Experience Sharing: Factors Affecting Standard Curve Establishment in Immunoassay!
In actual work practice, the preparation of the standard curve is very important. It can have a significant impact on the results of an entire batch of experiments. In the radioimmunoassay determination, the concentration of the tested substance is calculated according to the standard curve. The accuracy of the standard curve directly affects the accuracy of the assay results. However, how to accurately reflect the real functional relationship between B/B0% and the sample antigen concentration by the standard curve set up based on a few discrete points should be checked as much as possible from the experiment, so every step of making the standard curve The steps must be strict and precise. Now we will analyze the influencing factors of standard curve making in radioimmunity analysis. For colleagues reference.
1. Reagent components
(1) Standard products The activity, immunoreactivity and purity of standard products often affect the correctness of radioimmunoassay, thus greatly affecting the results.
The activity of the standard is required to be consistent with the immunoreactivity and the tested substance, and must be of a homologous system. It has good stability, is easy to store, does not contain cross-reactive substances and substances that interfere with immune reactions, and its quantification should be accurate and consistent with international standard products. The so-called standard product purity does not refer to chemical purity, it may contain impurities, but it should not contain substances that interfere with radioimmunoassay
(2) Protein
During the equilibrium reaction process between the standard product and the specific antibody, the protein has an inhibitory effect. If the test substance is used in plasma or serum, the same amount of protein should be added to the standard product, so that the protein will not interfere with the test substance and the standard product. consistent.
(3) PH value and ionic strength
The antigen-antibody reaction dissociates in an acidic or alkaline environment, so the reaction should be carried out in an environment close to neutral or slightly alkaline. Its pH value is preferably 7.4-7.8, and its ionic strength is 0.05-0.1mol/L. It must have buffering capacity (the ionic strength should not be too small), but too high ionic strength will prevent the antigen-antibody reaction.
(4) Reaction volume, temperature and time
Reducing the reaction volume can reduce the absolute amount of antibody and labeled antigen, so that the competitiveness of unlabeled antigen can be strengthened accordingly, the sensitivity can be improved, and the consumption of reflected reagents can be reduced. On the other hand, due to the corresponding reduction in the absolute amount of complexes formed in small volumes, the radioactivity measurement error increases, and because the sample volume becomes smaller, it is also easy to cause operational errors. Generally, the reaction volume of 0.3-1.2ml should be used first.
It takes a certain amount of time for the antigen-antibody reaction to reach a dynamic equilibrium. At the same time, it is affected by the temperature. For every increase in the reaction temperature of 10°C (not exceeding 37°C), the speed of antigen-antibody binding to reach equilibrium will be doubled, but the increase in temperature will make the antigen-antibody reaction slow down. Affinity decreases; and when the temperature is too high (60°C), the antigen and antibody will be inactivated.
Usually, the content of the sample to be tested is high and the affinity constant of the antiserum is large, so a higher temperature (37°C) can be selected for a shorter incubation time.
However, the binding fastness of the complex is poor; if the content of the analyte is low and the affinity constant K of the antiserum is small, it should be incubated at 4°C for a long time, and the binding fastness of the complex is high. Not easy to dissociate. Therefore, the radioimmunoassay room should be equipped with an air-conditioned environment, and the temperature of the cryogenic centrifuge should be controlled at 15°C to ensure that the precipitate generated by the reaction is not easy to dissociate.
(5) Peptidase, complement
Certain substances such as TRI-I, glucagon and other biological products can be degraded by peptidases, so a certain amount of aprotinin should be added to the assay buffer to inhibit the decomposition of peptide hormones. In addition, protein hormones Complement can affect the antigen-antibody response when peptide hormones are used as standard.
(6) Antiserum
The quality of antiserum directly affects the establishment of the standard curve. Its main quality indicators should have high titer, high affinity and high specificity, and the non-specific cross-reaction rate should be small. The dilution of the antiserum should be appropriate. If the dilution is too large, the dose-response curve will shift to the right. In the RIA analysis, the dilution of the antibody should be as large as possible, and it should have a high sensitivity.
(7) Others
The kit must be used within the validity period, and reagents of different batch numbers cannot be mixed. After the kit is taken out of the refrigerator, it should be equilibrated to room temperature before use. All reagents, especially the separating agent, must be thoroughly mixed by inversion before adding samples. All freeze-dried products can be used after reconstitution for 10-15 minutes.
2. The process of adding samples
(1) Personnel
Operators must undergo training in basic theory and basic operating techniques before taking up their jobs, including passing the sample addition consistency assessment. The error caused by non-compliance with the operation rules, because the operation accuracy decreases with the increase of the number of inspection tubes, it is advisable to operate no more than 500 tubes per person per day.
(2) Test tube
Uniform texture, good surface finish, low non-specific absorption, and no radioactive nuclides, so that the self-absorption and geometric position of radioactivity can be easily consistent.
(3) Adding samples
The amount of sample addition should be as accurate as possible (especially when adding labeled antigens). After a long period of use, the elasticity and sealing of the spring of the sampler will decrease, which will affect its accuracy. The sampler should be calibrated frequently. In order to reduce errors, the same injector should be used when adding the standard and the sample, and avoid mutual contamination through the adhesion of the tube wall and the tip of the pipette when adding the labeled antigen and antiserum. At the same time, disposable suction devices should be used. The order of adding specimens, iodine markers and antibodies cannot be reversed arbitrarily. The inclination angle of sample addition should be the same, and choose forward or reverse sample addition according to needs.
(4) A standard curve must be established for each batch of tests at the same time. It is not recommended to use the standard curve. If the standard curve made before is used, the measurement results of the whole batch will be higher or lower. In special cases, if it is really necessary to call the standard curve, the quality control product attached to the medicine box and the patient’s normal sample specimen can be used for strict quality control. In addition, 2-3 multiple tubes should be set up for each concentration standard product, and multiple tubes should be set up for the samples (cutting This is an important guarantee to reduce errors.
3. Measuring instruments
It is necessary to use high efficiency, low background, and good stability radioactive measuring instruments, and to use sufficient measurement time. The radiation counter should be maintained and maintained frequently. When measuring, the inner and outer walls of the upper part of the test tube should be dry to prevent contamination of the probe. The selection of the best working conditions of the detector can make a better standard curve, so as to obtain reliable measurement results. The method of selecting the working conditions usually includes the determination of the plateau curve or the determination of the quality factor.
(1) Determination of plateau curve
The photomultiplier tube of the detector must work under a certain high voltage state. This high voltage should be selected as a high voltage value that has little influence on the count rate when the voltage fluctuates slightly. The selection of the high voltage value is determined by the plateau curve. , from the results of multiple measurements, select a curve with a longer plateau section and a smaller slope, and take the high voltage corresponding to the first 1/3 of the plateau section of the curve as the working voltage.
(2) Determination of quality factors
When the sample is used for relative counting measurement, it is expected to obtain a high sample count rate and a low background count rate, and the relationship between the sample count rate and the background count rate is: quality factor (F)=E2/nb (E is the measurement efficiency, IIb is the background count rate) the higher F is, under this measurement condition, a higher sample count rate and a lower background count rate can be obtained. Application of plateau curve determination and quality factor determination, the latter considers more factors and the results are more reliable.
4. Curve Fitting Mode
The same dose-response curve can be fitted by different models, and a little comparison shows that the degree of fitting is different, and the more complicated the degree of fitting, the better. The correlation coefficient R2 can be used as an index to evaluate the fitting accuracy. The closer R2 is to 1, the higher the fitting accuracy is. R2>0.990 is required. DEV% is the fitting percentage deviation, from which we can see the deviation of each standard product point, generally requires DEV%<10%, and the common indicators of curve fitting goodness are R2 and DEV%.
It should be emphasized that the fitting of the curve is only a mathematical treatment of the curve segment determined by the standard product and its corresponding reaction parameters. Within the range given by the standard product, the fitted function relationship should ensure its Monotonicity, the curve fit cannot be extrapolated. At the same time, it should be noted that no matter what method is used for fitting, it is impossible to change a bad result into a good result. Only by keeping the experiment closed can the accuracy of the experimental results be truly improved.
