Immunoassay Kit Catalog #KNA0012 SAA. Multispecies

Transcription

1 Immunoassay Kit Catalog #KNA0012 Multispecies SAA Invitrogen Corporation 542 Flynn Rd, Camarillo, CA Tel:

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3 TABLE OF CONTENTS Intended Use... 4 Principle of the Method... 5 Reagents Provided... 6 Supplies Required but Not Provided... 6 Storage and Stability... 7 Safety... 7 Method 1 Serum, Plasma or Milk Assay... 7 Specimen and Reagent Preparation... 7 Sample/Conjugate Diluent Buffer... 8 Wash Buffer... 8 Biotinylated Anti-SAA... 9 Streptavidin-HRP... 9 Dilution of SAA Standards Procedure Interpretation of Test Results Method 2 Cell Culture Supernatant Assay Specimen and Reagent Preparation Sample/Conjugate Diluent Buffer Wash Buffer Biotinylated Anti-SAA Streptavidin-HRP Dilution of SAA Standards Procedure Interpretation of Test Results Limitations of the Procedure Performance Characteristics Sensitivity Rev. A7 06/16/08 PR105

4 INTENDED USE The Serum Amyloid A (SAA) family of acute phase proteins is named because of their immunological and biochemical similarity to Amyloid A, the fibril protein in reactive systemic amyloidosis. The liver produces several different isoforms of SAA following stimulation by immune system modulators including interleukin-1, interleukin-2 and tumor necrosis factor. In its native form, SAA generally consists of a 104 amino-acid polypeptide (12 kda) in association with the HDL 3 subclass of plasma lipoproteins. Circulating SAA concentrations may increase up to 1000-fold following inflammation, infection, tissue injury and cell necrosis and decline rapidly following recovery. This assay is designed to detect Serum Amyloid A (SAA) in serum, solubilized plasma, or milk. The test can also be used to detect SAA levels in cell culture and other biological fluids. This kit has been configured for research use only and is not to be used in diagnostic procedures. Read entire protocol before use. 4

5 PRINCIPLE OF THE METHOD The Invitrogen Multispecies SAA ELISA Kit is a solid phase sandwich Enzyme Linked-Immuno-Sorbent Assay (ELISA). A monoclonal antibody specific for SAA has been coated onto the wells of the microtiter strips provided. Samples, including standards of known SAA content, are added into microwells along with biotinylated anti-saa monoclonal antibody. Any SAA present in the well is both captured on the plate by the immobilized antibody and labeled with the conjugated antibody in a one-step procedure. After washing to remove all of the unbound material, Streptavidin-Horseradish Peroxidas (Streptavidin-HRP) is added to the well to complete the four-member sandwich. Following the second incubation, TMB substrate solution is added. The intensity of the color produced is proportional to the concentration of SAA present in the original specimen. 5

6 REAGENTS PROVIDED SAA antibody coated wells 2 x 96 well plates Wash buffer concentrate 1 x 50 ml (20x concentrate) Sample/conjugate diluent buffer 1 x 50 ml (10x concentrate) SAA Standard 1 freeze dried vial Biotinylated anti-saa 1 x 0.2 ml Streptavidin-HRP 1 x 0.1 ml TMB substrate 1 x 25 ml Stop reagent 1 x 25 ml SUPPLIES REQUIRED BUT NOT PROVIDED 1. Serum/plasma collection equipment. 2. Microtiter plate reader capable of measurement at 450 nm with reference at 630 nm, if available. 3. Accurate micropipettes and disposable tips to deliver 0-10 µl, µl and µl. 4. A repeat or multi-channel pipette ( µl) for large assays. 5. Deionized or distilled H 2 O. 6. Plate washer (automated or manual). 7. Data analysis and graphing software. Graph paper: linear (Cartesian), log-log, or semi-log, as desired. 8. Glass or plastic test tubes. 9. Absorbent paper towels well dust plate cover C incubator. 6

7 STORAGE AND STABILITY The kit components are stable when stored at 2 to 8 C until the expiration date indicated on the kit label. SAFETY Never pipette by mouth. Wear disposable latex gloves and eye protection where appropriate. The TMB substrate contains reagents that may irritate the skin or mucous membranes. Any reagent which comes in contact with skin should be washed off with water immediately. METHOD 1: SERUM, PLASMA OR MILK ASSAY SPECIMEN AND REAGENT PREPARATION Serum or plasma samples should be collected by venipuncture into serum or plasma collection tubes. Blood samples may be kept for up to 24 hours before separation of serum or plasma. However, it is best to remove serum from the clot or cells and debris from plasma or other fluids as soon as possible after collection. In general, serum or plasma may be stored at 2 to 8 C for up to 24 hours or stored frozen at 20 C for longer periods without loss of SAA. Repeated freeze-thaw cycles do not appear to affect the SAA concentration. It is important that all refrigerated samples are brought to room temperature and vortexed vigorously to assure accurate determination of the SAA concentration. Do not use grossly hemolyzed or lipemic samples. 7

8 All serum or plasma samples should be diluted 1:500 with 1x sample/conjugate diluent buffer prior to assay, by addition of 10 µl sample to 5.0 ml sample/conjugate diluent buffer. (Note: Equine serum or plasma samples should be diluted 1:2000 with 1x sample/conjugate diluent buffer.) Milk samples can be stored for up to 2 days at 2-8 C or stored frozen at -20 C for longer periods. All milk samples should be brought to room temperature and vortexed vigourously to assure accurate SAA determination. Milk samples should be diluted 1:50 with 1x sample/conjugate diluent buffer prior to testing. SAMPLE/CONJUGATE DILUENT BUFFER Dilute 1 volume of sample/conjugate diluent buffer (10x) with 9 volumes of distilled water. Prepared reagent is stable for one day at room temperature and should not be stored for extended periods. WASH BUFFER Dilute 1 volume of wash buffer concentrate (20x) with 19 volumes of distilled water. Store both the wash buffer concentrate and working wash buffer (1x) in the refrigerator. Diluted wash solution is stable for up to 2 weeks when stored at 4 C. Note: Ensure that any crystals that may have developed in the wash buffer or sample/conjugate diluent buffer have been completely dissolved prior to dilution for use. 8

9 BIOTINYLATED ANTI-SAA For all biological fluids, dilute Biotinylated Anti-SAA 1:100 in 1x sample/conjugate diluent buffer. Prepare appropriate volume of the diluted conjugate depending on the number of strips used. Fifty microliters of conjugate are used for each well (400 µl/strip). Discard any excess diluted conjugate. STREPTAVIDIN-HRP Dilute the Streptavidin-HRP to a 1:4000 final dilution by adding 5 µl of the stock solution to 20 ml of 1x sample/conjugate diluent buffer. At 100 µl per well, this is sufficient for twelve 8-well strips. Dilute only as much conjugate as will be used in the assay and return the excess Streptavidin-HRP stock to the refrigerator. Discard any excess diluted conjugate. 9

10 DILUTION OF SAA STANDARDS 1. Solubilize the standard by adding 200 µl of distilled water to the vial. Vortex vigorously to dissolve completely. 2. Dilute solubilized standard 1:5 (1 part standard to 4 parts 1x sample/conjugate diluent buffer). This is further diluted two-fold serially 4 times to provide the working standards as indicated in Table 1 below. Table 1. Preparation of Working Standard Curves Tube Volume of Volume of Serial Number Standards (µl) Diluent (µl) Dilution S S µl S1 S µl S2 S µl S3 S µl S4 S The final concentrations of the standards are indicated in Table 2. Table 2. Concentration of Standards for Serum, Plasma or Milk Standards Bovine (ng/ml) Porcine (ng/ml) Canine (ng/ml) Human (ng/ml) Equine (ng/ml) C C C C C C

11 PROCEDURE Allow test reagents and samples to reach room temperature before use. 1. Determine the number of 8-well strips needed for the assay. Re-bag extra strips, seal bag and store in a refrigerator. It is recommended that each sample or standard be analyzed in duplicate. 2. Add 50 µl of diluted Biotinylated anti-saa to each well. 3. Vortex the serum, plasma or milk samples to be examined by the test. Dilute serum and plasma samples 1:500 (except equine 1:2000) and milk samples 1:50 in 1x sample/conjugate diluent buffer. Add 50 µl of the diluted sample or standard to each well. Tap sides of the plate to mix gently. 4. Cover the plate with a dust cover. Incubate the plate for 1 hour at 37 C. 5. After incubation, aspirate or decant and wash the plate four times with diluted wash buffer. After the last wash, tap the plate dry on absorbent paper. 6. Add 100 µl of Streptavidin-Peroxidase to each of the wells. 7. Cover the plate and incubate at room temperature for 30 minutes. 8. Aspirate or decant and wash the wells four times. Tap the plate dry after the last wash. 9. Add 100 µl of TMB substrate. 10. Cover plate and incubate in the dark at room temperature for 30 minutes. 11. Add 50 µl of the stop solution. 12. Read the absorbance of each well at 450 nm using 630 nm as reference, if available. Alternatively, blank the plate reader against a chromogen blank composed of 100 µl of TMB substrate and 50 µl stop solution. 11

12 INTERPRETATION OF TEST RESULTS 1. Calculate the mean absorbance for each sample, control or standard. 2. Plot the absorbance of the standards against the standard concentration on semi-logarithmic graph paper. If necessary, the background absorbance for the 0 ng/ml standard may be subtracted from each of the data points, including the standards, unknowns and controls prior to plotting. Draw the best smooth curve through these points to construct the standard curve. 3. Determine the concentrations of the test samples and controls from the standard curve by multiplying the interpolated value by the appropriate dilution factor (e.g., SAA concentration values for sera or plasma diluted 1:500 should be multiplied by 500). Samples that have a signal greater than the highest standard should be further diluted in sample/conjugate diluent buffer and reanalyzed. METHOD 2: CELL CULTURE SUPERNATANT ASSAY SPECIMEN AND REAGENT PREPARATION The sensitivity of the cell culture supernatant assay may depend on the choice of cell line. Initial testing should be performed with undiluted cell culture supernatant, and sample diluted accordingly where higher levels are either obtained or suspected. SAMPLE/CONJUGATE DILUENT BUFFER Dilute 1 volume of sample/conjugate diluent buffer concentrate (10x) with 9 volumes of distilled water. Prepared reagent is stable for one day at room temperature and should not be stored for extended periods. 12

13 WASH BUFFER Dilute 1 volume of wash buffer concentrate (20x) with 19 volumes of distilled water. Store both the wash buffer concentrate and working wash buffer (1x) in the refrigerator. Diluted wash solution is stable for up to 2 weeks when stored at 4 C. Note: Ensure that any crystals that may have developed in the wash buffer or sample/conjugate diluent buffer have been completely dissolved prior to dilution for use. BIOTINYLATED ANTI-SAA Dilute Biotinylated Anti-SAA 1:100 in 1x sample/conjugate diluent buffer. Prepare appropriate volume of the diluted conjugate depending on the number of strips used. Fifty microliters of conjugate are used for each well (400 µl/strip). Discard any excess diluted conjugate. STREPTAVIDIN-HRP Dilute the Streptavidin-HRP to a 1:400 final dilution by adding 10 µl of the stock solution to 4 ml of 1x sample/conjugate diluent buffer. At 100 µl per well, this is sufficient for four 8-well strips. Dilute only as much conjugate as will be used in the assay and return the excess Streptavidin-HRP stock to the refrigerator. Discard any excess diluted conjugate. 13

14 DILUTION OF SAA STANDARDS 1. Solubilize the standard by adding 200 µl of distilled water to the vial. Vortex vigorously to dissolve completely. 2. Dilute solubilized standard 1:40 (1 part standard to 39 parts 1x sample/conjugate diluent buffer). This is further diluted two-fold serially 4 times to provide the working standards as indicated in Table 3 below. Table 3. Preparation of Working Standard Curves Tube Number Volume of Standards (µl) Volume of Diluent (µl) Serial Dilution S S µl S1 S µl S2 S µl S3 S µl S4 S

15 The concentrations of the standards are indicated in Table 4. Table 4. Concentrations of Standards for Cell Culture Standards Bovine (ng/ml) Porcine (ng/ml) Canine (ng/ml) Human (ng/ml) C C C C C C PROCEDURE Allow test reagents and samples to reach room temperature before use. 1. Determine the number of 8-well strips needed for the assay. Re-bag extra strips, seal bag and store in a refrigerator. It is recommended that each sample or standard be analyzed in duplicate. 2. Add 50 µl of diluted Biotinylated anti-saa to each well. 3. Add 50 µl of the neat or diluted culture supernatant or standard to each well. Tap sides of the plate to mix gently. Cover the plate with a dust cover. Incubate the plate for 1 hour at 37 C. 4. After incubation, aspirate or decant and wash the plate four times with diluted wash buffer. After the last wash, tap the plate dry on absorbent paper. 5. Add 100 µl of Streptavidin-HRP to each of the wells. 6. Cover the plate and incubate at room temperature for 30 minutes. 15

16 7. Aspirate or decant and wash the wells four times. Tap the plate dry after the last wash. 8. Add 100 µl of TMB substrate. 9. Cover plate and incubate in the dark at room temperature for 30 minutes. 10. Add 50 µl of the stop solution. 11. Read the absorbance of each well at 450 nm using 630 nm as reference, if available. Alternatively, blank the plate reader against a chromogen blank composed of 100 µl of TMB substrate and 50 µl stop solution. INTERPRETATION OF TEST RESULTS 1. Calculate the mean absorbance for each sample, control or standard. 2. Plot the absorbance of the standards against the standard concentration on linear graph paper. If necessary, the background absorbance for the 0 ng/ml may be subtracted from each of the data points, including the standards, unknowns and controls prior to plotting. Draw the best smooth curve through these points to construct the standard curve. 3. Determine the concentrations of the test samples and controls from the standard curve by multiplying the interpolated value by the appropriate dilution factor where appropriate. Samples that have a signal greater than the highest standard should be further diluted in sample/conjugate diluent buffer and reanalyzed. 16

17 LIMITATIONS OF THE PROCEDURE The influence of various drugs, aberrant sera (hemolyzed, hyperlipidemic, jaundiced, etc.) has not been investigated. PERFORMANCE CHARACTERISTICS Measuring Range The range covered by each calibration curve is dependent on the species under investigation. Examples of ranges are provided in the table below. Table 5. Reference Range for Multispecies SAA test Species Cell Culture Serum/Plasma Milk** (ng/ml) (µg/ml)* (µg/ml) Porcine Bovine Canine Human Equine *Ranges provided take into account the sample dilution of 1:500 (1:2000 for Equine). **For milk samples, the working range of µg/ml takes the 1:50 dilution into account. SENSITIVITY The current detection limit is 0.3 ng/ml for cell culture studies. For serum, plasma, or milk the sensitivity will depend on the species under investigation. Sensitivities are 1.9 µg/ml, 0.3 µg/ml and 0.15 µg/ml for porcine, bovine, and canine samples, respectively. 17

18 Important Licensing Information - These products may be covered by one or more Limited Use Label Licenses (see the Invitrogen Catalog or our website, By use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses. Unless otherwise indicated, these products are for research use only and are not intended for human or animal diagnostic, therapeutic or commercial use. 18

19 19 NOTES

20 Rev. A7 06/16/08 PR105 20