Human Francisella Tularensis IgM ELISA Kit

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Human Francisella Tularensis IgM ELISA Kit FOR RESEARCH USE ONLY. Not for clinical diagnosis use CATALOG #:95803 INTRODUCTION This kit allows for the determination of FT IgM expression in human serum, and other biological fluids. PRINCIPLE OF TEST The kit is for the qualitative determination of FT IgM in the sample, adopt purified antigen to coat microtiter plate, make solid-phase antigen, then pipette samples to the wells, with anti-human IgM conjugated Horseradish Peroxidase (HRP). Wash and remove non-combinative antibody and other components. Antibodies specific for the antigen will bind to the pre-coated antigen. After washing completely, add TMB substrate solution and color develops according to the amount of FT IgM. Reaction is terminated by the addition of a stop solution and the intensity of the color is measured at a wavelength of 450 nm. Compared with the CUTOFF value to judge if FT IgM exists in the sample or not. COMPOSITION OF THE KIT Reagent Quantity Microelisa Stripplate 12well×8strips Positive control 1×0.5ml Negative control 1×0.5ml HRP-Conjugate Reagent 1×6ml Sample Diluent 1×6ml Chromogen Solution A 1×6ml Chromogen Solution B 1×6ml Stop Solution 1×6ml Wash Solution 1×20ml User manual 1 Adhesive Strip 2 Sealed bag 1 STORAGE CONDITIONS  The unopened kit shall be stored at [2-8 ℃] .  For opened kit can be stored at [2-8 ℃] for up to 1 month. If not be used recently, the standard should be kept in -20 ℃. WASHING METHOD  Manually washing method: shake away the remained liquid in the enzyme plates; place some bibulous papers on the test-bed, and flap the plates on the upside down strongly. Inject at least 0.35ml after-dilution washing solution into the well, and marinate 1~2 minutes. Repeat this process according to your requirements.  Automatic washing method: if there is automatic washing machine, it should only be used in the test when you are quite familiar with its function and performance. SAMPLE PREPARATION 1. Serum-coagulation at room temperature for 10-20 min, centrifuge at the speed of 2000-3000 rpm for 20-min. Remove supernatant, if precipitation appeared, centrifuge again. 2. Extract as soon as possible after samples collection, and should be tested as soon as possible after the extraction. If not, samples can be kept in -20 ℃.Avoid repeated freeze-thaw cycles. 3. Don’t detect the samples which contain NaN3, because NaN3 inhibits HRP activity. ASSAY PROCEDURE Step 1: Number: determine the number of well to be used and store unused wells in 4 ℃. Set a blank well without any solution. Step 2: Prepare sample: pipette Positive control and Negative control 50μl to the well respectively. Controls need test in duplicate. Pipette sample dilution 40μl and testing sample 10μl to testing sample well. Pipette sample to the bottom of well, don’t touch the wall as far as possible, and mix gently. Step 3: Incubate: Cover with the adhesive strip provided, incubate for 30 min at 37℃. Step 4: Configurate liquid: Dilute wash solution 30-fold (or 20-fold) with distilled water. Step 5: Washing: Uncover the adhesive strip, discard liquid, pipette washing buffer to every well, still for 30s then drain, repeat 5 times. Step 6: Add enzyme: Pipette HRP-Conjugate reagent 50μl to each well, except blank well. Step 7: Incubate: Operation with 3. Step 8: Washing: Operation with 5. Step 9: Color: Pipette Chromogen Solution A 50ul and Chromogen Solution B to each well, avoid the light preservation for 15 min at 37℃ Step 10: Stop the reaction: Pipette Stop Solution 50μl to each well, stop the reaction (the blue change to yellow). Step 11: Calculate: take blank well as zero. Read absorbance at 450nm after pipette Stop Solution within 15min. CALCULATION OF RESULT Test validity: the average of Positive control well≥1.00; the average of Negative control well ≤0.10. Calculate Critical (CUT OFF): Critical= the average of Negative control well + 0.15. Negative control: sample OD< Calculate Critical (CUT OFF) is FT IgM Negative control Positive control: sample OD≥ Calculate Critical (CUT OFF) is FT IgM Positive control. EXPIRATION Six months [see label on the outer box for the specific date]. ATTENTION  The kit takes out from the refrigeration should be balanced 15-30 minutes in the room temperature, if the coated ELISA plates have not been used up after opening, the plate should be stored in sealed bag.  Washing buffer will Crystallization separation, it can be heated in water to dissolve.  Pipette sample with pipettors each step, and proofread its accuracy frequently to avoid the experimental error. Pipette sample within 5 min, if the number of sample is big, recommend using multichannel pipettor.  If the testing material concentration is excessively high (The sample OD is higher than the first standard well)，please dilute the sample (n-fold).  Adhesive Strip only limits the disposable use to avoid cross-contamination.  The substrate should evade the light to be preserved.  Please refer to the user instruction strictly, the test result determination must take the microtiter plate reader as a standard.  The preparation of samples and all the reagents should refer to infective material process.  Do not mix reagents with those from other lots.

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