WS/T 221-2002 Evaluation of application materials for immunoprecipitation analysis standard
Some standard content:
Health Industry Standard of the People's Republic of China
WS/T221-2002
Standard for Immunoprecipitation Analysis
Evaluation of Application Materials
Guidelines for immunoprecipitinanalyses-Procedures for evaluating the performance of materials2002-04-20 Issued
Issued by the Ministry of Health of the People's Republic of China
Implemented on 2002-07-01
WS/T221--2002
The formulation of this standard will help the relevant manufacturers of immunoprecipitation analysis reagents to unify the preparation methods of reagents, and will also help clinical laboratories to conduct standardized quality management of immunoprecipitation analysis, thereby changing the current chaotic situation in immunoprecipitation analysis. This standard will be implemented from July 1, 2002. This standard was proposed by the Medical Administration Department of the Ministry of Health.
The drafting unit of this standard: Huashan Hospital of Shanghai Medical University. The main drafters of this standard: Chang Yuan and Zhu Yusheng. This standard is entrusted by the Ministry of Health to the Ministry of Health Clinical Laboratory Center for interpretation. 1 Scope
Health Industry Standard of the People's Republic of China
Standard for Immunoprecipitation Analysis
Evaluation of Application Materials
Guidelines for immunoprecipitin analyses--Procedures for evaluating the performance of materialsWS/T221-2002
This standard establishes the evaluation procedures for the preparation of materials used in immunoprecipitation analysis, introduces the specificity issues of commonly used immunoprecipitation analysis methods, proposes the necessary information that should be included in the instructions of the kit, and recommends the preparation methods of reference products. This standard is applicable to clinical laboratory personnel engaged in immunoprecipitation analysis and related reagent manufacturers. 2 Evaluation of Antibody Mass Spectrometry and Evaluation of Immunochemical Specificity 2.1 Quality Evaluation of Immunogens
When preparing antiserum, it is usually not necessary to use monoclonal proteins or proteins from a single donor as immunogens, but normal protein molecules and/or isotype proteins from normal tissues and body fluids should be used to avoid interference of idiotype or allotype molecule specificity on detection. In this standard, various immunoglobulins are mainly used as examples for illustration, because these immunoglobulin molecules represent the largest antigenic variation among the proteins measured by the laboratory. The evaluation of immunoglobulin immunoprecipitation analysis can also be used as a reference for evaluating other serum protein analyses with lower specificity.
When preparing antibodies, the immunogens used should include all antigenic subtypes to avoid differences in the reactivity and specificity of the prepared antibodies as much as possible. For example: the antiserum produced by the immunogen immunized animals used to prepare anti-IgG antibodies can only react with IgG molecules after appropriate treatment, but not with other types of immunoglobulins or other proteins in serum, plasma, urine, cerebrospinal fluid, semen, tissue fluid and abnormal pathological body fluids. The antiserum produced should mainly contain antibodies against the common parts of the four IgG subclasses (IgGi, IgG2, IgG3, IgG,) molecules, and there should be no difference in the reactivity of these subclass molecules. The antiserum should not precipitate with the free or bound light chain of any immunoglobulin. Many serum proteins have polymorphism and different epitopes (such as α-antitrypsin and haptoglobin), and immunogens should include all forms of such protein molecules as much as possible. The preparation of anti-IgA should also basically comply with the above regulations, that is, the prepared anti-IgA should react with IgA, and IgA.
IgM subclasses have not yet been found, and the preparation of anti-IgM immunogens should use serum from individuals with Waldenstrom's macroglobulin and normal human serum.
2.1.1 Isotype Specificity
"When an immunogen is used to prepare antisera against abnormal immunoglobulin components, attention should be paid to isotype specificity. There are currently no commercial antisera that react with all clinical samples, so although these antisera are produced by immunizing animals with a series of purified proteins, occasionally a certain sample will not react completely with the antisera. 2.1.2 Degradation of Immunogens
When the immunogen may undergo degradation that significantly changes the molecular structure and the sample to be analyzed may degrade during storage, the most stable molecular form that has been degraded should be used as the immunogen instead of its natural form in the body. For example: when preparing antiserum against C3, a degraded and stable C3c (Bla) is used as the immunogen. Approved by the Ministry of Health of the People's Republic of China on April 20, 2002, and implemented on July 1, 2002
2.2 Evaluation of antibody specificitybzxz.net
2.2.1 Method
WS/T221-2002
The specificity evaluation of polyclonal antiserum should use highly sensitive methods such as cross-immunoelectrophoresis, immunoblotting, etc. Do not use methods with poor sensitivity and accuracy such as immunodiffusion, immunoelectrophoresis, etc. The specificity test of monoclonal antibodies should adopt the competitive inhibition mode, and the methodology can be selected from radioimmunoassay, enzyme immunoassay or immunoblotting. 2.2.2 Detection
The method of using cross-immunoelectrophoresis to evaluate the specificity of antiserum is to incubate the gel at room temperature for 1 hour after the second direction electrophoresis is completed and then observe the staining. Observe the results. When evaluating by immunoblotting, the transfer membrane should be incubated with the antiserum for 1.5 hours. The protein bands can be stained with Coomassie Brilliant Blue, silver stain, colloidal gold or other dyes with similar sensitivity. Ponceau S, Nitro Black, and Fast Green cannot be used. 2.2.3 Materials
Try to include all possible materials such as plasma, urine, and cerebrospinal fluid, and have different dilutions (1:20, 1:5, undiluted, optimal concentration, etc.). When using cerebrospinal fluid or urine, 10-fold or 100-fold concentrated samples should be used as quality control materials. The antigens used for the qualitative evaluation of antisera can be derived from fresh serum and plasma. If cross-reactions are suspected or known to exist, samples containing cross-reactive antigens should be used when conducting specific evaluations of antisera, and the degree of cross-reaction should be stated. 2.2.4 Immunity Proteins mainly detected by precipitation method 2.2.4.1 Immunoglobulins and light chains: IgA, IgD, IgG, IgM, light chains (n, a). 2.2.4.2 Complement: C1 (q, r, s), C3, C4, C5. 2.2.4.3 Acute phase reaction proteins: α1-acid glycoprotein, α1-antitrypsin, C-reactive protein, ceruloplasmin. 2.2.4.4 Hemoglobin and haptoglobin: hemoglobin A, hemoglobin F, haptoglobin. 2.2.4.5 Coagulation-related proteins: antithrombin ear, fibrinogen, plasminogen, prothrombin. 2.2.4.6 Lipoproteins and apolipoproteins: apolipoprotein (a), apolipoprotein AI, apolipoprotein A-1, apolipoprotein B (ApoB-100), apolipoprotein CI, apolipoprotein E.
2.2.4.7 Others: albumin, AFP, α2-HS globulin, cryoprecipitated globulin, fibronectin, Gc globulin, α2-macroglobulin, prealbumin, transferrin.
2.3 Preparation of antiserum
2.3.1 Selection of immune animals: The preparation of antiserum for immunoprecipitation analysis should not use large animals such as donkeys, horses, and rabbits, but small animals such as goats, rabbits, and chickens, because the antiserum produced by large animals is prone to excessive antigens. 2.3.2 Removal of fibrinogen
Antiserum with incomplete blood clots may contain a certain amount of fibrinogen, which can coagulate and produce precipitation during the diffusion process, so the fibrinogen in the antiserum should be removed.
2.3.3 Removal of lipids and particles
The prepared antiserum should be centrifuged (10,000g) to remove lipids and particles. The purification of immunoglobulins can be carried out by salting out, affinity chromatography, etc.
2.3.4 Removal of non-specific antibodies
Some antisera can form non-specific precipitation with certain serum or plasma specimens, especially abnormal specimens. Non-specific precipitation similar to specific precipitation reaction can be formed during the two-way diffusion process. Most immunoprecipitation analyses require the removal of unnecessary non-specific antibodies in antisera. Two methods are usually used: the first is absorption, that is, adding soluble antigens to form immune complexes with corresponding antibodies, and removing them by centrifugal precipitation and/or filtration. The second is adsorption, that is, adsorbing the corresponding antibodies through solid-phase antigens. The adsorption method may leave a small amount of antigen-antibody complexes in the antiserum. However, since this method is more practical, the adsorption method is more commonly used. 2.3.5 Removal of immune complexes
In the process of absorbing/adsorbing antisera to obtain relatively single-specific antisera, even if centrifugation or filtration is performed sufficiently, soluble antigen-antibody complexes cannot be removed. An appropriate amount of turbidity enhancer (such as PEG8000) can be added to the medium to remove soluble antigen-antibody complexes as much as possible.
2.3.6 Removal of IgM
WS/T221—2002
In some cases, reverse immunoprecipitation may occur. For example, the serum of patients with congenital IgA deficiency often contains a certain level of low-affinity human anti-antigen serum and milk protein antibodies, which mainly react with anti-antigen IgM and mouse immunoglobulin. When detecting low-level IgA, immune or mouse antisera with IgM removed should be used. 2.3.7 Interfering substances
Antiserum should not contain more than a certain level of interfering substances such as hemolysis and jaundice visible to the naked eye. 2.3.8 Quantitative evaluation of non-specific antibodies Antisera used for immunoprecipitation analysis are appropriately diluted and quantitatively evaluated for specificity by the one-way immunodiffusion (RID) method. In most cases, high-quality commercial antisera against human plasma proteins used for gel precipitation analysis should be diluted 1:3 to 1:8, or even higher.
2.3.9 Content of non-specific antibodies
Antiserum should have specific antibody content. If it cannot be quantified, the titer of the optimal antigen-antibody ratio and the antigen concentration when the hook effect occurs are required. When the antiserum is immunodiffused at a dilution higher than 1:5, its non-specific antibodies should not show visible precipitation and should not react directly with most plasma proteins at a concentration greater than 5 mg/L. 3 Kit instructions and product evaluation
From the user's perspective, the following issues should be considered when evaluating the product. 3.1 Specifications of the instructions
The purpose and limitations of the kit should be stated in the kit instructions. In fact, there is no absolute specificity of antiserum for antigens. Antiserum with nonspecific antibodies detected by RIA, EIA, and FIA can still be used for immunoprecipitation analysis. The materials derived from humans in the kit should be tested for HBsAg, HCV antibodies, and HIV antibodies and tested negative. The reagent instructions should clearly state this and indicate that all materials derived from humans should be treated as infectious. 3.2 Specificity
The specificity of antiserum is related to the determination method used. The kit instructions should indicate the method by which the specificity of antiserum is measured. 3.3 Source of immunogen
The source of immunogen should be described in general terms. 3.4 Antiserum treatment method
The treatment method of antiserum, such as absorption/adsorption method, should be described in the instructions and the evaluation section of the reagent. 3.5 Recommended methods for further treatment of antiserum For antiserum used in scattering assays and antiserum that may contain soluble antigen-antibody complexes, recommended methods for further treatment should be provided (see section 2.3.3).
3.6 Cross-species reaction
For some users, information about cross-species reaction may be important. If the antiserum has cross-species reaction, it should be stated in the instructions.
4 Analytical methods for immunoprecipitation reaction
Although many analytical methods have been designed based on the principle of antigen-antibody precipitation reaction, this standard only evaluates several widely used analytical methods. There are four main methods for in-gel immunoprecipitation analysis: one-way immunodiffusion (RID), rocket electrophoresis, immunoelectrophoresis (IEP) and immunofixation electrophoresis (IF). In addition, there are two quantitative analysis methods for liquid phase immunoprecipitation: transmission turbidimetry and scattering turbidimetry, including endpoint method and rate method.
4.1 One-way immunodiffusion (RID)
RID is related to the molecular weight and molecular configuration of the antigen. When performing immunoglobulin determination, it is also affected by the specificity of the antiserum. If the 3
WS/T221-2002
antiserum contains other contaminants or antigen degradation products of different molecular weights, multiple precipitation rings may appear during RID analysis.
4.2 Rocket electrophoresis
Rocket electrophoresis is less affected by changes in the relative molecular mass of the antigen, but is also susceptible to the influence of contaminants or class and subclass epitope specificity. When double or fuzzy rocket precipitation occurs, it indicates that the antibody specificity is poor or the antigen has a special structure. 4.3 Immunoelectrophoresis (IEP) and immunofixation electrophoresis (IF) IEP and IF are non-quantitative analysis methods with low sensitivity to low-level contaminants. In actual work, contaminants should not produce visible staining bands with most proteins in routine clinical samples (see 2.2.4). 4.4 Liquid immunoprecipitation analysis
Liquid immunoprecipitation analysis includes nephelometry and turbidimetry. Nonspecific antibodies in antiserum are not easy to detect in liquid phase immunoprecipitation analysis. Cross immunoelectrophoresis, countercurrent immunoelectrophoresis and immunofixation electrophoresis can be used to detect nonspecific antibodies in antiserum. In actual work, if the concentration of nonspecific antibodies in antiserum is low, it will generally not affect the results of liquid phase immunoprecipitation analysis too much. When performing liquid phase immunoprecipitation analysis, turbidity must be added to accelerate the formation of immune complex precipitation. Turbidity is generally added to the buffer, and the amount added should be strictly controlled.
5 Calibration/calibration products and quality control products
The use of automated instruments for serum specific protein determination techniques such as transmission turbidimetry and nephelometry has reached a level comparable to traditional colorimetric analysis. In addition, the precision of the results has reached or exceeded that of ordinary colorimetric analysis. Despite this, since the RID method does not require special instruments, a considerable number of laboratories still use the RID method. Regardless of the type of analysis, the purpose of calibration is the same, that is, to convert physical or photoelectric detection signals into mass units. There are two basic types of calibration: multi-point calibration and single-point calibration. Regardless of the method, the reagent manufacturer should test each batch of reagents to ensure the batch-to-batch stability and precision of the reagents. On the other hand, users should perform multi-point or single-point calibration according to different requirements. The following are some general calibration methods for commonly used immunoprecipitation analyses. The calibration methods for other types of immunoprecipitation analysis techniques are similar. 5.1 One-way immunodiffusion: Calibrators and quality control products When performing one-way immunodiffusion, the diffusion of proteins in the gel is affected by the operator's own factors. In order to minimize the variability of the results, there should be a set of calibrators and quality control products on each gel. In addition, the gel plate should be incubated in a constant temperature and high humidity environment. 5.1.1 Calibrators
The calibrators should include a series of different concentrations from the lower limit to the upper limit of the normal reference range. Five concentrations are generally recommended. The manufacturer should set the concentration value of the calibrator in accordance with the provisions of "1FCC Preparation of Human Serum Protein Reference Material (RPPHS)/CAP/BCR/IFCC Lot91/0619".
5.1.2 Quality Control Products
Quality control products should include three different concentrations: high, medium and low. Quality control products can be mixed samples prepared by clinical laboratories, which are freeze-dried under certain conditions after being packaged, or commercial quality control products that have been accurately quantified. 5.2 Immunotransmittance turbidimetry and immunoscattering turbidimetry instruments accurately sample, dilute, mix with antiserum and oscillate to form immune complexes with light scattering under certain temperature and humidity conditions. Therefore, the errors caused by the operator themselves have little effect on the results measured by the instrument. Nevertheless, users should also check the instrument regularly to ensure its normal operation. 5.2.1 Calibration
Currently, all instruments are required to use standard calibration methods to convert photoelectric signals into mass units or national The relative value of the international unit. The ideal calibration curve should be a straight line, but in reality it is difficult to be completely straight. Good reagent stability and instrument precision can reduce the number of calibrations. However, no matter how advanced the reagent instrument is, the user should still frequently monitor the operating status of the instrument by testing the fixed value quality control products. 5.2.1.1 Multi-point calibration system
WS/T221—2002
The calibration of the multi-point system requires a 6-point curve calibration, including the 0 point. The reagent manufacturer should ensure that the calibration products are prepared according to the IFCC reference materials. The calibration curve is stored in the instrument and can be used multiple times. The accuracy of the results is ensured by testing quality control products of different levels. 5.2.1.2 Single-point calibration instrument
Single-point calibration is a simplified form of multi-point calibration, but each calibration should be repeated 2 to 3 times to ensure the accuracy of the measurement. If the relationship between dose and response in the dose-response curve is in good proportion, the calibration curve is still valid despite a certain drift range. 5.3 Quality control materials
Quality control materials should include three concentrations: high, medium and low. The quality control materials must be similar to the sample properties (i.e., the medium is similar) and the concentrations must be in a certain ratio. The reagent manufacturer should ensure that the linearity of the quality control materials should be similar to that of fresh serum. 6 Reference materials
The reference materials used for protein determination by immunoprecipitation must be the latest and most authoritative reference materials available. IFCC has prepared a new and widely accepted human serum protein reference material, which is prepared based on the World Health Organization's reference material in IU and the US Centers for Disease Control and Prevention's National Reference Material for Serum Protein (RPSP) or purified protein in g/L. Apolipoprotein reference materials are not included in the above reference materials. Since most methods require clarified serum, lipoproteins must be removed. All manufacturers of commercial human serum albumin reference materials should refer to the IFCC reference material RPPHS/CAP/BCR/IFCC Lot 91/0619, which is available in Europe from the Bureau of Community References (BCR) in Brussels and in North America from the College of American Pathologists (CAP). WS/T221-2002
Health of the People's Republic of China
Industry Standard
Standard for Immunoprecipitation Analysis
Evaluation of Application Materials
WS/T221-2002
Published by China Standards Press
No. 16, Sanlihebei Street, Fuxingmenwai, Beijing
Postal Code: 100045
Tel: 6852394668517548
China Standards Press Qinhuangdao Printing Printed by Xinhua Bookstore in Beijing Distributed by Xinhua Bookstores in various places Sold by Xinhua Bookstores in various places*
Size 880×12301/16 Printing sheet 3/4 Word count 12,000 words First edition in August 2002 First printing in August 2002 Print run 1-800
Book number: 155066·2-14529
Website: bzcbs.com
Subject 613--387
Copyright reserved. Infringements must be investigated
Report phone number: (010) 68533533
Tip: This standard content only shows part of the intercepted content of the complete standard. If you need the complete standard, please go to the top to download the complete standard document for free.