Diagnostic Reagent Response Curve "Point-taking" Method

Diagnostic Reagent Response Curve "Point-taking" Method

1. End point method (END POINT METHOD)

According to the absorption spectrum characteristics and absorbance of the reaction product when the reaction reaches equilibrium, the method of quantitative analysis of the substance is carried out. For general chemical reactions, when the reaction is complete (or forward and reverse reaction dynamic equilibrium) and the reaction product is stable, it is the end point of the reaction. For the antigen-antibody reaction, the end point is when the antigen and antibody react completely and form the largest and stable immune complex. On the reaction time course curve is a line segment parallel to the X axis. In terms of measurement and calculation methods, it is generally divided into two types: one-point method and two-point method.

1. One point method (ONE POINT) The absorbance value of the air blank (GB), water blank (WB) or reagent blank (RB) before the reagent and sample are mixed is used as the base point for determination calculation, and the absorbance reading at the end of the reaction is subtracted from the blank reading to obtain Reaction absorbance. By comparing with the reaction absorbance of the calibration solution under the same conditions, the determination result is obtained. It is often used in conjunction with the one-point calibration method, that is, a calibration concentration is used, and the calibration curve passes

through the zero point and becomes linear. Multi-point calibration is also applied.

2. Two-point end point method (TWO POINT END), that is, end-point-start point method, takes a certain time point after the reagent and sample are mixed as the starting point, and subtracts the starting point reading from the absorbance reading at the end of the reaction. Under certain conditions, the specificity of the sample to the reaction or the reaction itself can be reduced (mainly refers to chromaticity interference). Double reagents are often used, and a point before the addition of R2 is usually used as the starting point of the measurement; in some cases, a point after the addition of R2 can also be used as the starting point of the measurement. Difficult to use when using a single reagent, the main reaction starts too quickly or the instrument has limited initial readout points.

The difference between the fixed time method (FIXED METHOD) and the two-point endpoint method is that the end point of the measurement reading is not in the reaction equilibrium section, but is selected according to the methodology. Such as serum creatinine (picric acid method) determination.

3. The three-point endpoint method is the double-end method, and two reaction-related endpoint methods are determined in one channel at a time. For example, free fatty acids and triglycerides are measured together. Some instruments (such as HITACHI series) set this method.

Second, the continuous monitoring method (CONTINUOUS MONITORING METHOD)

Also known as the rate method (RATE ASSAY). That is, the method of continuously monitoring the reaction process and performing quantitative analysis according to the measured rate of product formation or substrate consumption. On the reaction time course curve, there is a constant-speed section of the reaction (the slope remains constant), which is often used for the determination of the linear reaction period of enzyme activity.

1. The continuous monitoring method is the zero-order reaction rate method, also known as the slope method. In a longer reaction time period (at least 90-120 seconds), read the absorbance value every certain time (usually 2-30 seconds), read at least 4 points, and get 3 △A; The least square method is used for multiple readings, and the multi-point method with rate time (TR) is used for those whose reading interval is too short. The readings of the linear reaction part are all taken, and the reaction rate △A/MIN per unit time is obtained. This method must be based on the zero-order reaction, because only under the zero-order reaction, the change in absorbance per unit time (reaction rate △A/MIN) is directly proportional to the enzyme activity. This method relatively reduces the analysis error and greatly improves the analysis speed and accuracy. However, semi-automatic biochemical analyzers use single-sample continuous monitoring, which is quite time-consuming. To apply the continuous monitoring method, first of all, samples with high, medium and low concentrations within the linear range should be prepared, and the reaction time course curves should be drawn respectively to understand the whole process of the reaction at different concentrations, taking into account the determination of the delay time and the linear monitoring period.

2. The two-point rate method is the so-called pseudo-first-order rate method. Select two time points T1 and T2 in the reaction, read the absorbance A1 and A2, and calculate (A2-A1), (T2-T1)=△A, △T. There are two main differences between this method and the endpoint two-point method: the reaction at the latter reading point does not reach the endpoint, and the result is calculated based on the rate. Compared with the continuous monitoring method, its disadvantage is that T 1 and T 2 are determined artificially, and there are many uncertain factors, which cannot guarantee that the reaction is linear during the T 1-T 2 period, which affects the accuracy of the results; a preliminary test should be done before the routine measurement to determine the linear time period. If the reaction is not linear within the selected time period (such as the zero-order reaction period is short, the instrument cannot be set or measured), the end point method can only be used instead. The advantage is that the method is simple; when the enzyme activity is low and the measured absorbance value is small, the measurement time period can be increased without being limited by the continuous monitoring time point of the instrument, and the reading error can be reduced.

3. The rate B method performs two reaction-related rate method determinations in one channel at a time. It can be used for the determination of two tests, and can also be used for automatic compensation of interference or/and sample blank. The continuation effect of the rate of the first reaction can be subtracted from the rate of the second reaction (main reaction). For example, it is used to eliminate the decrease in absorbance of bilirubin converted to biliverdin, and the negative interference to the determination of creatinine picric acid method, etc. Some instruments (such as HITACHI series) set this method.

Three, blank (BLANK) correction

In spectrophotometry, the blank solution is often used to adjust the absorbance zero point of the instrument, or to offset some interference factors in the determination. In the determination of biochemical analyzers, in addition to the use of dual or multi-wavelength, two-point methods, etc. to eliminate background interference, it is often necessary to use a special blank measurement in order to deduct its influence from the absorbance of the sample. Correct selection of blank calibration plays an important role in improving accuracy.

1. Reagent blanks are generally divided into two categories: with or without reagent blanks, in the method type and calibration mode. Reagent blanks are measured separately or in conjunction with calibration, and pre-loaded deionized water sample cups or reagent blank racks are required. The absorbance of each measuring point of the calibration or patient sample shall be subtracted from the reagent blank absorbance or blank rate value of the corresponding measuring point. For methods without a reagent blank, the water blank in the cuvette is often used as the reference value for determination.

In many instruments, the reagent blank determination is similar to a calibration determination and is not performed in real time while the patient sample is being measured. Therefore, attention should be paid to its measurement frequency to avoid calculation errors caused by changes in the reagent blank due to changes in the reagent batch number or quality.

2. The sample blank is mainly to eliminate the interference of the turbidity or chromaticity of the sample itself. The blank channel method is often used, and the measurement calibration result = the result of the chromogenic reaction channel - the result of the blank channel. Most instruments have to occupy additional measurement channels, and the analysis speed is halved, but the accuracy of interference removal should be higher than the two-point end point.