1. ELISA test effectiveness and three controls
What is "Validity"? To obtain an accurate "Validity" evaluation of the test result, it will be related to: Why are the three indispensable controls such as "negative, positive and blank" set up? What kind of The substance serves as a "negative, positive and blank control (product)"? How to set up the "three controls", need to set a few well positions? How to "approval, trade-off and calculation" of the measured value obtained? Then, how to follow the positive control What is the difference between the mean value (PCx) and the negative control mean value (NCx) (PN, or NP) to make a final decision on the "validity of the test results"?… And so on The ELISA reagent "Instruction Manual" has detailed and detailed instructions. There are both theoretical key points and specific and detailed explanations. It is a pity that when the ELISA detection boom started in China around 90 years, in the voice of "foreign reagent instructions are too detailed, too complicated, not easy to understand, ..." and so on, it just caters to domestic businesses. "Will". Since then, the instructions for domestic ELISA reagents have become increasingly simple. There are two "benefits" among merchants: one of the benefits is to use A4, or B5, or even a piece of paper smaller than B5, instead of several pages of "instructions" for international reagents (equivalent to a "operation" "Manual"), can save a large amount of printing costs every year; the second advantage is that, as the international reagent instructions explain to the users the detailed content indicating the quality of this reagent, the so-called "instructions" for domestic reagents can be " "Delete, avoid, reject" to inform and promise users. It is a pity that I have collected the original manuals of the relevant reagents such as <美> Abbott, <French> Pasteur, <Dutch> Aksu, etc. that I collected around 90 years ago. Recently, a friend of the Hangzhou Quarantine Bureau sent a Chinese version of "Human Immunodeficiency Virus Antigen Antibody Diagnostic Kit (Enzyme-linked Immunoassay) User's Manual" in Chinese. After comparing and reading, I deeply feel that this is a real "User's Manual"! I can find answers to several "concepts and questions" mentioned below. I now refer to the relevant content of my 1993 "Hepatitis ELISA Reagent Quality and Test Results Effectiveness Evaluation" conference presentation, and express the following understanding and discussion on "Three Controls and Test Results Effectiveness". 2. Three control requirements and use ELISA test, according to the requirements of the reagent instructions, each test, each board must be set up the following three control and use: 5ZJ8Yh>
1. Blank control: only use the diluent instead of the test sample to observe the "background" color of the final reaction, and use it to eliminate and deduct the "background" of the microplate reader, that is: read with the background as "zero" Absorbance values ​​of positive and negative control wells and sample detection wells (A); or, subtract the background absorbance of the blank control when calculating the negative control mean (NCx), positive control mean (PCx), and S / CO value of the sample reading value. The microplate reader in the early years did not automatically deduct the reading of the blank control well. This kind of enzyme labeling is no longer used. The meaning of this paragraph is to supplement the purpose of setting the blank control. How to read the blank hole according to the instructions. For example, the Chinese version of the "Human Immunodeficiency Virus Antigen and Antibody Diagnostic Kit (Enzyme-linked Immunoassay) User's Manual" in the Chinese version of BioMerieux China Co., Ltd. prompts: read blanks in air (without placing racks and slats) at Single wavelength), or 450nm and 620-700mn (substrate is TMB) reference wavelength for reading.
2. Negative control (product): o7 ~ 4 jAA
(1) The concept and requirements of the negative control (product): The so-called negative control (product) should be homologous and homologous to the items to be picked up (samples, human reagents, human serum, etc.) used in this test. It is qualitative and does not contain the substance to be tested, and can objectively compare and identify the differences between processing factors (specific antigens and antibodies react in the serum immunological test). Therefore, the use of animal serum or its products (such as bovine serum albumin, etc.) instead of negative human serum as a control substance is undesirable in theory and practice, and has many disadvantages and does not require detailed description. Note: As far as I know, some of the domestic ELISA reagents are diluted with animal serum products or mixed with some human serum; second, the negative control reading is not "lower is better". The NCx value is close to 0.00X level, which is an illusion! Imagine that the NCx value of a negative control that actually uses "negative human serum" will be so low? For example, the NCx values ​​of Abbott ’s six EIA reagents are: : 0.010 (HBsAg), 0.027 (anti-HBs), 0.035 (HBeAg), 1.083 (anti-HBe), 1.065 (anti-HBc), 0.045 (anti-HBc-IgM). The HIV reagent NCx of BioMerieux is 0.091. A very simple method to distinguish whether the reagent NCx value is reasonable, as long as the actual readings of a large number of negative samples are compared, it will be "at a glance"! Vx / cX $
An extra question: an important statistical sign indicating the inherent quality of ELISA reagents is to see: the upper limit of negative samples should not be greater than, must be less than COI = 1.00; on the contrary, the lower limit of positive samples should not be less than, should be greater than COI = 1.00 ! In the early years, I used SPSS software to output three histograms of Aksu HIV and domestic A and B reagents to detect serum of about 600 healthy human samples. It can show the different distribution characteristics of negative samples, which is quite interesting. Why should I elaborate on NCx in this way? Because the NCx value is quite important in the calculation of Cut off value (more on this later). pPLOCBh
(2) Negative controls (products) are divided into two categories. One is the reference level of the test substance that is not contained in the serum of the subject, or the method sensitivity cannot be detected, and is the result judgment value (Cut off) The formula determines the only variable value of "yes" (positive) or "no" (negative). A high negative control value results in a false negative; otherwise, it results in a false positive. Such as: HBsAg, HBeAg, anti-HBc, etc. The second is the setting of negative control. The methodology requires the addition of indicator quantitative standards, such as neutralizing agent HBeAg, quantitative HBcAg, etc., to detect anti-HBe and anti-HBc. Alternatively, select a normal human serum that represents an average level to use as a negative control, such as detecting anti-HBc IgM. When calculating such Cut off values, most of them include two variable values: negative and positive control values.
3. Positive control (product): 4j, <10K
(1) The concept and requirements of the positive control (product): It is the same as the negative control (product), except that it is prepared using "selected" human serum containing the substance to be tested. The so-called "selection" means that the collected positive human serum is "screened" after several tests, and the serum containing "interfering substances" is removed and not used to avoid the occurrence of "false positive" interference test results. km / 0R>) i
(2) The setting of positive control (product) can also be divided into two types and uses: P> + |; Cp /) 8
First, the positive control is mainly used to evaluate whether the test result is effective and the stability and comparability of the test result. The measured value of the set positive control is not included in the Cut off value calculation, but it must be done. Such as HBsAg, anti-HBs, HBeAg, etc. \ Z%) `0Rj
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