 90.98% > 1.528 83.21 78.21% to 87.25% 83.33 71.26% to 90.98% > 1.532 82.82 77.79% to 86.91% 83.33 71.26% to 90.98% > 1.536 82.44 77.38% to 86.57% 83.33 71.26% to 90.98% The optimal cutoff concentration for DR-70 is 1.513 µg / mL, with a sensitivity of 84.35% (95% CI: 79.46–88.25%) and a specificity of 83.33% (95% CI: 71.26–90.98%). Other embodiments

[0042] All features disclosed in this specification can be combined in any combination. Each feature disclosed in this specification can be replaced by an alternative feature used to achieve the same, equivalent or similar purpose. Unless otherwise expressly stated, each feature disclosed is merely an example of a series of general equivalent or similar features.

[0043] Based on the above description, those skilled in the art can easily determine the basic features of the present invention, and various changes and modifications can be made to the present invention to adapt it to various uses and conditions without departing from the spirit and scope of the present invention. Therefore, other embodiments are also within the scope of the following patent application. [Simplified Explanation of the Diagram]

[0012] The following description refers to the accompanying drawings, wherein: Figure 1A shows a graph of the expression levels of the DR-70 antigen in control dogs and dogs with tumors, and Figure 1B shows a recipient operating characteristic curve calculated from the data presented in Figure 1A and constructed from a series of cutoff values.

Claims

1. A method for quantifying fibrin / fibrinogen degradation products (FDP) in a blood sample, the method comprising: obtaining a blood sample from a non-human animal; centrifuging the blood sample at 10×g to 2,000×g to obtain a first supernatant; collecting the first supernatant; and centrifuging the first supernatant at 5,000×g to 50,000×g to obtain a second supernatant; Collect the second supernatant; dilute the second supernatant 1:5 to 1:10,000; contact the diluted second supernatant with a reagent containing an antibody that specifically binds to FDP; The method removes antibodies that do not bind to FDP and detects the amount of antibodies that specifically bind to FDP, thereby quantifying the FDP in the blood sample.

2. The method of claim 1, wherein the blood sample is anticoagulated.

3. The method of claim 1, wherein the non-human animal is a dog, a cat, a rabbit, a guinea pig, a ferret, or a pig.

4. The method of claim 2, wherein the anticoagulated blood sample is centrifuged for 30 seconds to 30 minutes.

5. The method of claim 4, wherein the first supernatant is centrifuged for 30 seconds to 30 minutes.

6. The method of claim 5, wherein the second supernatant is diluted 1:50 to 1:5,000 prior to the contact step.

7. The method of claim 6, wherein the second supernatant is diluted 1:100 to 1:1,000 prior to the contact step.

8. A method for quantifying fibrin / fibrinogen degradation products (FDP) in a blood sample, the method comprising: obtaining a blood sample from a non-human animal; centrifuging the blood sample at 10×g to 2,000×g to obtain a first supernatant; collecting the first supernatant; centrifuging the first supernatant at 5,000×g to 50,000×g to obtain a second supernatant; collecting the second supernatant; diluting the second supernatant at a ratio of 1:5 to 1:10,000; subjecting the diluted second supernatant to a quantitative immunoassay specifically detecting FDP, thereby quantifying the amount of FDP in the blood sample.

9. The method of claim 8, wherein the blood sample is anticoagulated.

10. The method of claim 8, wherein the quantitative immunoassay is an enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, immunofluorescence assay, flow cytometry, or microfluidic immunoassay.

11. The method of claim 10, wherein the quantitative immunoassay is an ELISA using a multi-strain rabbit serum containing an antibody that specifically binds to FDP.

12. The method of claim 10, wherein the quantitative immunoassay is an ELISA using a monoclonal antibody that specifically binds to FDP.

13. The method of claim 9, wherein the non-human animal is a dog, a cat, a rabbit, a guinea pig, a ferret, or a pig.

14. The method of claim 9, wherein the anticoagulated blood sample is centrifuged for 30 seconds to 30 minutes.

15. The method of claim 14, wherein the first supernatant is centrifuged for 30 seconds to 30 minutes.

16. The method of claim 15, wherein the second supernatant is diluted 1:50 to 1:5000 prior to the undergoing step.

17. The method of claim 16, wherein the second supernatant is diluted 1:100 to 1:1000 prior to the undergoing step.

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