Sandwich enzyme immunoassay for in vitro quantitative measurement of CRP in human serum, plasma, tissue homogenates, cell lysates, urine, cerebrospinal fluid, cell culture supernates and other biological fluids

The kit is a sandwich enzyme immunoassay for in vitro quantitative measurement of CRP in human serum, plasma, tissue homogenates, cell lysates, urine, cerebrospinal fluid, cell culture supernates and other biological fluids. [ RAW MATERIAL ] Standard Capture Ab Immunogen of capture Ab Detection Ab Immunogen of detection Ab Native protein mAb Native protein mAb Native protein [ REAGENTS AND MATERIALS PROVIDED ] Reagents Quantity Reagents Quantity Pre-coated, ready to use 96-well strip plate 1 Plate sealer for 96 wells 4 Standard 2 Standard Diluent 1×20mL Detection Reagent A 1×120μL Assay Diluent A 1×12mL Detection Reagent B 1×120μL Assay Diluent B 1×12mL TMB Substrate 1×9mL Stop Solution 1×6mL Wash Buffer (30 × concentrate) 1×20mL Instruction manual 1 [ MATERIALS REQUIRED BUT NOT SUPPLIED ] 1. Microplate reader with 450 ± 10nm filter. 2. Single or multi-channel pipettes with high precision and disposable tips. 3. Microcentrifuge Tubes. 4. Deionized or distilled water. 5. Absorbent paper for blotting the microplate. 6. Container for Wash Solution. 7. 0.01mol/L (or 1×) Phosphate Buffered Saline(PBS), pH7.0-7.2. [ STORAGE OF THE KITS ] 1. For unused kit: All the reagents should be kept according to the labels on vials. The Standard, Detection Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20oC upon receipt while the others should be at 4oC. 2. For used kit: When the kit is used, the remaining reagents need to be stored according to the above storage condition. Besides, please return the unused wells to the foil pouch containing the desiccant pack, and zip-seal the foil pouch. Note: It is highly recommended to use the remaining reagents within 1 month provided this is prior to the expiration date of the kit. For the expiration date of the kit, please refer to the label on the kit box. All components are stable up to the expiration date. [ SAMPLE COLLECTION AND STORAGE ] Serum - Use a serum separator tube and allow samples to clot for two hours at room temperature or overnight at 4oC before centrifugation for 20 minutes at approximately 1,000×g. Assay freshly prepared serum immediately or store samples in aliquot at -20oC or -80oC for later use. Avoid repeated freeze/thaw cycles. Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1,000×g at 2-8oC within 30 minutes of collection. Remove plasma and assay immediately or store samples in aliquot at -20oC or -80oC for later use. Avoid repeated freeze/thaw cycles. Tissue homogenates - The preparation of tissue homogenates will vary depending upon tissue type. 1.Tissues were rinsed in ice-cold PBS to remove excess blood thoroughly and weighed before homogenization. 2.Minced the tissues to small pieces and homogenized them in fresh lysis buffer (catalog: IS007, different lysis buffer needs to be chosen based on subcellular location of the target protein) (w:v = 1:20-1:50, e.g. 1mL lysis buffer is added in 20-50mg tissue sample) with a glass homogenizer on ice (Micro Tissue Grinders woks, too). 3.The resulting suspension was sonicated with an ultrasonic cell disrupter till the solution is clarified. 4.Then, the homogenates were centrifugated for 5 minutes at 10,000×g. Collect the supernatant and assay immediately or aliquot and store at ≤-20oC. Cell Lysates - Cells need to be lysed before assaying according to the following directions. 1. Adherent cells should be washed by cold PBS gently, and then detached with trypsin, and collected by centrifugation at 1,000×g for 5 minutes (suspension cells can be collected by centrifugation directly). 2. Wash cells three times in cold PBS. 3. Resuspend cells in fresh lysis buffer with concentration of 107 cells/mL. If it is necessary, the cells could be subjected to ultrasonication till the solution is clarified. 4. Centrifuge at 1,500×g for 10 minutes at 2-8oC to remove cellular debris. Assay immediately or aliquot and store at ≤-20oC. Urine - Aseptically collect the first urine of the day (mid-stream), voided directly into a sterile container. Centrifuge to remove particulate matter, assay immediately or aliquot and store at ≤-20oC. Avoid repeated freeze-thaw cycles. Cerebrospinal fluid (Csf)- Remove particulates by centrifugation and assay immediately or aliquot and store samples at ≤-20oC. Avoid repeated freeze-thaw cycles. Cell culture supernatants and other biological fluids - Centrifuge samples for 20 minutes at 1,000×g. Collect the supernant and assay immediately or store samples in aliquot at -20oC or -80oC for later use. Avoid repeated freeze/thaw cycles. Note: 1. Samples to be used within 5 days may be stored at 4oC, otherwise samples must be stored at -20oC (≤1 month) or -80oC (≤2 months) to avoid loss of bioactivity and contamination. 2. Sample hemolysis will influence the result, so hemolytic specimen should not be used. 3. When performing the assay, bring samples to room temperature. 4. It is highly recommended to use serum instead of plasma for the detection based on quantity of our in-house data. [ REAGENT PREPARATION ] 1. Bring all kit components and samples to room temperature (18-25oC) before use. If the kit will not be used up in one time, please only take out strips and reagents for present experiment, and leave the remaining strips and reagents in required condition. 2. Standard - Reconstitute the Standard with 1.0mL of Standard Diluent, kept for 10 minutes at room temperature, shake gently(not to foam). The concentration of the standard in the stock solution is 40,000pg/mL. Please firstly dilute the stock solution to 4,000pg/mL and the diluted standard serves as the highest standard (4,000pg/mL). Then prepare 7 tubes containing 0.5mL Standard Diluent and use the diluted standard to produce a double dilution series according to the picture shown below. Mix each tube thoroughly before the next transfer. Set up 7 points of diluted standard such as 4,000pg/mL, 2,000pg/mL, 1,000pg/mL, 500pg/mL, 250pg/mL, 125pg/mL, 62.5pg/mL, and the last EP tubes with Standard Diluent is the blank as 0pg/mL. Detection Reagent A and Detection Reagent B - Briefly spin or centrifuge the stock Detection A and Detection B before use. Dilute them to the working concentration 100-fold with Assay Diluent A and B, respectively. 4. Wash Solution - Dilute 20mL of Wash Solution concentrate (30×) with 580mL of deionized or distilled water to prepare 600mL of Wash Solution (1×). 5. TMB substrate - Aspirate the needed dosage of the solution with sterilized tips and do not dump the residual solution into the vial again. Note: 1. Making serial dilution in the wells directly is not permitted. 2. Prepare standards within 15 minutes before assay. Please do not dissolve the reagents at 37oC directly. 3. Please carefully reconstitute Standards or working Detection Reagent A and B according to the instruction, and avoid foaming and mix gently until the crystals are completely dissolved. To minimize imprecision caused by pipetting, use small volumes and ensure that pipettors are calibrated. It is recommended to suck more than 10μL for one pipetting. 4. The reconstituted Standards, Detection Reagent A and Detection Reagent B can be used only once. 5. If crystals have formed in the Wash Solution concentrate (30×), warm to room temperature and mix gently until the crystals are completely dissolved. 6. Contaminated water or container for reagent preparation will influence the detection result. [ SAMPLE PREPARATION ] 1. We are only responsible for the kit itself, but not for the samples consumed during the assay. The user should calculate the possible amount of the samples used in the whole test. Please reserve sufficient samples in advance. 2. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their particular experiments. 3. Serum/plasma samples require about a more than 500 or 500 fold dilution. For example, to prepare a 1:500 dilution of sample, transfer 20μL of sample to 180μL PBS. This yields a 1:10 dilution. Next, dilute the 1:10 sample by transferring 10μL to 490μL PBS. You now have a 1:500 dilution of your sample. Mix thoroughly at each stage. Sample should be diluted by 0.01mol/L PBS(PH=7.0-7.2). 4. If the samples are not indicated in the manual, a preliminary experiment to determine the validity of the kit is necessary. 5. Tissue or cell extraction samples prepared by chemical lysis buffer may cause unexpected ELISA results due to the impacts from certain chemicals. 6. Due to the possibility of mismatching between antigen from other origin and antibody used in our kits (e.g., antibody targets conformational epitope rather than linear epitope), some native or recombinant proteins from other manufacturers may not be recognized by our products. 7. Influenced by the factors including cell viability, cell number or sampling time, samples from cell culture supernatant may not be detected by the kit. 8. Fresh samples without long time storage is recommended for the test. Otherwise, protein degradation and denaturalization may occur in those samples and finally lead to wrong results. [ ASSAY PROCEDURE ] 1.Determine wells for diluted standard, blank and sample. Prepare 7 wells for standard, 1 well for blank. Add 100μL each of dilutions of standard (read Reagent Preparation), blank and samples into the appropriate wells. Cover with the Plate sealer. Incubate for 1 hour at 37oC. 2. Remove the liquid of each well, don’t wash. 3. Add 100μL of Detection Reagent A working solution to each well, cover the wells with the plate sealer and incubate for 1 hour at 37oC. 4. Aspirate the solution and wash with 350μL of 1× Wash Solution to each well using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher, and let it sit for 1~2 minutes. Remove the remaining liquid from all wells completely by snapping the plate onto absorbent paper. Totally wash 3 times. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against absorbent paper. 5. Add 100μL of Detection Reagent B working solution to each well, cover the wells with the plate sealer and incubate for 30 minutes at 37oC. 6. Repeat the aspiration/wash process for total 5 times as conducted in step 4. 7. Add 90μL of Substrate Solution to each well. Cover with a new Plate sealer. Incubate for 10 - 20 minutes at 37oC (Don't exceed 30 minutes). Protect from light. The liquid will turn blue by the addition of Substrate Solution. 8. Add 50μL of Stop Solution to each well. The liquid will turn yellow by the addition of Stop solution. Mix the liquid by tapping the side of the plate. If color change does not appear uniform, gently tap the plate to ensure thorough mixing. 9. Remove any drop of water and fingerprint on the bottom of the plate and confirm there is no bubble on the surface of the liquid. Then, run the microplate reader and conduct measurement at 450nm immediately.