For ELISA testing, according to the requirements of the reagent instructions, the following three controls should be set for each test and each pull:
1. Blank control: only use the diluent to replace the test sample, to observe the "background" of the color of the final reaction, and to eliminate the "background" by the microplate reader, or to calculate the mean value of the negative control (NCx), positive control The mean (PCx) and the S / CO value of the sample reading minus the background absorbance of the blank control. How to read the blank hole according to the instructions. Such as: reading blank with air (without racks and slats), reading at 450nm (single wavelength), or 450nm and 620-700mn (substrate is TMB) reference wavelength.
2. Negative control (product):
(1) The concept and requirements of the negative control (product): The so-called negative control (product) should be homologous and homogenous to the items to be picked up (samples, human serum, etc.) in this test. 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 serum immunological tests). 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. The negative control reading is not "lower is better". The NCx value is close to 0.00X level, this is an illusion! Imagine that the NCx value of a negative control that actually uses "negative human serum" is so low? An extra question: an important statistical sign that shows 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 ! The NCx is so elaborated here because the NCx value is quite important in the Cut off calculation.
(2) Negative controls (products) are divided into two categories. One is to represent 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) calculation The type 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. The second is the setting of negative control. Methodologically, it is required to add an indicator quantitative standard, or select a normal human serum representing an average level as a negative control. When calculating such Cut off values, most of them include two variable values: negative and positive control values.
3. Positive control (product):
(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.
(2) The setting of positive control (product) can also be divided into two types and uses:
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. The second is the setting of positive control, which is not only used to evaluate whether the test result is valid and the stability and comparability of the test result, but also included in the calculation of Cut off. The Cut off value at this time represents the upper limit of the normal value range of the marker in healthy (normal) people. When the test value of the sample to be tested exceeds the upper limit (Cut off), it means that it has diagnostic significance.
Positive and negative gray bands refers to the percentage of positive and negative gray bands here, which is not only the quality evaluation index of the reagent itself, but also whether there is a suspicious sample range (Sample Retest Range) in the sample to be tested and the batch / Suspicious sample occupancy rate during each test.
Cake Decorating Tools,Baking Tools,Baking Utensils,Cake Baking Utensils
Yangjiang Yado Kitchen Industry Co., Ltd. , https://www.yadokitchen.com