Determination of non-human mammal tk1 protein levels

A non-human mammal, TK1 technology, applied in the field of kits for determining the TK1 protein level of non-human mammals, can solve the problem of no immunochemical method

Pending Publication Date: 2019-03-01
ALERTIX VETERINARY DIAGNOSTICS AB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Currently, there are no immunochemical methods available for the study of oncology in non-human mammals

Method used

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  • Determination of non-human mammal tk1 protein levels
  • Determination of non-human mammal tk1 protein levels
  • Determination of non-human mammal tk1 protein levels

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0119] A new TK1-ELISA was developed for the determination of serum TK1 (STK1 ) protein levels in dogs with hematological and solid tumors. Both STK1 activity and STK1 protein levels were measured in sera from healthy dogs, dogs with leukemia and lymphoma, and solid tumors. In addition, serum samples from six dogs with lymphoma were followed during treatment to test the monitoring efficiency of these two TK1 assays.

[0120] Materials and methods

[0121] serum sample

[0122] Serum samples from healthy dogs, dogs with haematological and solid tumors were collected and stored at the University Animal Hospitalat the Swedish University of Agricultural Sciences, Uppsala, Sweden At -20 °C until analysis. The project was approved by the Swedish Animal Ethics Committee. The study included samples from healthy dogs (n=30), dogs with hematological tumors (n=36: lymphoma, n=31, leukemia, n=5) and dogs with solid tumors (n=40) . Mean and median ages were 6 years (range 3 to 10 yea...

Embodiment 2

[0161] The following experiments demonstrate the reactivity of dog TK1 antisera raised to peptides from different regions of the dog and feline TK1 sequence. Except for a few amino acid differences, most of the peptide sequences of TK1 are conserved among human, dog, cat and equine TK1 sequences, see figure 1 . However, there is considerable divergence between the TK1 sequences at the C-terminus of the TK1 proteins.

[0162] TK1 antiserum

[0163] Two different polyclonal rabbit antisera were raised using peptides from the C-terminal region of dog TK1 (CTK1p-211, CTK1p-215) and one antiserum was raised from the active site region of dog TK1 (CTK1p-161) . The first canine TK1 peptide sequence (CTK1p-215) antiserum was directed against a synthetic peptide of 16 amino acids (amino acids 215-230 in dog TK1; figure 1 ) to which an N-terminal cysteine ​​(CGKPGEGKEATGVRKLF, SEQ ID NO:5) was added. The second antiserum (CTK1p-211) uses a 15 amino acid peptide (amino acids 211-225...

Embodiment 3

[0170] The procedure in Example 1 was repeated with the following differences.

[0171] Sandwich ELISA procedure for detection of TK1 in dog serum

[0172]Maxisorp, NUNC (Denmark) microtiter plates were coated with 100 μL of CTK1p-215 anti-TK1 rabbit polyclonal antibody (4 μg / mL) in carbonate buffer (pH 9.6) and incubated overnight at 4 °C . Plates were washed using a Tecan Hydro flexible microplate washer four times with wash buffer (Tris 0.1M, pH 7.4, NaCl 0.3M, Tween 0.05% and BSA 1%). Thereafter, all wells were blocked with 5% nonfat dry milk diluted in TBST for 1 hour at room temperature. Dilute 50 μL of dog serum samples in 50 μL sample dilution buffer and pre-incubate for 1 h at room temperature. Plates were washed again and pre-incubated serum samples were added. Plates were then incubated for 2 hours at room temperature on a rocking platform. Plates were washed as above and biotinylated CTKlp-161 antibody (4 μg / mL in wash buffer) was added. After incubation for ...

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Abstract

A kit useful for determination of non-human mammal TK1 in a sample is disclosed. The kit comprises a first antibody immobilized to a support or intended to be immobilized to a support. The kit also comprises a second antibody. One of the antibodies has specificity for a peptide consisting of a first amino acid sequence from a C-terminal region of non-human mammal TK1 and the other antibody has specificity for a peptide consisting of an amino acid sequence from an active site of TK1or for a peptide consisting of a second amino acid sequence from the C-terminal region.

Description

technical field [0001] Embodiments of the invention relate generally to the determination of TK1 protein levels, and in particular to kits and methods comprising the use of anti-TK1 antibodies to determine TK1 protein levels in non-human mammals. Background technique [0002] Dogs are often affected by various neoplastic diseases such as lymphoma, leukemia and mammary tumors. Lymphoma is the most common form of blood cancer and accounts for 5% of all dog cancers. The estimated annual incidence is 13 to 40 cases per 100,000 dogs. Canine lymphoma is similar to human non-Hodgkin lymphoma in terms of genetic and environmental factors that contribute to disease progression. Early diagnosis combined with effective chemotherapy can control malignancy. Several proliferation markers, including argyrophilic nucleolar organizing region (AgNOR), proliferating cell nuclear antigen (PCNA), and Ki-67, have been studied as prognostic markers in canine lymphoma, but their use is limited t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/574C07K16/40C12Q1/48G01N33/573
CPCG01N33/573G01N33/574C07K2317/34G01N2800/52G01N2800/54C07K16/40C07K16/18C07K2317/20G01N33/541G01N33/5434
Inventor S·埃里克松H·罗恩伯格K·K·贾加拉穆迪
Owner ALERTIX VETERINARY DIAGNOSTICS AB
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