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    Home > Biochemistry News > Microbiology News > Instructions for the use of color-showing substation kits (end-point color-showing method, with ad parity reagents)

    Instructions for the use of color-showing substation kits (end-point color-showing method, with ad parity reagents)

    • Last Update: 2021-01-23
    • Source: Internet
    • Author: User
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    color-
    reagent
    (Chromogenic End-point Tachypleus Amebocyte Lysate, CE TAL) is used for quantitative testing of endobacterial endotoxins in the body and is prohibited from entering the body in any way.Principle
    The reagent is a frozen
    drine
    product of the blood deformed cell dissolution of the oriental dragonfly of the animal, which contains C factor, B factor, coagulation enzyme, coagulation proteogen, etc. Under suitable conditions (temperature, pH and non-disturbing substances), bacterial endotoxin activates the C factor, causing a series of enzymatic reactions, activates the coagulase to form a coagulase, and the coagulase breaks down the synthetic color-showing substate, breaking it down into
    peptides
    and yellow nitrobenzene (pNA, xenx s 405 nm). Over a period of time, the amount of pNA produced is positively related to the concentration of bacterial endotoxins, according to which the endotoxin concentration of the test products can be quantified. At the same time, nitrobenzene (pNA) can also be dyed rose red with a pair of anitride reagents (max s 545 nm), avoiding interference with the absorption peak at 405 nm by the color of the test itself.
    detection limit
    0.1EU/ml-1EU/1EU and 0.01EU/ml-0.1EU/ml can be detected by reaction time (reaction time T1 and T2 can be found in the factory inspection report).(Kit composition)
    bacterial endotoxin work products, 2
    reagents, 1.7 ml/branch, 2
    color-showing substations, 1.7 ml/branch, 2
    auspicturic reagents 1,10 ml/branch, 2
    -atriums 2,10 ml/branch, 2
    -a-coupled nitriding reagents 3,10 ml/branch, 2
    HCl (reaction terminator), 50 ml/bottle, 1 bottle
    bacterial endotoxin inspection water, 50 ml/bottle, 2 bottlesstorage
    shade, optimum 2-8 degrees C, light storage.usage:1.Materials and equipment
    (1) reagents
    radon reagents, bacterial endotoxin work products, color-showing substations, atrial nitriding reagents 1, atrial nitriding reagents 2, atrial nitriding reagents 3, HCl (reaction terminator), bacterial endotoxin inspection water.
    (2) equipment
    heat-free test tube, no heat-raw suction head. Vortex mixer,
    pipe pipe
    , multi-channel piper, seal film, test tube rack.
    temperature
    water bath (37±1 degrees C), gradometer.
    note: contact reagents and all utensils for testing must be heat-free (our company provides heat-free
    supplies
    ). Glassware can be dried at 250 degrees C for at least 60 minutes to remove the heat.
    2.Storage and pre-treatment of test products
    Note: If the test products for blood, please refer to our factory supporting blood-related treatment reagents instructions.
    (1) the pH of the test should be between 6 and 8, and if this range is exceeded, it should be regulated with a heat-free buffer, 0.1 N sodium hydroxide, or 0.1 N hydrochloric acid.
    (2) If there may be interference substances in the test subject, the treatment can be found in the interference test of the test.
    (3) If the test products may contain β-glucosaccharides (β-glucosaccharides will produce g-factor bypass reaction, interfere with endotoxin detection), the use of specific reagents (provided by our company).
    (4) If the test is available for some antibiotics such as cephalosporins and sulfonamide preparations will produce a conjoined reaction to aloin nitrogen dyes and interfere with atrial nitride coloring, need to choose a kit free of amerinous nitride reagents (provided by our company).
    (5) If the background absorbent value of the test product is greater than 0.5, the test product must be diluted before detection.
    (6) If the color of the test is dark (e.g. hemolytic), or if the color is darker under acidic conditions (e.g. some
    tissues
    training
    media), a test subject blank tube must be set up to deduct the color background of the test subject itself.
    the test tube is not added to the reagent, to the same volume of radon reagent solvent (where the bacterial endotoxin inspection water) instead. The rest of the operation is the same as the test tube.
    3. Experimental operation
    (1) Bacterial endotoxin standard solution preparation
    the standard curve can be used endotoxin concentrations of 0.01, 0.025, 0.05, 0.1 EU/ml or 0.1, 0.25, 0.5, 1.0 EU/ml. The dilution method is as follows (in 0.1, 0.25, 0.5, 1.0 EU/ml for example: take 1 bacterial endotoxin work product, dilute the endotoxin solution to 10EU/ml according to the instruction manual of bacterial endotoxin work product, and then dilute to 1.0EU/ml endotoxin solution, dilute to 0.1,0.25 for the parent table with 1.0EU/ml endotoxin solution. 0.5, 1.0 EU/ml concentration gradient. The preparation of the endotoxin standard solution should be used up within 4 hours. (2) negative control for bacterial endotoxin inspection water.
    (3) dissolved
    reagents such as anti-lycolor reagents, color-showing substations, azithromid reagents, etc.: according to the indicated amount plus bacterial endotoxin inspection water in the radon reagent, gently shake so that the radon reagent completely dissolved, pay attention not to use the vortex mixer to shake violently. Dissolved radon reagents should be used up within 10 minutes.
    color-showing substate: according to the indicated amount plus bacterial endotoxin inspection water in the color-showing substate, gently shake so that the color-showing substate completely dissolved. The color-showing substation solution can be stored for up to 8 hours at 4 degrees C under non-polluting conditions.
    ades atrium nitriding reagent 1: according to the indicated amount plus reaction terminator hydrochloric acid in adage nitrid reagent 1;
    A2: Water for agatoxic endotoxin inspection is used in agatoxic reagent 2 according to the indicated amount;
    A-nitrid reagent 3: Water for agatoxic endotoxin inspection is used for abacterium endotoxin 3 by indicated amount; and
    A-nitrid reagent solution can be stored for 1 week at 4 degrees C.
    (4) experimental operation
    take the thermogen-free test tube, add 100 ml of bacterial endotoxin to check water, endotoxin standard solution, or for test products. Add another 100 ml of reagent solution, mix well, and warm T1 minutes at 37 degrees C.
    the end of warmth, add 100 ml of color-showing substation solution, mix well, 37 degrees C temperature-breeding T2 minutes. At the end of the temperature, add 500 ml anitridization reagent 1 solution, mix well, add 500 ml anitrium reagent 2 solution, mix and add 500 ml anitrium reagent 3 solution, mix well, sit still for 5 minutes, read the absorbent value at 545 nm wavelength. (see table 2) Mix well, set aside for 5 minutes, and read the absorbent value at 545 nm
    4. Data processing
    establishes a standard curve: Y s b X s a, where Y is the absorbance value at 545nm, X is the concentration of endotoxins, b is the straight line slope, and a is the Y-axis intercept.
    the experiment is valid when the experimental data meet three conditions at the same time:
    1. The correlation coefficient of the standard ≥ is 0.980,
    2. The Y value at the lowest point of the standard curve is greater than the Y value of the negative control,
    3. The average value of the parallel tubes for the test is within the interval of the standard curve.
    interference test for test products
    can be found in the "photometric interference test" of the Bacterial Endotoxin Inspection Law in the 2005 edition of the Pharmacopoeia of the People's Republic of China. Interference tests shall be carried out in the event of changes in the reagents, the source of the test, the formulation, changes in the production process or any changes in the test environment that may affect the test results, in the following steps: 1. Select the endotoxin concentration (set to xym) near the mid-point of the standard curve, as the endotoxin concentration added in the test interference test, prepare a positive control of the test containing the xym endotoxin, and measure the endotoxin concentration of the solution, called Cs;
    2. Measured the concentration of endotoxins in the solution of the test subject without the addition of exon endotoxins,
    3. It is calculated that the recovery rate under the test conditions is R (Cs-Ct)/m× 100%
    4. When R is between 50% and 200%, it is considered that there is no interference in the solution of the test under this test condition.
    5. When R is outside 50% to 200%, the series dilution (dilution multiplication shall not exceed the maximum effective dilution multiplication MVD) or other treatment to eliminate interference, each dilution solution repeats steps 1-3, until the recovery rate of endotoxin R is between 50% and 200%. Select a dilution multiply with a recovery rate of R closest to 100% for endotoxin detection.
    No. SJ-02
    Sales Enterprise, Shanghai Yubai Biotechnology Co., Ltd. 021-51602548
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