procedure for lab animal 2 plate elisa assays
Contents
1. freezing thawing of samples Samples should not contain sodium azide B Dilute the serum 1 50 in Sample Diluent For example add 5 ul of serum sample to 245 ul of 1X Sample Diluent If not assayed immediately diluted samples should be stored at 20 C or below Procedural Notes 1 Review the complete instructions before performing the test 2 Strips of the ELISA plate are removable Remove unused strips and store as described in Storage and Stability Instructions Before testing begins the user should inspect the ELISA strip holders and ensure that all strips are secure A white stabilizer residue is normally observed in the bottom of unused wells Strip holders should be handled with care to ensure that no strip is dislodged during testing It is recommended that each strip be numbered with a laboratory marker prior to use Additionally since the strips are pre coated with positive viral antigen and negative control antigen alternating 6 positive and 6 negative antigen coated strips strip holder it is recommended that each strip be labeled with a or to indicate type of antigen coat in each well NOTE Assembled strip holders always start with a positive antigen coated strip Subsequent strips alternate between negative control antigen and positive antigen so that strips 1 3 5 7 9 and 11 are pre coated with positive antigen and strips 2 4 6 8 10 and 12 are pre coated with negative control anti2. in the test 1 Make a 1 50 dilution of the test serum in 1X Sample Diluent in a small dilution tube and mix well EXAMPLE Add 5 ul of serum to 245 ul of 1X Sample Diluent Fit the strip holder with the required number of pre coated Positive Viral Antigen and Negative Control Antigen strips Mark the appropriate strips with a or Allow one well to be used for the Negative Control Sera and one well for the Positive Control Sera Pipette 100 ul each of the diluted serum sample the Negative Control and positive Control into the appropriate and marked wells Cover the wells and incubate at 37 C for 45 1 minutes After incubation wash each well five 5 times with 1X Wash Solution refer to Wash Procedure 10 11 Pipette 100 ul of liquid ready to use Peroxidase Conjugate into each test well Cover the wells and incubate at 37 C for 45 1 minutes After incubation wash each well five 5 times with 1 X Wash Solution refer to Wash Procedure Pipette 100 ul of liquid ready to use ABTS Peroxidase Substrate into each test well Incubate the plate at room temperature 20 25 C for 30 minutes Do not cover the plate Blank the micro reader on air and read the absorbance of the colorimetric reaction in each well at 405nm If the plate is not read immediately pipette 25 ul of Stop Solution into each test well Read the plate at 405 nm within 15 minutes INTERPRETATION OF RESULTS 1 2 It is reco
3. 028 This difference is less than 0 300 This sample is considered negative Express Biotech International P O BOX 576 Frederick MD 21705 USA Tel 301 228 244 Fax 301 560 6570 Toll Free 888 562 8914 www expressbiotech com info expressbiotech com XpressBio Life Science Products Marsuniversalinsert_2PLATE 09222009MH
4. XpressBio Life Science Products PROCEDURE FOR LAB ANIMAL 2 PLATE ELISA ASSAYS Research Reagents for the Detection of Antibodies in animal sera by ELISA Store at 2 8 C For Research Use Only REAGENTS Sufficient reagents are supplied to run 96 tests NOTE Kit plate and conjugate lots are matched and must be used as a set All reagents are ready to use EXCEPT the wash concentrate is supplied as a 20X concentrate ELISA Strips 2 strip holders containing 6 positive viral antigen coated strips and 6 negative antigen coated strips each alternating and antigen strips Sample Diluent 2 bottles 30 ml each Peroxidase Conjugate 2 vials 12 ml each ABTS Peroxidase Substrate 2 vials 12 ml each Stop Solution 1 vial 10 ml Wash Concentrate 20X 2 vials 60 ml each Positive Control Serum 1 vial of 1ml Negative Control Serum 1 vial of 1ml STORAGE AND STABILITY INSTRUCTIONS 1 Store all reagents at 2 8 when not in use The expiration date printed on the box label indicates the limit of stability of the product 2 The foil packs containing the ELISA strips should be allowed to warm to room temperature 20 25 C before opening to prevent condensation Once opened microtitration strips may be stored at 2 8 C until the expiration date on the label provided that desiccated conditions are maintained INSTRUCTIONS FOR USE Reagent Preparation Prepare the following reagents and samples before beginning the assay proce
5. dure All reagents and samples should be at room temperature 20 25 C prior to beginning the assay and may remain at room temperature during testing Return reagents to 2 8 C immediately after use Sample Diluent 2 vials 30 ml vial Contains normal goat and bovine serum in phosphate buffered saline and Proclin as a preservative The Sample Diluent is stable for a minimum of 1 year when stored at 2 8 C POSITIVE Control Serum 1 vial 1 ml This vial contains positive control serum ready to use at the dilution to be used in the test No further dilutions are required The Control Serum is stable for a minimum of one year when stored at 2 8 C NEGATIVE Control Serum 1 vial 1 ml This vial contains negative control serum ready to use at the dilution to be used in the test No further dilutions are required The Control Serum is stable for a minimum of one year when stored at 2 8 C Wash Concentrate 20X 2 vials 60 ml vial Contains tris buffer with surfactant Check the Wash Concentrate for the presence of salt crystals If crystals have formed in the solution resolubilize by warming at 37 C until crystals dissolve Wash Solution is stable for 3 weeks from date of preparation if stored at 2 8 C Therefore dilute Wash Concentrate as needed Dilute the Wash Concentrate 1 20 with deionized or distilled water in a clean glass or plastic screw cap container for example add 50 ml of wash concentrate to 950 ml of
6. gen A schematic representation of this is below Reed Pi late Pre coated a OK Ta ee a Ce ps i BS ua Hs Waal iN a f i pen eni IAN ENY SEs ji P a EW T a k E ey ha 3 C gt pa Positive Viral Antigen 5 z Negative Control Antigen Avoid touching bottom surfaces of wells as this may affect readings ELISA strips must be used only once Strip holders may be used again Dispose of all used materials as biohazardous waste A new pipette tip must be used for each sample never touching pipette tip to the bottom of the well If plastic troughs are used ensure that they have a dedicated purpose do not use the same trough for Peroxidase Conjugate and ABTS Peroxidase Substrate Wash Procedure Proper washing and aspiration of the stripwells is required to obtain accurate reliable results Wells should be washed 5 times after both Sample incubation and after Conjugate incubation 1 2 3 4 9 Aspirate wells into a waste flask Fill each well with Wash Solution Aspirate wells Repeat steps 2 and 3 for an additional 4 cycles Total 5 washes Invert plate and tap firmly on absorbent paper to remove excess liquid Take care not to dislodge strips Test Procedure All samples and Controls should be tested on both the positive viral antigen and the negative control antigen wells Use the enclosed record sheet to identify the location of each serum and type of strip or antigen used
7. mmended that each laboratory establish their own criteria for performance of these Research Reagents In our quality control testing we use the following criteria a The Negative Control Serum after subtracting the absorbance in the negative control antigen well should produce a net absorbance on the Positive Viral Antigen of lt 0 250 at 405 nm b The Positive Control Serum after subtracting the absorbance in the negative control antigen well should produce a net absorbance on the Positive Viral Antigen of 2 0 600 at 405 nm c A sample may be considered positive by the following criteria Determine the difference A between the sample absorbance at 405 nm on the Positive Viral Antigen well and the absorbance at 405 nm on the Negative Control Antigen well This difference A should be greater than or equal to 0 300 for a sample to be considered positive Example 1 Positive Sample Given a sample absorbance of 1 101 at 405 nm on the Positive Viral Antigen well and a sample absorbance of 0 190 at 405 nm on the Negative Control Antigen well The difference A between the above absorbances is 0 911 This difference is greater than or equal to 0 300 This sample is considered Positive Example 2 Negative Sample Given a sample absorbance of 0 347 at 405 nm on the Positive Viral Antigen well and a sample absorbance of 0 319 at 405 nm on the Negative Control Antigen well The difference A between the above absorbances is 0
8. water Mix gently by inverting several times to avoid excessive foaming When using an automated plate washer to ensure sufficient volume 1 x 60 ml of Wash Concentrate has been provided to allow for excess Wash Solution to prime the plate washer 2400 ml total Wash Solution after dilution Peroxidase Conjugate 2 vials 12 ml vial The Conjugate is provided ready to use One vial provides enough Conjugate for 12 strips 1 plate If more than 1 vial is required pool the contents of both vials in a clean glass or plastic screw cap container and mix genily by inverting several times to avoid excessive foaming Opened Conjugate is stable for 30 days Label and date the vial and store at 2 8 C ABTS Peroxidase Substrate 2 vials 12 ml vial Each vial contains 12 mls of 2 2 Azino di 3 ethyl benzthiazoline sulfonate solution The substrate is ready for use ABTS is stable for a minimum of one year at 2 8 C Stop Solution 1 vial 10 ml Contains 1 25 sodium fluoride CAUTION Avoid contact with eyes and skin If contact is made wash area with copious amounts of water and seek immediate medical attention SPECIMEN COLLECTION AND PREPARATION A Obtain blood and allow clot to form Insoluble materials should be removed by centrifugation Remove the serum aseptically Serum samples should be refrigerated as soon as possible after collection If not assayed within 48 hours the samples should be aliquotted and frozen Avoid repeated
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