Application PishtazTeb Company ELISA CHECK is used for ELISA washing machine performance verification, accuracy and linearity of microplate reader and also to check precision of micropipettes. This kit provides necessary documents for equipment calibration and monitoring of ELISA kit process and also useful for detection and troubleshooting of problems during test procedure. Introduction Equipment like ELISA washer, microplate reader and micropipettes has critical role in ELISA test performance with direct influence on accuracy and precision and eventually reliability of results. Equipment performance should be monitor on time schedule interval, after repair and also in follow up of unacceptable results. ELISA microplate reader for example should be checked monthly for linearity and its accuracy evaluated on 3 months time schedule period. Test principle ELISA microplate reader accuracy The test used to determinee the difference between TRUE OD values with OD value obtained by microplate reader. The test principle is based on color reagents with known OD value (adjusted by calibrated readers). ELISA microplate linearity The test goal is to determine relationship between concentration and OD value by using color reagents with linear relationship between concentration and OD value at 450 nm. ELISA microplate and Micropipette precision The goal of this test is to determine any sensor malfunctioning or problem in light pathway of reader by using color reagent with certain OD value at 450 nm. Washing process assessment The test is based on nonspecific binding. If washing take place inadequately the OD value will increased more than acceptable limit. If washing performed appropriately, unacceptable OD may be due to ELISA washing machine malfunctioning. Pishtaz Teb Diagnostics 1 MA_ELISA CHECK_02
Kit contents 1. ELISA microplate (96 wells) 2. Yellow color reagents (A1, A2, A3) for ELISA microplate reader accuracy. TRUE OD value is printed on vials; each vial contains 4 ml of reagent. 3. One red color reagent (code L) to investigate ELISA microplate reader linearity; 2 ml volume. 4. One vial containing ready to use enzyme conjugate, 4 ml volume 5. One vial containing chromogen, 4 ml volume. 6. One vial containing stop solution, 4 ml volume. 7. One vial containing 50 ml of concentrated washing solution (20 X). To prepare working solution dilute the reagent 1:20 with distilled water. Materials not provided with kit 1. ELISA microplate reader with 450 nm reading filter (those filter are preferred) 2. Precise 100 l micropipette 3.Distilled water with 630 nm reference Storage condition 1. Kit should be stored at 2-8 o C upon receipt and when it is not in use. 2. Do not use expired date reagents. 3. Do not freeze. 4. Protect from light and moisture. General Information 1. Do not mix kit reagents from different batch/lot numbers. All kit components must be used only in their original kit. 2. Before preceeding with the test, bring all reagents and samples to room temperature. 3. Once the assay has been started, all subsequent steps should be performed without interruption. 4. The results should be read within 30 minutes of adding the stop solution. Test Procedure 1. ELISA microplate reader accuracy check Remove three strips and add 100 l of A1 reagent into all wells of first strip, followed by 100 l of A2 reagent and 100 l of A3 reagent into all wells of strip 2 and 3 respectively. Read OD of each strip at 450/630 nm and compare the results with value printed on each vial. Significant changes need further study. Pishtaz Teb Diagnostics 2 MA_ELISA CHECK_02
To evaluate ELISA microplate reader accuracy, we test each color reagent separately on three calibrated ELISA microplate reader machine. Table 1 is showing mean OD value for each machine. A1 A2 A3 Acceptable limit (OD) 0.20-0.29 0.9-1.1 2.4-2.8 Bio Tek (ELx800) 0.24 0.97 2.65 ASYS HITECH H 0.25 1.0 2.66 TECAN (Sunrise) 0.24 0.97 2.55 2. Micropipette and ELISA microplate reader precision Calculate mean and standard deviation of ODs for all above color reagent (A1-A3) and determined Coefficient of Variation (CV). Acceptable CV is lower or equal to 3% ( 3%). 3. Linearity of ELISA microplate reader Take two strip of microplate and add 100 l of red color reagent code L into two first wells of first strip and also 100 l of distilled water to the remaining wells. Add 100 l of red color reagent code L into the third and fourth wells of first strip and after mixing transfer 100 l to the next two wells, mix again and make a serial dilution in duplicate format until end of wells and discard the final 100 l. Make sure that each well contains 100 l of color reagent and also take care to avoid bubble formation during serial dilution preparation. Read OD of wells at 450 nm with 630/620 nm filter as reference filter and calculate mean of each duplicate at each dilution level. The microplate reader should be linear at each manufacturer claimed dilution level. The first two wells OD used as reference for calculation of expected OD of remaining wells. Recovery limit ranged between 90 to 110%. To study linearity of ELISA microplate reader the test procedure run on three different ELISA reader and results are shown on Table 2. Table 2. Dilution 1 1:2 1:4 1:8 1:16 1:32 TECAN (Sunrise) 2.58 1.29 0.665 0.339 0.182 0.09 ASYS HITECH 2.438 1.222 0.610 0.306 0.154 0.077 Bio Tek 2.498 1.272 0.638 0.329 0.164 0.085 Pishtaz Teb Diagnostics 3 MA_ELISA CHECK_02
Estimation of Recovery a. Assume mean OD value of first two wells (red color reagent code L) as 100% referencee value. b. Dividing this OD value by 2, 4, 8, 16 and 32 to obtain expected OD values. c. Divide mean OD value of each dilution level into related above expected OD value 100. Recovery formula ObtainedOD Recovery percentage = x 100 ExpectedOD For example mean OD value of Biotech ELISA microplate reader in table 2 is 2.498 which consider as 100% reference value. The expected OD at 1:2 dilution level is 2.498 2 = 1.249 but in table the read value is 1.272. To calculate the recovery percentage: Recovery percentage: 1.272 1.249 100 = 101% 4. Washing machine monitoring Remove one strip and add 100 l conjugate solution into all wells of one strip. Incubate wells for 5 minutes at room temperature then wash the wells 4 times with working wash solution. Add 100 l of chromogen substrate and incubate 5 minutes at room temperature in dark. Stop the reaction by adding 100 l of stop solution into each well. The OD value should be less than 0..1 in each well. Higher values indicate improper washing. To find possible causes, check the washing solution preparation, washing aspiration and discharge status. Pishtaz Teb Diagnostics 4 MA_ELISA CHECK_02
It is recommended to run one strip separately in each working run as kit internal control to ensure kit performance. For this case to ensure kit reagent working properly run one strip with two times washing which should resulted in OD value of greater than 0.1. Warning: This kit is not used for calibration of ELISA microplate reader and unacceptable results obtained with this kit need to be evaluated by instrument manufacturer service department. Pishtaz Teb Diagnostics 5 MA_ELISA CHECK_02