OF ASSAYS AVAILABLE TO INVESTIGATORS IN THE BIOCHEMISTRY CORE LABORATORY ANALYTE SPECIMEN ACE - Angiotensin Converting Plasma Tissue ACE2 Enzyme Plasma Tissue Angiotensin I1 Plasma,Urine Tissue Angiotensin II1 Plasma,Urine Tissue Angiotensin-(1-7)1 Plasma,Urine Tissue Angiotensin-(1-12)1 Plasma,Urine Tissue Angiotensinogen Plasma.Tissue Urine Bradykinin Plasma Tissue Catecholamines Plasma, tissue Collagen (ICTP) Serum Corticosterone Serum c-AMP Tissue media c-GMP Tissue media Urine c-Reactive Protein Serum, media Endothelin Plasma Urine Estradiol Serum 8-lsoprostane Plasma Serum Insulin Serum, urine Leptin Serum, urine Neutral Endopeptidase (NEP) Tissue PLGF Plasma, tissue, urine, media Procollagen (PICP) Serum PHS 398/2590 (Rev.11/07) Page 417 METHOD Measurement of hydrolysis rate of ^H- Hip-Gly-Gly (Alpco Kit) Measurement of hydrolysis rate of 125I Ang I. Measurement of Activity using Fluorescent Tag ( Anaspec Custom Tag) Measurement of hydrolysis rate of 125I Ang II. Extraction with C18 chromatography followed by RIA (PerkinElmer Kit) Extraction with Ci8 chromatography followed by RIA (Alpco Kit) Extraction with ds chromatography followed by RIA Extraction with C18 chromatography followed by RIA RIA measurement of angiotensin I produced by complete hydrolysis of substrate (DiaSorin kit) after addition of exogenous renin Extraction with Ci8 chromatography followed by EIA ELISA (ALPCO kit) for NE, EPI, and/or DA RIA measurement of type 1 collagen (DiaSorin Kit) ELISA (Alpco) RIA (PerkinElmer Kit) RIA (PerkinElmer Kit) ELISA (ALPCO kit) human and rat EIA (ALPCO Kit) Ethanol extraction of sample followed by RIA (Polymedco Kit) EIA (Cayman Chemical Kit) RIA (Linco) RIA (Linco) Kinetic assay (Microplate-based) ELISA kit (R&D) RIA measurement of type 1 procollagen (DiaSorin Kit) Continuation Format Page Program Director/Principal Investigator (Last, First, Middle): FerrariO, Progesterone Serum Prostaglandin E2 Urine Tissue media 6 keto-Prostaglandin F1a Urine Tissue media Prostaglandin F2a Urine Tissue media Prorenin Plasma Tissue Renin activity/concentration Plasma Tissue sVEGFRI Plasma, tissue, media TNF-a Plasma FERUM Thromboxane B2 Plasma, serum Urine VEGF Plasma, urine Tissue media Carlos Maria Ethanol extraction of sample followed by RIA (Polymedco Kit) EIA (R&D Kit) EIA (R&D Kit) EIA (R&D Kit) RIA of trypsin-activated angiotensin I (DiaSorin Kit) RIA of plasma-generated angiotensin I (DiaSorin Kit) ELISA (R&D) ELISA (Biosource Kit) EIA Kit (Cayman Chemical Kit) ELISA (R&D kit) 1Two protocols are available: a - samples analyzed directly for total immunoreactivity of each peptide after semipurification on C18 chromatography columns b - samples are semipurified on C18 columns, eluates further purified on HPLC to isolate the three peptides. The collected fractions are then quantitated by the peptide RIAs listed above. CHARACTERISTICS OF ASSAYS AVAILABLE IN THE BIOCHEMISTRY CORE LABORATORY GENERAL PRINCIPLES: A) Only technologists experienced in each assay are scheduled to analyze research samples. If a new assay is introduced, the technologist is supplied with sufficient reagents and practice time to become skilled in the described protocol. Wherever possible, this practice includes samples from """"""""normal"""""""" subjects or animals in which expected values are documented in the literature. Otherwise, control material and previously assayed samples will be used. B) All routine assays for the analysis of research samples include control samples to validate the assay precision. C) All assay standards, controls, and specimens are pipetted in duplicate (sample volumes permitting). Occasionally, the reproducibility of the low range of an assay may be enhanced with the pipetting of triplicates. D) Once an assay protocol has been established, the technologists are required to adhere to the protocol unless requested to do otherwise by the Laboratory Director. E) All assays performed and all results released by the Biochemistry Core Laboratory are individually reviewed by the Laboratory Director. Samples are retested (sample volume permitting) in cases where the reliability of the result is in question. 1. ACE - ANGIOTENSIN CONVERTING ENZYME: Alpco. Windham. NH. 03087 Principle: Serum or supernatants of tissue homogenates are incubated with the radioactive tripeptide, H-Hip- Gly-Gly, at pH 8.0 for 60 minutes at 37? C. After incubation, the sample is acidified and the tritiated hippuric acid is separated from unreacted substrate by the scintillation fluid. The radioactivity present is determined with a beta counter. The quantity of liberated hippuric acid, and hence the enzyme activity, is calculated from the radioactivity of a measured known mass of unhydrolyzed peptide and expressed as nmol/ml/minute. PHS 398/2590 (Rev. 11/07) Page 418 Continuation Format Page Program Director/Principal Investigator (Last, First, Middle): FerrariO, Carlos Maria Specificity: The nature of the enzymatic reaction measured by this protocol has been characterized by studies which demonstrated the inhibition of hippuric acid release in incubated serum by angiotensin I and EDTA. Sensitivity: The minimal detectable quantity is 3.8 units of ACE. Precision: The precision of the assay is as follows: intraassay, 3.9% CV (N=25, Mean = 26.8 U);interassay, 5.9% and 7.0 CV %, respectively with means of 39.9 U and 13.0 U (N = 10). 2. ACE2 - ANGIOTENSIN CONVERTING ENZYME2: Pretreatment of samples: Frozen tissues are homogenized at 4?C in HEPES buffer (10 mmol/L). The homogenate is spun at 30,000 g for 20 minutes at 4?C. The pellet is resuspended in HEPES, rehomogenized and recentrifuged. Membrane pellets are resupended in HEPES buffer (10 mmol/L). Principle: Serum or supernatants of tissue homogenates are incubated with the fluorescent substrate [MIPH- 1 (MCA-Ala-Pro-Lys(DNP)-OH), custom synthesis, Anaspec, San Diego,CA] with appropriate inhibitors. The intensity of MCA fluorescence will be measured using a Perkin-Elmer LS50 fluorometer (excitation wavelength 320 nm, emission wavelength 405 nm) and compared to a known standard stock. Specificity: Assays will be done in the presence of a cocktail of inhibitors including: lisinopril (5 umol/L) and SCH-39370 (5 Mmol/L) at 37?C for 60 min. Blank values will be obtained by parallel incubation of the above mentioned reaction mixture in the presence of 1 mmol/L of EDTA or the specific ACE2 inhibitor (ML00106791, 5 umol/L). The reaction will be stopped by adding 0.5% TFA. Standards: MCA-Ala-Pro Standards from Sigma with range of 0.16 - 15nmoles. Incubation: 1 hour substrate at 37?C Sensitivity: The minimal detectable activity is 0.1 nmoles/ml/min Precision: To be determined. 3. ANGIOTENSIN I: Peninsula. Belmont.CA Principle: Endogenous angiotensin I is measured by RIA after plasma or tissue homogenates have been extracted by chromatography on Ci8 columns. Recoveries are monitored by the addition of trace quantities of l-angiotensin II to each sample prior to extraction. The column eluates are dried, reconstituted to a known volume and quantitated by RIA for total immunoreactivity. This preanalytical purification step removes interfering substances and permits concentration of samples to enhance sensitivity. For those studies where greater specificity is required, the C18 column eluates will be further purified by HPLC and each of the isolated peptide fractions are quantitated by RIA using a specific antibody. Specificity: Antibody specificities listed in Table 2. Standards: Concentrations are lot specific. However, the standards typically range from 1.25 to 1000 pg/tube. Incubation: 17 hours at 2-8?C. Separation: Separation of protein bound from free ligand is achieved by the addition of a second antibody and decanting supernatant after centrifugation. Sensitivity: Sensitivity varies with sample size and extraction reconstituted volumes. Generally, the lowest detectable concentration is 2.5 pg/tubel in the direct RIA. Precision: The precision of the assay is as follows: intraassay, 12.1% CV (N = 8, Mean = 632.8);interassay, 15.6 CV% (N = 37, Mean = 573.6). 4. ANGIOTENSIN II: Alpco Diagnostics Kit.Windham. NH03087 Principle: Endogenous angiotensin II is measured by RIA after plasma or tissue homogenates have been extracted by chromatography on C18 columns. Recoveries and extraction are as described for Ang I above. Specificity: Antibody specificities listed in Table 2. Standards: Concentrations are lot specific. However, the standards typically range from 4 to 400 pg/ml. Incubation: This sequential assay has 2 incubations at 2-8?C;one for 6 hours and one for 18 hours. Separation: Separation of protein bound from free ligand is achieved by the addition of a second antibody and decanting supernatant after centrifugation. Sensitivity: Sensitivity varies with sample size and extraction reconstituted volumes. Generally, the lowest detectable concentration is 0.8 pg/tube for the direct RIA. PHS 398/2590 (Rev. 11/07) Page 419 Continuation Format Page Program Director/Principal Investigator (Last, First, Middle): FerrariO, Carlos Maria Precision: Intraassay, 10.6% CV (N = 8, Mean = 28.6);interassay, 23.9% CV at a mean of 60.9 pg/ml. 5.ANGIOTENSIN-M-7): Principle: A modified version of the RIA method described by ChappelL et aL (1) is used to measure endogenous angiotensin (1-7). Recoveries and extraction procedures are as described for Ang I above. Tracer: Radioiodinated angiotensin-(1-7) is routinely prepared by the chloramine T procedure and then the preparation is purified by HPLC using the HFBA solvent system described for the isolation of angiotensin peptides (method #25). The resultant specific activity is usually near 2000 Ci/mM. Antibody: Produced in rabbits at the Cleveland Clinic and at Wake Forest University School of Medicine Specificity: Antibody specificities listed in Table 2. Standards: 2.5 to 2,000 pg/tube. Incubation: 17 hours at 2-8? C. Separation: Separation of protein bound from free ligand is achieved by the addition of a second antibody and decanting the supernatant after centrifugation. Sensitivity: Sensitivity varies with sample size and extraction reconstituted volumes. Typically, the lowest detectable concentration is 2.5 pg/tube in the direct RIA. Precision: The precision for this assay is as follows: intraassay, 8.4% CV (N = 5, Mean = 158.3 pg/ml); interassay, 14.4% CV with a mean of 554.1 pg/ml. 6. Angiotensin-M-12): Principle: A modified version of the RIA method is used to measure endogenous angiotensin (1-12). For the RIA, the primary antibody is n Ang1-7 diluted to 1:20,000 in 50 mM HEPES (pH 7.4) 125 mM NaCI, 5 mM I o Ang 1-9 o Ang II EDTA containing 0.5% BSA and 0.5% Triton-X to reduce non-specific ? Ang I absorption. Varying concentrations of standard Ang-(1-12) [0.1to I 500 fmols] are incubated with the antibody and 10,000 cpm of125I- Ang-(1-12) in a total volume of 0.5 ml overnight at 4?C.Recoveries and extraction procedures are as described for Ang I above. Tracer: Radioiodinated angiotensin-(1-12) is routinely prepared by ? Ang 1-12 the chloramine T procedure and then the preparation is purified by HPLC using the HFBA solvent system described for the isolation of angiotensin peptides (method #25). The resultant specific activity is usually near 2000 Ci/mM. Antibody: The Ang-(1-12) antibody was produced at Wake Forest Univeristy to the unique C-terminus of rat Ang-(1-12) by coupling the N-terminal region to KLH via cysteine linkage. For the RIA, the primary antibody is diluted to 1:20,000. Specificity: Antibody specificities listed in Table 2. Standards: 0.1 to 500 fmols. Incubation: 17 hours at 4? C. Separation: Separation of protein bound from free ligand is achieved by the addition of a second antibody and decanting the supernatant after centrifugation. Sensitivity: Sensitivity varies with sample size and extraction reconstituted volumes. Typically, the lowest detectable concentration is 5 fmols in the direct RIA. Precision: TBD. 7. ANGIOTENSINOGEN: DiaSorin.. Stillwater. MN. 55082 Principle: Plasma containing ACE inhibitors is incubated with exogenously added rat renin prepared by the method of Sen et al. (2). To determine rAGN, 50 ul plasma will be diluted 1:100 in assay buffer and incubated with 445 ul of Tris-HCL (pH 7.4) containing 0.025 mol/L Na-EDTA, 1.0 g/L BSA and purified rat renin with 5 ul PMSF for 1 hour at 37?C. For human angiotensinogen, 25 ul plasma will be diluted 1:100in assay buffer and incubated with 470 ul of citrate-phosphate buffer (pH 5.7) containing 0.025 mol/L Na-EDTA, 1.0 g/L BSA and 50 ng human renin (Sigma-Aldrich, Inc, S. Louis, MO) and with 5ul PMSF for 1 hour at 37?C. When all the substrate has been consumed, the angiotensin I formed during the incubation is quantitated by the RIA method PHS 398/2590 (Rev. 11/07) Page 420 Continuation Format Page Program Director/Principal Investigator(Last, First, Middle): FeiTario, CarlOS Maria as described in Method #19. To test the specificity of the reaction these incubations will be done with and without remmikiren, a REN inhibitor that blocks human but not rat renin. The methods are modified from Bohlanderetal.(138). 8. BRADYKININ: Peninsula. Belmont. CA Principle: EIA measurement is performed after extraction by chromatography on Cia columns. Recoveries are monitored by the addition of trace quantities of radiolabelled peptide. The eluates are evaporated and reconstituted in assay buffer. Specificity: Antibody specificities are listed in Table 2. Standards: 50 to 50 000 fmol/ml. Incubation: Two overnight incubations at 4?C are needed for antibody binding followed by 2 incubations totaling 2.5 hours for separation. Separation: Bradykinin present in the sample will bind to specific antibodies coated on the wells. Any unbound peptide is discarded by washing the well. Sensitivity: 0.1 fmol/ml. Precision: Intra-assay = 4-8%CV, inter-assay = 10%CV. 9. PROCOLLAGEN (PICPV. DIASORIN. Stillwater. MN. 55082 Principle: Serum samples are assayed directly by RIA Specificity: To be determined. Standards: 25 to 500 ng/mL [ncubation: 2 hours at 37 ?C, incubate later with precipitating complex for 30 minutes at room temperature Separation: After the addition of a second antibody complex and centrifugation, the unbound portion is decanted and discarded. Sensitivity: 1.2ug/L Precision: Intra-assay = 2.1-3.2%CV, inter-assay = 4-6.6%CV. 10. COLLAGEN (ICTP): DIASORIN. Stillwater. MN.55082 .Principle: Serum samples are assayed directly by RIA Specificity: To be determined. Standards: 1.0 to 50 ug/L Incubation: 2 hours at 37?C, incubate later with precipitating complex for 30 minutes at room temperature Separation: After the addition of a second antibody complex and centrifugation, the unbound portion is decanted and discarded. Sensitivity: 0.5 ug/L Precision: Intra-assay = 2.8-6.2%CV, inter-assay = 4.1-7.9%CV. 11. c-AMP: PerkinElmer.Boston. MA. 02118 Principle: Samples submitted for c-AMP are assayed directly by RIA and do not require extraction. Specificity: Antibody specificities are listed in Table 2. Standards: 0.05 to 4.0 pmol/ml. Incubation: 18 hours at 2-8?C. Separation: After the addition of a second antibody complex and centrifugation, the unbound portion is decanted and discarded. Sensitivity: 0.025 pmol/ml. Precision: To be determined. 12. c-GMP: PerkinElmer. Boston. MA. 02118 Principle: Samples submitted for c-GMP will be assayed by RIA directly and do not require extraction. Specificity: Antibody specificities are listed in Table 2. Standards: 0.05 to 10.0 pmol/0.1ml. [ncubation: 18 hours at 2-8?C. PHS 398/2590(Rev. 11/07) Page 421 Continuation Format Page Program Director/Principal Investigator (Last, First, Middle): Ferrario, Carlos Maria Separation: After the addition of a second antibody complex and centrifugation, the unbound portion is decanted and discarded. Sensitivity: 0.05 pmol/0.1ml. Precision: Interassay precision is 10.3% CV at a mean of 56.4 pmol/0.1ml. 13. ESTRADIOL: Polvmedco. Inc.. Cortlandt Manor. NY. 10566 Preassay Purification: The reagent standards are provided in a human serum matrix which contains significant concentrations of sex hormone binding globulin. Because rat serum lacks this binding protein for estrogens, a significant matrix effect is avoided by extraction of both standards and rat serum into ethanol before analysis. After drying the extracts, the materials are then redissolved in steroid-free serum before assay. Specificity: Antibody specificities listed in Table 2. Standards: 15 to 5,000 pg/ml. Incubation: 1 hour at 37? C. Separation: Separation of the protein bound from the free ligand is achieved by the addition of a second antibody, goat anti-rabbit gamma globulin bound to iron particles. The protein bound material is precipitated using a magnetic surface and the supernatant containing the free radioactivity can be discarded. Sensitivity: 5 pg/ml. Precision: The precision of the assay is as follows: intraassay, 8.3% CV (N= 20, Mean = 21.0 pg/ml), interassay, 9.3, 8.9 and 9.3 % CV for mean estradiol values of 59, 255 and 518 pg/ml, respectively. 14. ISOPROSTANE: Cayman Chemical. Ann Aabor. Mi. 48108 Principle: The measurement of isoprostane is preformed by Competitive Enzyme Immunoassay Preassay Purification: Plasma samples most be passed over a Isoprostane Affinity Column using Kit from Cayman Specificity: Isoprostane = 100% Standards: 4 to 500 pg/ml Incubation: 18 hr at room temperature Sensitivity: 5 pg/ml Precision: To be determined. 15. PROGESTERONE: Polvmedco. Inc.. Cortlandt Manor. NY. 10566 Preassav Purification: Although progesterone is poorly bound to sex hormone binding globulin, the reagent standards are supplied in a human serum matrix. To avoid any possible matrix affects due to differences in albumin or other protein components of the two systems, the standards and the rat samples will also be extracted into ethanol and the dried extracts reconstituted in steroid-free serum before analysis. Specificity: Antibody specificities listed in Table 2. Standards: 0.1 to 50 ng/ml. Incubation: 3 hours at room temperature. Separation: Separation of the protein bound from the free ligand is achieved by the addition of a second antibody, sheep anti-rabbit gamma globulin bound to iron particles. The protein bound material is precipitated using a magnetic surface, and the supernatant containing the free radioactivity can be discarded. Sensitivity. 0.1 ng/ml. Precision: Reported precision is 16.7, 6.4 and 6.9 % CV for mean progesterone values of 0.24, 2.35 and 6.95 ng/ml (N=10) for interassay precision. 16. PROSTAGLANDIN E.:R&D Systems. Minneapolis. MN. 55413 Principle: Samples submitted for prostaglandins do not require extraction and will be assayed directly by EIA. Specificity: Antibody specificities listed in Table 2. Standards: 39 to 5000 pg/ml. Incubation: 2 hours on horizontal shaker. After addition of conjugate, 45 minutes at room temperature. PHS 398/2590 (Rev. 11/07) Page 422 Continuation Format Page Program Director/Principal Investigator (Last, First, Middle): FeiTario, CarlOS Maria Separation: Antibody bound Prostaglandin E2 present in standards or samples will bind to a second antibody coating the wells of a microplate. The unbound substances are removed by washing the wells with assay buffer. Sensitivity: Although sensitivity varies with sample size and reconstituted volumes for samples which are extracted, the lowest detectable concentration is 6.2 pg/ml. Precision: To be determined 17. 6 keto-PROSTAGLANDIN Fi,,: R&D Systems. Minneapolis. MN. 55413 Principle: Samples submitted for prostaglandins do not require extraction and will be assayed directly by EIA. Specificity: Antibody specificities listed in Table 2. Standards: 16 to 60,000 pg/tube. Incubation: 2 hours on a horizontal shaker. After the addition of the conjugate, 45 minutes at room temperature. Separation: Antibody bound Prostaglandin F1a present in standards or samples will bind to a second antibody coating the wells of a microplate. The unbound substances are removed by washing the wells with assay buffer. Sensitivity: Although sensitivity varies with sample size and reconstituted volumes for samples which are extracted, the lowest detectable concentration is 1.4 pg/ml. Precision: To be determined. 18. PROSTAGLANDIN F^: R&D Systems. Minneapolis. MN. 55413 Principle: Samples for prostaglandins do not require extraction and will be measured by EIA directly. Specificity: Antibody specifics listed in Table 2. Standards: 12.2 to 50,000 pg/ml. Incubation: 2 hours on a horizontal shaker. After addition of conjugate, 45 minutes at room temperature. Separation: Antibody bound Prostaglandin F2a in standards or samples will bind to a second antibody coating the wells of a microplate. The unbound substances are removed by washing the wells with assay buffer. Sensitivity: Although sensitivity varies with sample size and reconstituted volumes for samples which are extracted, the lowest detectable concentration is 4.6 pg/ml. Precision: To be determined. 19. RENIN ACTIVITY: DiaSorin. Stillwater. MN. 55082 Principle: Plasma renin activity (PRA) and concentration (PRC), defined as the rate of angiotensin I generation from endogenous and exogenous substrate, respectively, is measured in incubated plasma treated with EDTA and PMSF to prevent the degradation of the generated peptide. The Ang I is quantitated by RIA using a clinical human renin kit which the Core laboratory has had long experience. By convention PRC is measured in the presence of exogenous substrate obtained from nephrectomized rat/sheep plasma (which provides a suitable substrate for human renin). This method of assay has the advantage that with excess exogenous substrate only renin determines the rate of Ang I generation. The samples and substrate are incubated for a suitable period in the presence of ACE inhibitors and the generated angiotensin I is measured by RIA. For the transgenic rats, which have both human and rat renin, PRA as defined as the combined production of Ang I by both human and rat renins will be measured as described above but at pH 7.4 with and without remmikiren, a human renin inhibitor which blocks human but not rat renin as described by Bohlender et al (3). For PRC, one set will be incubated with rat nephrectomized plasma as exogenous substrate and another set, will be incubated with sheep nephrectomized plasma as exogenous substrate for human renin. These incubations will be done with and without remmikiren. For the human renin determination, the amount of activity inhibited by the remmikiren will be designated as human renin concentration;for the rat renin determination, the amount of activity not inhibited by remmikiren will be designated as rat renin concentration. Specificity: Antibody specificities listed in Table 2 following. Standards: 0.02 to 5.0 ng/tube. Incubation: RIA tubes are incubated 3 hours at room temperature. PHS 398/2590 (Rev. 11/07) Page 423 Continuation Format Page *%??- Program Director/Principal Investigator (Last, First, Middle): FeiTario, Carlos Maria Separation: The method is a solid phase RIA with the anti-angiotensin I antibody attached to the walls of the assay test tube. Separation of the protein bound from the free ligand is achieved by decanting the assay media. Sensitivity. The lowest standard in the RIA is 0.02 ng/tube but total assay sensitivity varies with size of sample pipetted and the amount of inhibitors or exogenous substrate added. For samples which are incubated for 90 minutes and use the volumes listed in the manufacturer's protocol, the minimal detectable activity is 0.1 ng/ml/hr. Precision: The precision for the assay is as follows: intraassay, 10.0% CV for a mean of 1.6 ng/ml/hr; interassay, 14.8% CV for a mean of 5.8 ng/ml/hr. 20. PRORENIN : Diasorin., Stillwater. MN.55082 Principle: The measurement of prorenin is performed after trypsin activation of the sample renin as described by Glorioso et al (4). Trypsin is added to the rat plasma sample at a concentration of 6 mg/ml. The sample is incubated for 1 minute at 4?C. The activation is halted by the addition of soy bean trypsin inhibitor. Angiotensinase inhibitors are then added and the assay proceeds as described in method #19. 21. THROMBOXANE B7: Cayman Chemical. Ann Aabor. Mi. 48108 Principle: The measurement of Thromboxane 62 is preformed by Competitive Enzyme Immunoassay Specificity: To be determined. Standards: 7.8-1000 pg/ml Incubation: 18 hours on a horizontal shaker. After addition of Ellman's reagent, 85 minutes at room temperature Separation: Standards and sample after competition between TXB2 and ACHE conjugate for binding sites occurs and produces an inversely proportional relationship of TXB2 in the well to rabbit antiserum. The rabbit antiserum-TXB2 complex is bound to a monoclonal on the walls of the well. The unbound substances are removed by washing the wells with assay buffer. Sensitivity. 13pg/ml Precision: To be determined. 22. RAT TUMOR NECROSIS FACTOR-ALPHA (TNFa): Biosource International. lnc..Camarillo. Ca. 93012 Principle: Elisa Kit with antibody that specifically measures TNFa Specificity: to be determined Standards: 15.6-1000 pg/ml Incubation: 1.5 hours on a horizontal shaker, after addition of streptavidin-hrp reagent, incubate 45 minutes at room temperature, after addition of chromogen incubate for 30 minutes at R.T. Separation: Standards and sample are added to a plate coated with antibody for TNF@, a Biotinylated second antibody for TNFa is added, and then HRP is added which is activated by a chromogen . The unbound substances are removed by washing the wells with assay buffer. Sensitivity: TNFa = <4 pg/ml Precision: Inter-assay precision is 3.9% CV at a mean of 259.6 pg/mL, Intra-assay precision is 2.7%CV at a mean of 267.7 pg/mL 23. Endothelin: Alpco Diagnostics Kit.Windham. NH 03087 Principle: The Endothelin kit is an enzyme immunoassay to measure directly from plasma and urine. There is a polyclonal used for capture and a monoclonal used for detection. The unbound substances are removed by washing the wells with assay buffer. Specificity: Cross reactivity: ET 1(1-21);100%: ET 2(1-21);100%: ET 3(1-21);<5%: Big Endothelin;<1% Standards: 0-10fmol/ml Incubation: 16-24 hours at 18 ?C-26 ?C, incubate later at 37 ?C for 60 minutes with shaking, incubate with substrate for SOminutes Separation: After the addition of a second antibody complex , the unbound portion is decanted and discarded. Sensitivity. 0.05fmol/ml Precision: Intra-assay = 4.5 %CV, inter-assay = 6.9-7.6 %CV. PHS 398/2590(Rev. 11/07) Page 424 Continuation Format Page Program Director/Principal Investigator (Last, First, Middle): FerrariO, Carlos Maria 24. Neutral endooeptidase fNEP): Principle: This is a two-staged enzymatic reaction. In reaction 1, NEP cleaves the substrate (succinyl-Ala-Ala- Phe-4-nitroanilide) on the amino side of phenylalamine. In reaction 2, Phe-4-NA thus formed is subsequently degraded by aminopeptidase M to phenylalamine and 4-nitroaniline. The increase in absorbance at 405 nm is due to release of 4-nitroaniline. This kinetic assay is measured in microtiter plate continuously for 3 hrs. Specificity. Assays will be done in the presence of a cocktail of different compounds including: lisinopril (1umol/L) and with and without SCH39370 inhibitor (neprilysin inhibitor) (10 umol/L). Standards: A standard curve will be plotted from known amounts of NEP. Incubation: 3 hours at 37?C Sensitivity: The minimal detectable activity is ~ 1.2 Units Precision: To be determined. 25. HPLC FOR SEPARATION OF ANGIOTENSIN PEPTIDES PRIOR TO RIA QUANTITATION: For those studies which require greater characterization of the angiotensins than can be provided by RIA alone, the biologic sample (plasma, tissue extract or tissue culture fluid) will be fractionated by HPLC to obtain individual peptides and subsequently quantitated by RIA. This method was developed by Chappell and associates (5). Preliminary purification: The angiotensin peptides of biologic samples are first isolated from their matrices by using Ci8 extraction columns. The peptide eluates are dried and then redissolved in mobile phase buffer before being injected into the HPLC system. Recoveries are monitored by the addition of trace quantities of 125 l-angiotensin peptides to each sample prior to extraction. Equipment: 3 HPLC units [Shimadzu SCL-10A, 2 pumps Shimaduze LC-10AD with auto sampler (Shimadzu SIL-10AF], [Shimadzu Prominence CBM-20A, 2 pumps ShimadzuProminence LC-20AD, degasser, column oven auto sampler] [Shimadzu Prominence CBM-20A and 2 pumps Shimadzu Prominence LC-20AD] UV detector/vis detector-Shimadzu SPD-10A Gamma detector-Bioscan Flowcount serial columns - Aquapore C8 guard column (2x15 mm) to retain proteins and filter particles Nova Pack C18 column - (2x150 mm) Fraction Collector (Gilson FC204, and 2 Gilson FC20A) Mobile phase: A: 0.1% heptafluorobutyric anhydride (HFBA) (v/v) in water, pH 3.0 B: 80 % acetonitrile in 0.1 % HFBA, pH 3.0 Elution systems: 30 to 50% mobile phase B 20 mins, linear gradient, flow rate 0.35 ml/min 50% mobile phase B 10 mins, isocratic Fractions collected at 0.5 ml/min Standards: Standards of pure synthetic peptides (angiotensin I, II, 1-7,1-12) are used to determine peak positions and retention times of the desired peptides on the chromatogram. Quantitation of the peptides is performed subsequently by RIA on the collected peptide fractions. Since the antibodies used for the measurement of angiotensins I, II, 1-, 1-12 cross react with angiotensin peptides sharing the same C-terminal amino-acid (Table 2 following), additional information can be obtained by quantitating other identifiable chromatographic peaks with the following scheme: Anti-angiotensin I antibody: Ang-l, Ang-(2-10), Ang-3-(1-10) Anti-angiotensin II antibody: Ang II, Ang- (2-8), Ang-(3-8).Ang-(4-8) Anti-angiotensin- (1-7) antibody: Ang-(1-7), Ang-(2-7) Anti-angiotensin-(1-12) antibody. Ang-(1-12) Sensitivity: The lowest detectable mass of peptide per collected fraction tube is as follows: angiotensin I 1.25pg angiotensin-(1-7) 2.5pg angiotensin II 0.8 pg angiotensin-(1-12) pg PHS 398/2590 (Rev. 11/07) Page 425 Continuation Format Page Program Director/Principal Investigator (Last, First, Middle)'. FerrariO, CarlOS Maria 26. Mass Spectrometer Facility. The Mass spectrometer facility specializes in the analysis of lipids like phospholipids (Lipidomics) and the analysis of modified peptides from protein digests (Proteomics). The laboratory is equipped with 1) a Waters Q-TOF API US tandem mass spectrometer with a Waters CapLC and nanospray source. The system can perform data directed de novo protein identification including 2-D chromatography for complex samples. Database searching is performed on a dedicated server located in the laboratory. This instrument is currently the state-of-the-art mass spectrometer for protein identification having both high sensitivity (<5 fmole) and high mass accuracy (<5 ppm). 2) In 2007 a Quantum TSQ triple quadrupole mass spectrometer interfaced to an Agilent model 1100HPLC was installed in the laboratory. The system has a dynamic range of greater than 105 with fmole or better detection sensitivity for many applications. The primary use of this instrument is for the analysis of complex lipids and their mixtures. 3) Finnigan Trace GC mass spectrometer equipped with an autosampler that is used to analyze volatile lipids like fatty acid methyl esters and oxysterols. 4) A Micromass Quattro II triple quadrupole mass spectrometer interfaced to a Symbiosis chromatography system is used for more routine analysis. The Q-TOF and the TSQ are fitted with an Advion Triversa source to improve sensitivity and increase throughput. Analyses are performed using fee-for-service. Dr. Michael J. Thomas, our consultant, directs the laboratory. Dr. John Owen and Mr. Michael Samuel perform the day-to-day operations of the laboratory. TABLE 2 ANTIBODY SPECIFICITIES OF RIA ASSAYS ANGIOTENSIN I (ANGIOTENSINOGEN. PLASMA RENIN ACTIVITY. PRC. and PRORENIN) DiaSorin, Stillwater MN 55082 Peptide % Cross Reactivity Asp1-lleu5-Angiotensin I 10? Asp1-lleu5-Angiotensin II
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