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Place of Origin
HS-CODE
30-
Package & Delivery Lead Time
Detailed Description
Human Clostridium difficile (CD-IgG) ELISA Kit
FOR RESEARCH USE ONLY. Not for clinical diagnosis use
CATALOG #95786
INTRODUCTION
This kit allows for the determination of CD-IgG expression in human serum, and other biological fluids.
PRINCIPLE OF TEST
The kit is for the qualitative determination of CD-IgG in the sample, adopt purified antigen to coat microtiter plate, make solid-phase antigen, then pipette samples to the wells, with CD-IgG antigen 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 CD-IgG. 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 CD-IgG 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. Plasma-use suited EDTA or citrate plasma as an anticoagulant, centrifuge at the speed of 2000-3000 rpm for 20-min. Remove supernatant, if precipitation appeared, centrifuge again.
3. Urine-collect sue a sterile container, centrifugation 20-min at the speed of 2000-3000 rpm. Remove supernatant, if precipitation appeared, centrifugal again.
4. Cell culture supernatant-Detect secretory components, remove particulates by centrifugation for 20-min at the speed of 2000-3000 rpm. Remove supernatant detect the composition of cells, dilute cell suspension with PBS (PH7.2-7.4) to make cell concentration 1 million / ml, repeated freeze-thaw cycles, damage cells and release intracellular components, centrifugation 20-min at the speed of 2000-3000 rpm. Remove supernatant, if precipitation appeared, centrifugal again.
5. Tissue homogenates-after cutting samples, check the weight, pipette PBS (PH7.2-7.4), frozen with liquid nitrogen, maintain samples at 2-8℃ after melting. Pipette PBS (PH7.4), homogenized by hand or Grinders, centrifugation 20-min at the speed of 2000-3000 rpm. Remove supernatant.
6. 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.
7. 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 CD-IgG Negative control
Positive control: sample OD≥ Calculate Critical (CUT OFF) is CD-IgG 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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