a promising marker of heart failure in dogs

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1 TechNotes Canine NT-proBNP a promising marker of heart failure in dogs NT-proBNP measurement has been introduced into veterinary practice during the last decade. NT-proBNP levels are elevated in dogs with mitral valve disease and dilated cardiomyopathy. Highest concentrations are observed in dogs developing congestive heart failure. NT-proBNP measurement helps to discriminate congestive heart failure from primary respiratory tract disease as an underlying cause of respiratory signs in dogs (Boswood et al, 2008). NT-proBNP concentration in blood correlates with the severity of disease and reflects the risk of following complications. Growing number of studies shows that NT-proBNP is successfully used for diagnosing cardiac disease in dogs, assessing the severity of disease in dogs with cardiac disease and prognosis in dogs with heart disease (reviewed in Oyama and Singletary, 2010). One of the main challenges with canine NT-proBNP is that it is not very stable at room temperature. Degradation of NT-proBNP makes the use of current immunoassay kits complicated; sample processing protocols are inflexible and may require additional work by the practitioner in order to minimize degradation of NT-proBNP before the samples can be assayed (Collins, 2013). One way to improve the immunoassays and their clinical utility would be to select antibodies that are less sensitive to analyte degradation (also suggested by Collins et al., 2010). We have previously shown which epitopes in human NT-proBNP are the most stable (Katrukha et al., 2005). However, the amino acid sequences of human and canine NT-proBNP differ significantly, so the epitope stabilities may be at least partially different for human and dog NT-proBNP. Monoclonal antibodies and calibrator from HyTest For development of canine specific NT-proBNP immunoassays we can now offer both the calibrator and various mabs with pair recommendations. These tools allow development of highly specific immunoassays for determining canine NT-proBNP concentration in blood. Our preliminary results indicate that plasma samples could be stored at least for 72 hours at +4 C or 24 hours at +20 C with little to no loss in immunoreactivity of NT-proBNP. NT-proBNP and BNP are established biomarkers of heart failure in humans B-type natriuretic peptide (BNP) is a cardiac hormone involved in the maintenance of blood pressure, water, and electrolyte balance. It reduces vascular resistance and increases both diuresis and sodium excretion thus lowering systemic blood pressure. BNP is synthesized as prohormone. Specific cleavage of the precursor molecule results in formation of active BNP and N-terminal fragment of probnp (NT-proBNP). Both BNP and NT-proBNP are secreted to blood in equimolar amounts. More information can be found in reviews by Goetze (2012) and Potter (2011). In humans, BNP and NT-proBNP concentrations in blood are increased in different cardiovascular pathologies, but the most prominent growth is observed in heart failure. Nowadays both proteins are used as biochemical markers of heart failure. Quantification of either BNP or NT-proBNP in blood improves the diagnostic accuracy compared to standard clinical judgment in the diagnosis of acute heart failure among patients presenting to the emergency department with acute dyspnea. Both BNP and NT-proBNP are also powerful prognostic indicators for patients with heart failure or acute myocardial infarction.

2 Anti-canine NT-proBNP monoclonal antibodies Host animal: Mice Balb/c Cell line used for fusion: Sp2/0 Antigen used for immunization: Recombinant canine NT-proBNP with tag (CaNT90), recombinant canine NT-proBNP produced as a fusion protein with carrier (CaNT89, CaNT19, CaNT46, CaNT925, and CaNT930), and synthetic peptides corresponding to 3-26 a.a.r. (CaNT73), a.a.r. (CaNT611), and a.a.r. (CaNT49, CaNT53, CaNT59) of canine NT-proBNP Purification method: Protein A affinity chromatography Presentation: PBS, ph 7.4, 0.1% sodium azide HyTest offers monoclonal antibodies specific to different regions of canine NT-proBNP (Fig. 1). Antibodies bind with high affinity recombinant NT-proBNP expressed in E. coli (Cat.# 8CNT9) as well as native NT-proBNP from canine plasma samples. Figure 1. Anti-canine NT-proBNP monoclonal antibodies: Location of epitopes. Canine NT-proBNP quantitative sandwich immunoassays Selection of best pairs. A panel of more than sixty specific monoclonal antibodies has been produced and used to select the best antibody pairs for immunoassay of NT-proBNP in canine plasma. All monoclonal antibodies were tested as capture and detection antibody in a sandwich immunoassay. Capture antibodies were absorbed onto a 96-well EIA plate. Detection antibodies were labeled with stable europium chelate. Recombinant canine NT-proBNP expressed in E. coli (Cat.# 8CNT9) was used as a calibrator. A number of combinations demonstrated high sensitivity in sandwich immunoassay for detecting both recombinant and endogenous NT-proBNP in our experiments. The best MAb combinations were: Capture CaNT90 CaNT19 CaNT930 CaNT90 Detection CaNT89 CaNT89 CaNT49 CaNT53 Sensitivity of these immunoassays for recombinant NT-proBNP was 25 pg/ml 1. Calibration curves for recommended combinations are provided in Fig To obtain concentration in pmol/l please divide concentration in pg/ml by TechNotes Canine NT-proBNP

3 Please note that an immunoassay performance depends on a number of factors including the diagnostic platform, type of label conjugated with detection antibody and labeling protocol. Therefore, other combinations of anti-nt-probnp antibodies could demonstrate better performance in our customers immunoassays than those listed above. For a complete list of two-site antibody combinations for detection of canine NT-proBNP in plasma please contact our customer support at hytest@hytest.fi. Figure 2. Calibration curves for NT-proBNP sandwich immunoassay. A Calibration curves of the best immunoassays B Parallelism between the calibration curve and curve of serial dilution of pooled canine plasma sample Two-step sandwich type fluoroimmunoassays in streptavidin coated plates. Capture MAb (CaNT90, CaNT930 and CaNT19): 200 ng/well, biotinylated Detection MAb (CaNT89, CaNT53 and CaNT49): 200 ng/well, labeled with europium chelate Antigen: canine recombinant NT-proBNP (Cat.# 8CNT9) Sample volume: 50 µl Incubation time: 40 minutes at room temperature Quantification of NT-proBNP in canine plasma of healthy dogs and dogs with heart disease NT-proBNP concentrations in plasma samples from healthy dogs and dogs with diagnosed cardiac disease were determined using a 2-step sandwich type fluoroimmunoassay (Fig. 3). The results clearly demonstrate that NT-proBNP concentrations were significantly higher in the group of dogs with heart disease than in group of healthy dogs. Even with high NT-proBNP concentrations no dilution step was required due to the wide dynamic range of the assay used. Figure 3. NT-proBNP concentration in EDTA plasma of healthy dogs and dogs with heart disease. Two-step sandwich type fluoroimmunoassay. Capture MAb CaNT90: 1 µg/well Detection MAb CaNT89: 200 ng/well, labeled with europium chelate Calibrator: canine recombinant NT-proBNP (Cat.# 8CNT9) Sample volume: 50 µl Incubation time: 40 minutes at room temperature TechNotes Canine NT-proBNP 3

4 Improved apparent stability of endogenous NT-proBNP in plasma samples One of the main challenges for a reliable measurement of concentration of NT-proBNP in samples is degradation of the protein over time. While proper sample handling and storage are critical in decreasing degradation, another important factor is the selection of antibodies in the assay. We have previously shown that the apparent stability of human NT-proBNP depends on the specificity of the antibodies utilized (Katrukha et al., 2005). We tested the ability of our antibodies to detect endogenous canine NT-proBNP during sample storage. Pooled EDTA plasma of dogs with heart disease was incubated at two different temperatures (+4 C and +20 C). At +4 C NT-proBNP remained stable for at least 72 hours (95-105% of initial immunoreactivity was detected in samples, Fig. 4A). When plasma was incubated at +20 C, 89-98% of initial immunoreactivity was detected in samples after 24 hours (Fig. 4B). This preliminary data indicates that with our recommended antibody pairs plasma could be stored at +4 C for at least 72 hours with little to no loss in the immunoreactivity of endogenous canine NT-proBNP. When stored at room temperature, the signal decreased but not dramatically during the first 24 hours. Please note that the stability of native NT-proBNP in individual plasma samples or in serum samples could differ from the results obtained with the pooled dog EDTA-plasma. When developing a canine NT-proBNP assay special attention should be paid to the selection of antibodies. Choosing antibodies that are less sensitive to the degradation of NT-proBNP might allow less stringent and complicated instructions for sample handling and storage. Such a robust assay would greatly improve the clinical utility of canine NT-proBNP assay. Figure 4. Stability of endogenous canine NT-proBNP in pooled EDTA-plasma. A Immunoreactivity of NT-proBNP in the plasma sample incubated at +4 C for 24, 48 and 72 hours. B Immunoreactivity of NT-proBNP in the plasma sample incubated at +20 C for 4, 8, 24, and 48 hours. EDTA plasma was collected without protease inhibitors; samples were centrifuged, separated and stored frozen at -70 C before use. Pooled EDTA plasma was incubated at +4 C or +20 C with the addition of 0.1% of sodium azide to prevent bacterial growth. After incubation samples were stored at -70 C until measurements. NT-proBNP concentration in the pooled plasma was 9 ng/ml (determined by CaNT90-CaNT89 immunoassay). Two-step sandwich type fluoroimmunoassay. Capture MAbs CaNT90 and CaNT19: 1 µg/well Detection MAbs CaNT53 and CaNT89): 200 ng/well, labeled with europium chelate Antigen: canine recombinant NT-proBNP (Cat.# 8CNT9) Sample volume: 50 µl Incubation time: 40 minutes at room temperature 4 TechNotes Canine NT-proBNP

5 Canine NT-proBNP immunodetection in Western blotting HyTest antibodies can be used for NT-proBNP immunodetection in Western nlotting (Fig. 5). Figure 5. Detection of canine recombinant NT-proBNP in Western Blotting by different monoclonal antibodies. NT-proBNP was transferred to nitrocellulose membrane after tricine-sds-page in reducing conditions and probed by HRP-conjugated monoclonal antibodies (direct detection). In cases of CaNT59 and CaNT73, non-labeled primary antibodies were used for probing followed by anti-mouse IgG antibodies conjugated with HRP. Antigen: Canine recombinant NT-proBNP with tag (Cat.# 8CNT9), 0.1 µg/lane Substrate: Diaminobenzidine CaNT19 CaNT611 CaNT89 CaNT49 CaNT53 CaNT930 CaNT46 CaNT59 CaNT925 CaNT73 CaNT90 Ordering information: MAb Cat. # Epitope Subclass Application CaNT73 4CNT a.a.r. IgG2a Sandwich type immunoassay, direct ELISA, WB CaNT89 4CNT a.a.r. IgG1 Sandwich type immunoassay, direct ELISA, WB CaNT925 4CNT a.a.r. IgG1 Sandwich type immunoassay, direct ELISA, WB CaNT930 4CNT a.a.r. IgG1 Sandwich type immunoassay, direct ELISA, WB CaNT611* 4CNT a.a.r. IgG1 Sandwich type immunoassay, direct ELISA, WB CaNT90* 4CNT a.a.r. IgG1 Sandwich type immunoassay, direct ELISA, WB CaNT19 4CNT a.a.r. IgG1 Sandwich type immunoassay, direct ELISA, WB CaNT46 4CNT a.a.r. IgG1 Sandwich type immunoassay, direct ELISA, WB CaNT49 4CNT a.a.r. IgG1 Sandwich type immunoassay, direct ELISA, WB CaNT59 4CNT a.a.r. IgG2b Sandwich type immunoassay, direct ELISA, WB CaNT53* 4CNT a.a.r. IgG1 Sandwich type immunoassay, direct ELISA, WB * These products and some applications in which these products may be used are covered by patents issued and applicable in certain countries. Because purchase of these products does not include a license to perform any patented application, users of these products may be required to obtain a patent license, depending on the particular application and country in which the product is used. Canine recombinant NT-proBNP expressed in E. coli Source: Recombinant protein with N-terminal proprietary affinity tag expressed in E. coli Purity: >95% according to Tricine-SDS-PAGE Application: Standard for the calibration of immunoassays Number of amino acids: 101 a.a.r. Molecular weight: Sequence: Tag 1 HPLGGRSPASEASEASEASGLWAVQELLGRLKDAVSELQAEQLALEPLHRSHSPAEAPEAGGTPRGV LAPHDSVLQALRRLRSPK 85 Presentation: Frozen protein solution in PBS Storage: -70 C Figure 6. Tricine-SDS-PAGE of canine recombinant NT-proBNP (10 μg) in reducing conditions. Gel was stained with Coomassie brilliant blue R-250. kda Recombinant NT-proBNP corresponds to the fragment 1-85 a.a.r. of canine probnp and contains additional affinity tag sequence of 16 a.a.r. at the N-terminus. The protein is purified to homogeneity using tag affinity chromatography (Fig. 6). TechNotes Canine NT-proBNP 5

6 TechNotes Canine NT-proBNP In order to confirm that N-terminal tag doesn t interfere with antibody binding, immunochemical activities of recombinant canine NT-proBNP with and without tag were compared using sandwich type fluoroimmunoassay. The immunochemical activities of both recombinant proteins were highly similar in all immunoassays (n=9) performed. Representative calibration curves are given in Fig. 7. This shows that the recombinant NT-proBNP containing an N-terminal tag is a suitable calibration material for canine NT-proBNP immunoassays. Figure 7. Comparison of immunochemical activity of recombinant canine NT-proBNP with and without an N-terminal proprietary tag. Two-step sandwich type fluoroimmunoassay Capture MAb CaNT19: 1 µg/well Detection MAb CaNT89: 200 ng/well, labeled with europium chelate Antigens: canine recombinant NT-proBNP with tag (Cat.# 8CNT9) and canine recombinant NT-proBNP without tag Sample volume: 50 µl. Incubation time: 40 minutes at room temperature. Ordering information: Product Cat. # Purity Canine recombinant NT-proBNP with tag (E. coli) 8CNT9 >95% References: 1. Boswood A, Dukes-McEwan J, Loureiro J, James RA, Martin M, Stafford- Johnson M, Smith P, Little C, Attree S. The diagnostic accuracy of different natriuretic peptides in the investigation of canine cardiac disease. J Small Anim Pract. 2008;49(1): Oyama MA, Singletary GE. The use of NT-proBNP assay in the management of canine patients with heart disease. Vet Clin North Am Small Anim Pract. 2010;40(4): Collins, S. Measuring NT-proBNP in small animal practice. Royal College of Veterinary Surgeons Diploma in veterinary cardiology Collins SA, Patteson MW, Connolly DJ, Brodbelt DC, Torrance AG, Harris JD. Effects of sample handling on serum N-terminal prob-type natriuretic peptide concentration in normal dogs and dogs with heart disease. J Vet Cardiol Apr;12(1): Katrukha A, Tolstaya A, Antopolskii M, Koshkina E, Krasnoselskii M, Kharitonov A, Seferian K. NT-proBNP stability assessed by several sandwich immunoassays utilizing monoclonal antibodies with different epitope specificity. Poster presented at AACC Annual Meeting, July 24 28, 2005, Orlando, FL. 6. Goetze JP. B-type natriuretic peptide: from posttranslational processing to clinical measurement. Clin Chem. 2012;58(1): Potter LR. Natriuretic peptide metabolism, clearance and degradation. FEBS J. 2011; 278(11): Intelligate 6 th floor, Joukahaisenkatu 6 FI Turku, FINLAND Tel Fax hytest@hytest.fi Internet: HyTest June 2013