Example of Semi-automatic Operation for Shibayagi s ELISA kits

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1 Example of Semi-automatic Operation for Shibayagi s ELISA kits Features and Caution in ELISA MeritYou can attain uniform operation process, and you can assay small volume and many samples with high sensitivity. You can choose your instruments depending on your financial situation. Of course it is necessary to use your automatic machines that are regularly maintained as for pipetting, reaction temperature, and washing which affects your results. Demerit Automatics are generally expensive and the regular maintenance are needed. Please consider any possibility of outsourcing of assay. Caution Our kits are not always same in assay protocols for standards, well-dispensing, reagents-dispensing, washing, and color former. Please check our instructions beforehand in our website, or read the instructions attached to kits carefully. Different ELISA kits from other companies have different protocols from ours. Handling of reagents Shibayagi s ELISA kits contain reaction stopper (diluted sulphuric acid: 1M H2SO4in them. Please read caution on the bottle label, and follow it. Also please read the instruction carefully about other reagents. FrontBack How to dispose reagent solutions and well-plate after assay Please dispose reagent solutions after assay according to the rules enacted by the country, state, city ou your institution. As reference: If you refer to the disposal rules suggested in our website, please discuss with your research safety leader or group leader and confirm if it is applicable. 1

2 Pipetting Please read instruction paper attached to your pipette and follow it strictly, and select an optimum volume pipette as recommended by the pipette maker. You can find information in catalogues provided by the makers about following items. Basic knowledge on dispensing, technique of dispensing, calibration, and hints in pipetting Please select your tip which is provided by your pipette maker, and use tip with minor protein adsorption. Before your assay, please confirm that the CV is within 1 % by your pipette for standards or samples when repeating ten times (calculate CV from each weight ). Please repeat pipetting at a constant pace. Especially, serum and plasma samples have higher viscosity, and as piston-operated tip-exchangeable pipette is used for sampling, please pipette at a slow pace. Please do not scratch the bottom or wall of wells by getting nervous. We don t recommend to use multi-channel pipettes (8 or 12 channels). If you prefer to use them, please make sure that all the tips are attached to the pipette equally and tightly before use, and that all the channels can work within permissible variation. Some part of the liquid in the tips pipette may be leaked if the tips are not attached to it correctly. Maintain your multi- channel pipette regularly to avoid the systematic error. Hold and treat multi-channel pipette in parallel with the plate. If not, the pipette may scratch the side or bottom of the wells and remove the coated antibody. Spilled liquid because of incorrect attachment of tip Tanning-up of bubbles and unequal liquid volumes Sample Collecting condition Please choose the appropriate way of collection from laboratory animals following the regulation of your institution. Sampling should be carried out to avoid various factors influencing assay system, such as hemolysis, chyle, bilirubin and fibrin. These factors may affect your sample assay. Treatment of serum or plasma samples should be done quickly. (Temporary storage in ice is also recommended.) Please make sure to check permissible anticoagulant and its concentration beforehand. Heparin is not recommended for collecting samples for Rat TSH ELISA KIT, anti-ds/ss DNA ELISA KIT Citric acid is not recommended for collecting samples for Human Apo B-48 ELISA KIT We recommend you to preserve serum or plasma at lower than -35 o C (if possible -80 o C). Depending on assay substances, storage condition may be different. Therefore, please check it beforehand. Please notice that some samples are inactivated by freezing-and-thawing cycles If you use collect blood under ether anesthesia, the antigen-antibody reaction might be affected by ether contamination in blood in some assay system. In such case, ether should be evaporated off before assay. 2

3 Sometimes insulin is contained in culture medium, so please prepare a standard curve using culture medium alone, and compare with a standard curve using assay buffer to check influence of culture components. Tissue extracts (with acid or ethanol) contain organic solvents. Evaporate any organic solvents in sample, and make the ph neutral. Please refer to How to Operate Shibayagi's ELISA Kits for details (power point Example of operation and apparatus needed When ELISA kits arrive from distributor First, check the condition of kits like temperature or any damage of container. Store kits in a refrigerator at 2-8 o C. Please keep kits away from cool air nozzle in the refrigerator because it may freeze kits. If you want to use a kit partly, take it out from the refrigerator and separate the necessary amount of reagents into glass or PP tubes. Then bring the reagents to room temperature. Please return the rest of reagents in the refrigerator right away. However, original standard, labelled antibody, and avidin-hrp have to be diluted with buffer. Therefore, let these reagents stay in a refrigerator until the buffer fully gets back to room temperature, and after that take out the reagents from a refrigerator and prepare them using the buffer. If the undiluted solution is left, cap the bottle tightly and put it in the refrigerator as soon as possible. If you use only a part of the well-plate, cut off the part of clear seal of 96 well plate with a cutter as you need, and take out the strips for use. Put the rest of strips in a refrigerator soon and store When you use a whole ELISA kit, take out the whole kit parts from the container and put on a table to warm up to room temperature. Sometimes temperature around laboratory table and air conditioner s setting are different. So, please confirm whether reagents are back to room temperature by touching with your hand before starting assay. 3

4 Example of necessaries for ELISA operating (Some can be replaced by automatic apparatus.) Example of operation of Rat Insulin ELISA KIT (TMB) Preparation of reagents Washing buffer (concentrated) ( I )Dilute washing buffer (concentrated) to x10 by purified water. Take a necessary volume of concentrated washing buffer. Transfer it to a volumetric cylinder containing purified water (a little less than a necessary volume). Add some water to the necessary volume. Transfer the mixture to a beaker, put a stirrer, and mix. Transfer the diluted washing buffer to a jet bottle. Standard solution (B)Dilute insulin standard by buffer (C) Please refer to Pippetting caution on page 2. 4

5 Dilute insulin standard by buffer. Conc. (ng/ml) Standard(µl)Orig.: * 100* 100* 100* 100* 100* 0 Buffer (µl) Standard solution of one rank higher concentration An example of Work station which can be used for preparation of standard solutions, biotin-conjugated anti-insulin antibody solution and HRP-conjugated streptavidin solution Also for sample dilution Regular maintenance needed. (For details, ask maker or vendor.) Special tips from manufacturer will fit to the pipette. Operation (A) Anti-insulin coated 96 well plate Remove the clear seal of plate after getting back to room temperature. If you peel it while it is still cool, some white stickiness remains on the plate. Each strip should be numbered in order to get it back in case it gets out of frame. Washing 4 times Use an automatic washe as is described below. Ask a technician of the machine vendor to adjust the suction / exhaust pressure most suitable for Shibayagi s well-plate (Nunc, MaxiSorp). Ask the technician to install the operation program that shall not make wells dry. The flow-rate should be adjusted, if possible, to be 5-25 ml/min. It is dependent on the diameter of the nozzle, and often is not adjustable. The performance of the machine should give the OD of blank well is obviously smaller than that of 5

6 the lowest standard. After treatment with the washing machine, check the plate if all the wells are clear of washing buffer. Maintain all the nozzles and exhaust pipes by checking and washing them regularly. If you find some drops of washing buffer in wells, remove the remaining liquid by striking the reverse held plate onto folded several sheets of Kimwipe (2-3 cm-thick). Be careful, too strong-striking may disintegrate well-plate. Taking care not to dry the well, proceed to the next step as soon as possible. You had better to use Kimtowel or paper towel whose thickness is 2-3 cm because they could soften the beating impact to hands and are useful to prevent wells from dropping out of frame. In the following picture, you can see wash buffer is still in the wells. After discarding the wash buffer and strike, if some washing buffer drops remain in the bottom of wells, remove them by touching small piece of kimwipe. (D) Biotin-labelled anti-insulin antibody 100µl/well Sample or each insulin standard solution 10µl/well Please refer to Pippetting of Caution. We recommend you to add 1-2 quality control (QC) serum to your samples. As QC, you can use stored samples the contents of which are already known through previous assays. An example of Work station which can deliver standard solutions to wells, and also reagent solutions. Refer to statements about work station described above for the caution. 6

7 Mixing Mix rpm, 10 seconds x 3times with some dynamics. Incubation at room temperature for 2 hours Check the temperature of incubator beforehand and incubate well-plate at o C for 2 hours. Evaporation of solvent of reaction mixture during incubation caused by air stream on microplate,especially in warm and dry ventilation air in winter, may influence on reactions and non-specific coloration. To avoid such effects, cover wells with Prafilm and/or attached plate cover. Incubator Thermometer Timer Washing 4 times Refer to statement of washing above for caution. (E) HRP-conjugated streptavidin 100µl/well Refer to Sample or Standard above for 96 wells dilution. 7

8 Mix Refer to Mixing above. Incubation at room temperature for 30 minutes Refer to Incubation at room temperature for 2 hours above. Washing 4 times Refer to How to remove wash buffer above. (F) Chromogenic substrate reagent (TMB) 100µl/well Refer to Sample or Standard for 96 well dilution above. When TMB reagent reacts with enzyme (HRP), the color of the reaction mixture becomes pale blue. Mixing Refer to Mixing above. Incubation at room temperature for 30 minutes Refer to Incubation at room temperature for 2 hours above. (H) Reaction stopper (1M H2SO4) 100µl/well Please be careful in treating this reagent since it is dilute sulfuric acid. Please refer to Handling and caution of HRP-conjugated streptavidin above. When reaction stopper is added, the color turns to yellow or orange from blue. 8

9 Mixing Please refer to Mixing above. Some 96 wells plate readers have a mixing function. Measurement of absorbance (Wave: 450nm, sub.620nm) It is possible to change sub wave length in range of nm. Sample absorbance = Absorbance at 450nm-Absorbance at 620nm. 96 wells micro plate reader Useful information For our products list, please visit: How to operate Shibayagi s ELISA kits, please visit: ** We have more of useful information! Please visit: If you have questions, please feel free to ask us: syc-info@shibayagi.co.jp Ver. 1: Shibayagi Co., Ltd Ishihara, Shibukawa, Gunma, Japan Tel: Fax: syc-info@shibayagi.co.jp 9