GB 15997-1995 Diagnostic criteria and treatment principles for diphtheria

This standard specifies the diagnostic criteria and treatment principles for diphtheria. This standard applies to the diagnosis, reporting and treatment of diphtheria patients by medical, health and health care institutions and personnel at all levels and types. GB 15997-1995 Diagnostic criteria and treatment principles for diphtheria GB15997-1995 standard download decompression password: www.bzxz.net

GB15997—1995
Diphtheria is an acute respiratory infectious disease caused by Corynebacterium diphtheriae. Its infection characteristics are congestion and swelling of the mucous membranes of the pharynx, larynx, and nose, as well as systemic poisoning symptoms caused by bacterial exotoxins. In severe cases, myocarditis and peripheral nerve paralysis are often combined. Due to the widespread use of diphtheria vaccines (DTP triple preparations), typical diphtheria has gradually decreased, while atypical diphtheria or mild diphtheria has increased. In particular, the incidence of adult diphtheria should be paid attention to.
During the formulation of this standard, the "Law of the People's Republic of China on the Prevention and Control of Infectious Diseases" and the "Implementation Measures for the Law of the People's Republic of China on the Prevention and Control of Infectious Diseases" formulated by the Ministry of Health in 1989 were referred to, and the epidemiology, clinical practice and local conditions of my country were combined as much as possible to facilitate implementation and application. Appendix A of this standard is the appendix to the standard;
Appendix B and Appendix C of this standard are both suggestive appendices. This standard was proposed by the Ministry of Health of the People's Republic of China. Drafting units of this standard: Institute of Epidemiology and Microbiology, Chinese Academy of Preventive Medicine, Ditan Hospital, Beijing, and Capital Institute of Pediatrics.
The main drafters of this standard are Zhang Rongzhen, Yang Lixin and Wang Shushan. The Ministry of Health entrusted the technical unit, the Office of Supervision and Administration of Communicable Disease Prevention and Control of the Ministry of Health, to interpret this standard. 1192
1 Scope
National Standard of the People's Republic of China
Diagnostic criteria and principles of management of diphtheria
Diagnostic criteria and principles of management of diphtheria This standard specifies the diagnostic criteria and management principles of diphtheria. GB15997—1995
This standard applies to the diagnosis, reporting and management of diphtheria patients by medical, health and health care institutions and personnel at all levels and types. 2 Diagnostic principles
Clinical diagnosis should be made based on epidemiological data, clinical manifestations and the results of bacteriological smears of pharyngeal swabs; for a confirmed diagnosis, a positive culture of diphtheria bacteria is required, and it is proved that toxins can be produced or diphtheria-specific antibodies can be detected. 3 Diagnostic criteria
3.1 Epidemiological history
In areas where diphtheria is prevalent, there is a history of direct or indirect contact with confirmed diphtheria patients. 3.2 Clinical symptoms
Fever, sore throat, nasal congestion, hoarseness, and barking cough. There are gray-white pseudomembranes that are not easy to peel off in the nose, pharynx, and larynx, and they bleed easily when peeled off. 3.3 Laboratory diagnosis
3.3.1 Corynebacterium diphtheriae isolation and culture are positive and proven to produce exotoxins, see Appendix A. 3.3.2 Direct smear microscopy of throat swabs shows Gram-positive corynebacterium and metachromatic particles. 3.3.3 The specific antibodies in the patient's double serum increase by more than four times, see Appendix C. 3.4 Case classification
3.4.1 Suspected cases
Those with 3.2.
3.4.2 Clinically diagnosed cases
Suspected cases plus 3.3.2, refer to 3.1.
3.4.3 Confirmed cases
Suspected cases plus any of 3.3.1 and 3.3.3. 4 Principles of prevention and treatment
4.1 Isolation of patients
Suspected or diagnosed cases should be isolated immediately. The isolation period lasts until the symptoms disappear and the throat swab culture is negative twice, or 14 days after the symptoms disappear.
4.2 Terminal disinfection
After the patient is isolated, the patient's home and the ward after the patient is discharged from the hospital should be terminally disinfected. Fluoride-containing disinfectants can be used. 4.3 Principles of treatment
Approved by the State Administration of Technical Supervision on December 15, 1995 and implemented on July 1, 1996
GB15997--1995
4.3.1 Give diphtheria antitoxin as soon as possible and in one dose. The first choice of antibiotics is penicillin to kill pathogens and reduce the secretion of toxins. 4. 3.2 Take symptomatic treatment and prevent complications, especially the prevention and treatment of myocarditis. 4.4 Emergency measures for susceptible people
4.4.1 Emergency vaccination of diphtheria toxoid should be taken for susceptible people around the patient who have not developed the disease. 4.4.2 For weak and sickly susceptible children, intramuscular injection of diphtheria antitoxin can be performed after the Sick test (diphtheria toxin skin test) is positive. 4.4.3 Close contacts should be quarantined for 7 days. Carriers with positive bacterial culture should be given antibiotic treatment. 4.5 Immunization and prevention of diphtheria
In areas where diphtheria is prevalent, emergency vaccination with diphtheria toxoid is very effective in controlling the epidemic. According to the current immunization procedures in my country, routine basic immunization and booster immunization are carried out well, and the quality of vaccination is guaranteed, so that the occurrence of diphtheria can be controlled. 3.9.1
A1 Isolation, culture and identification of Corynebacterium diphtheriae
GB15997—1995
Appendix A
(Standard Appendix)
Methods for etiological diagnosis of diphtheria
Collect specimens from the patient's pharyngeal pseudomembrane or nasopharynx to isolate pathogens. A2 Collection of specimens
A2.1 Patients
Use a sterile cotton swab or a sterile cotton swab to absorb freshly prepared potassium tellurite and glycerol saline preservation solution to dip specimens from the edge of the suspected patient's pharyngeal pseudomembrane or take a nasopharyngeal swab at the same time, put it in a sterile test tube and send it for examination at the same time. A2.2 Carriers or suspected patients
Since no obvious pseudomembrane is seen, secretions from the nasopharyngeal or tonsil mucosa should be collected for examination. A3 Nasopharyngeal swabs are inoculated with Lu's serum slant or potassium tellurite medium for bacterial culture observation and identification A3.1 Bacterial type
Gram-positive elongated rods are rod-shaped, irregularly arranged, often in a herringbone or fence shape. Metachromatic granules can be seen in Ponzi staining. A3.2 Culture and biochemical characteristics
Diphtheria bacilli form grayish white, round and smooth colonies on blood agar plates with hemolysis rings. On potassium tellurite blood agar plates, the colonies are black or grayish black in the center and gray on the edges. On urea yolk disaccharide medium, preliminary identification can be made after culturing at 37℃ for 12 to 48 hours. Diphtheria bacilli ferment glucose, lactose, maltose, and fructose, produce acid but not gas, do not ferment lactose or mannitol, and generally do not decompose sucrose. Do not produce indole, can reduce nitrate, enzyme positive, oxidase negative, do not liquefy gelatin, and do not decompose urea. Severe diphtheria bacilli can decompose starch, glycogen and dextrin, and slowly decompose sucrose. Corynebacterium diphtheriae can be divided into three types: mild, moderate and severe according to morphology, culture and biochemical characteristics. A4 Virulence test
Corynebacterium diphtheriae is an acute infectious disease caused by Corynebacterium diphtheriae, and its pathogenic factor is exotoxin. It has strong antigenicity and severe toxicity. Not all strains of Corynebacterium diphtheriae are virulent, only those strains carrying β-corynebacterium phage can produce exotoxin. Virulence test can determine the ability of Corynebacterium diphtheriae to produce exotoxin. There are two commonly used methods. A4.1 Elek's plate toxicity test Heat and melt Flek culture medium, cool to about 50℃, add 2mL of normal sterile (rabbit or bovine) serum, mix well, and use a sterile spoon to take the pre-prepared self-antitoxin (containing 5000-10000u per ml), stick the dry filter paper strip to the central surface of the plate, place it in a 37C incubator for about 0.5h, and dry the surface moisture. The time should not be too long to avoid affecting the results. Inoculate the test strain on the plate in a straight line so that it intersects with the antitoxin filter paper at right angles. 4-5 specimens can be inoculated on the same plate at the same time. At the same time, a known toxic strain should be used as a positive control. Culture at 37C for 48-72 hours. If the development is good, the results can be observed. For positive cases, flocculent precipitation arcs begin to appear along the inoculation line slightly away from the paper strip. If no precipitation arcs appear after 72 hours, a negative report can be made. A4.2 Guinea pig intradermal inoculation toxicity test
Inoculate the strain to be tested on the Lu serum slope, culture at 37C for 16-18 hours, add 1mL of broth, scrape off the bacterial moss, make a suspension, absorb 0.5mL of this bacterial liquid, add it to 3.5mL of broth, mix well and use it. At the same time, a known toxic strain should be used as a control. Select two guinea pigs weighing about 250g, one of which is injected with 1000u of diphtheria antitoxin intraperitoneally 24 hours before the test as a control animal; the other is not injected with antitoxin and is used as a test animal. Before inoculation, fix the animal on the rack with its abdomen facing up. Wash the abdomen with warm water and shave the hair. After shaving, clean the area with sterile saline and wait until it is dry. Use a 1mL syringe to draw up the bacterial solution and accurately inject 0.1mL can be inoculated intradermally, and 395
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6~8 strains of bacteria can be inoculated at the same time. 4h after injection, inject 4000u of antitoxin to the test animal to avoid death due to the strong virulence of the strain. Observe the intradermal reaction once at 24, 48, and 72h after injection. The control animal should have no local reaction regardless of whether it is inoculated with toxic or non-toxic strains; the test animal will show redness and swelling at the site of injection of the toxic strain at 24h, purulent lesions at the edge of the red and swollen site at 48h, hard lumps can be seen at 72h, and gray-black necrotic spots will appear, while non-toxic strains will have no lesions. Animal toxicity tests can also use rabbits and chickens as test animals. Appendix B
(Suggested Appendix)
Diphtheria toxin test (Sick's test)
Diphtheria toxin test can be used to determine the body's susceptibility to diphtheria and determine whether immunity is produced after diphtheria vaccination. B1 Method
Inject 0.1mL of diphtheria toxin solution into the skin of the left arm and 0.1mL of control solution into the skin of the right arm at the upper 1/3 of the palmar side of both forearms. B2 Observation and judgment of reaction
Negative reaction: No reaction at the injection site of both arms is a negative reaction. There are slightly red lumps at the injection site of both arms, the color is lighter, the boundaries are unclear, the red color fades quickly, leaving no trace, and it is most obvious in 24 to 36 hours, and basically disappears in 48 to 72 hours, which is a false positive. Negative and false positive reactions indicate immunity to diphtheria, but the disease can still occur when the infection is large. Positive reaction: The injection site of the control solution shows a positive reaction or a false negative reaction (basically disappears in 48 to 72 hours), while the injection site of the test solution shows a red mass with clear boundaries and a diameter of more than 1 cm. It usually begins to appear 24 to 48 hours after injection, is most obvious in 72 to 96 hours, and gradually disappears after 7 days, leaving a brown spot, which can disappear completely after several weeks to months. A positive reaction indicates that there is no immunity to diphtheria. Appendix C
(Suggested Appendix)
Serological Diagnostic Methods
C1 Indirect Hemagglutination Method for Determination of Diphtheria Antibodies
C1.1 Principle
The indirect hemagglutination method uses red blood cells as carriers and, after certain treatments, allows protein antigens to be adsorbed on the surface of red blood cells. Such blood cells are called sensitized blood cells. Sensitized blood cells are then used to determine the corresponding trace antibodies. When specific antibodies are present, antigen-antibody binding occurs, and blood cells agglutinate, which is a positive reaction.
C1.2 Materials
C1.2.1 Antigen
Used as sensitized blood cells. The content of diphtheria toxoid is above 20~30 Lf/mL. C1.2.2 Standard Antitoxin
Each milliliter contains 10 international units, which is used to determine the titer of sensitized blood cells. It must be inactivated at 56℃ for 30 minutes before use. C1.2.3 Blood cells
Healthy sheep blood cells (preserved in blood cell preservation fluid). C1.2.4 Preparation of test solution
C1.2.4.1 Preparation of 0.15 mol/L phosphate and salt solution C1. 2. 4. 1. 1 0. 15 mol/L NazHPO4NazHPO.·12H,O(MW=358.16)
Distilled water to
C1.2.4.1.2 0.15 mol/L.KH,PO)
KH,PO,(M. W= 136. 09)
Distilled water to
C1. 2. 4. 1. 3 0. 15 mol/L NaClNaCI(M. W=58.45)
Distilled water to
GB 159971995
1000mL
1 000 ml
1000mL
C1.2.4.2pH7.2 Phosphate buffer (PBS) 0. 15 mol/L Na2HPO
0. 15 mol/L KH,PO,
0. 15 mol/L NaCl
Measure the pH value after preparation and sterilize for use.
C1.2.4.3pH6.4 Phosphate buffer (PBS) 0. 15 mol/L Na,HPO
0. 15 mol/L KH2PO,
0. 15 rmol/L NaCl
Measure the pH value after preparation and sterilize for use.
C1.2.4.4pH8.2 phosphate buffer (PBS) 0. 15 mol/L Na2HPO.
0. 15 mol/L KH,PO
Measure the pH value after preparation, sterilize and set aside.
C1.2.4.5 Glutaraldehyde diluent
pH8.2 phosphate buffer (PBS)
0. 15 mol/L NaCl
Distilled water
Sterilize and place in cold storage for later use.
C1.2.4.61% glutaraldehyde solution
25% glutaraldehyde solution
Glutaraldehyde dilution
C1.2.4.71% dry acid solution
Add distilled water to
100 mL
Store at 4C away from light and use for one week. Dilute to 1:20000 with physiological saline before use. C1.2.4.8 Blood cell preservation solution
Glucose
Sodium citrate
Add distilled water to
Measure pH, and use 10% citric acid to calibrate to pH6.15, and disinfect at 0.7kPa for 20 minutes. C1.3 Preparation of sensitized blood cells
C1.3.1 Collection and processing of blood cells
Select healthy sheep for venous blood collection, inject into blood cell preservation solution (the ratio of blood cells to preservation solution is 1:1), shake well and place in a cold storage (2~8℃) for 48h, centrifuge and discard the supernatant, wash the blood cells three times with normal saline, centrifuge (2000~2500r/min) for 20~25min each time, discard the supernatant and obtain 397
blood cells.
C1.3.2 Aldehydation of blood cells
GB15997—1995
Take 1 portion of the above packed blood cells, add it to 24 portions of cooled 1% glutaraldehyde solution, place it at 2-6℃, fix it for 30 minutes or more, shake it every 5-6 minutes, centrifuge it (2000r/min, 10min), discard the supernatant, wash it five times with physiological saline, then wash it five times with distilled water, finally make it into 10% blood cell suspension with physiological saline, add 1:10000 thimerosal for preservation, and place it in a cold storage at 2-8℃ for use. C1.3.3 Sensitization of blood cells
10% aldehydation blood cell suspension, dilute the white blood cell to 2.5% with pH7.2 phosphate buffer (PBS) and then treat it with acid. a) Gan acid treatment
2.5% hemocytes are added with one portion of acid at a ratio of 1:10000, mixed and placed in a 45℃ or 37℃ water bath for 30min (shake every 5-6min), then washed three times with pH7.2 phosphate buffer (PBS) (2000-2500r/min, 5-6min), and finally prepared into a 2.5% hemocyte suspension with pH6.4 phosphate buffer (PBS). b) Sensitization
2.5% hemocytes are added with one portion of purified diphtheria toxoid (25Lf/mL) and three portions of pH6.4 phosphate buffer (PBS), placed in a 45℃ or 37℃ water bath for 30min (shake frequently), centrifuged and discarded the supernatant, washed twice with 0.5% rabbit serum saline, and then prepared into a 2.5% hemocyte suspension with 0.5% rabbit serum saline. C1.3.4 Preparation of control blood cells
Add 4 parts of pH 6.4 phosphate buffer (PBS) to 2.5% purified blood cells and place in a 45℃ or 37℃ water bath for 30 minutes (shake frequently), centrifuge and discard the supernatant, wash the blood cells twice with 0.5% rabbit serum saline, and then prepare a 2.5% control blood cell suspension with rabbit serum saline.
C1.4 Titer titration
C1.4.1 Determination of sensitized blood cells and sensitivity Dilute the standard diphtheria antitoxin with 0.5% rabbit serum saline to make it contain 1 unit per milliliter, and then dilute it in multiple ratios, from 1:2 to 1:4096. Take one drop (0.025mL) of each dilution and add it to the well of the blood coagulation plate, then add one drop of sensitized blood cells (0.025mL), shake well and place at 37℃ for 2h, take out and read the results or put it in the refrigerator, read the results the next morning, and take "++" as the end point of the agglutination test. The sensitivity of diphtheria toxin sensitized blood cells cannot be lower than 0.003906IU/mL. At the same time, make two controls: a) add antiserum of different dilutions to the blood cells to check whether the antiserum has nonspecific agglutination; b) add sensitized blood cells to 0.5% serum-free saline to check whether the blood cells have autoagglutination. The above two controls must be negative for the test to be valid, otherwise the test should be repeated.
C1.4.2 Treatment of serum to be tested
a) Inactivation at 56℃ for 30 min.
b) Absorption by aldehyded blood cells. Absorption method: Add 1 part of serum to be tested to 2 parts of 50% aldehyded blood cells, place at 37℃ for 1-2 hours, centrifuge and precipitate, and aspirate the supernatant as the serum sample diluted 1:2. C1.4.3 Titration of serum to be tested
First, add one drop (0.025mL) of 0.5% rabbit serum saline to each well of the blood coagulation plate, then use a micropipette to drop the serum to be tested into the first well (0.025mL) to mix with 0.5% rabbit serum saline, and be careful not to generate bubbles. After mixing the serum, take 0.025mL from each well and add it to the second well. After mixing, aspirate 0.025mL from the second well to the third well, and so on, and take out 0.025mL from the last well and discard it. Or use a 0.025mL dilution stick to make a series of dilutions starting from the second well. Use a micropipette to draw 0.025mL of 0.5% sensitized blood cells and add them to each well. Do not touch the wall of the well when adding, so as not to be stained with antiserum. Then shake the hook, place at 37℃ for 2h, take it out, place it in a refrigerator at 2~～10℃, and read the results the next morning. "Ten plus ten" is the endpoint of the agglutination test. Each serum to be tested can be used as a control for three dilutions of antiserum plus acid blood cells. The dilution method is as described above (i.e. 1:2, 1:4, 1:8) to exclude non-specific agglutination in the serum. C1.4.4 Result calculation
Test serum unit = test serum endpoint dilution × number of units contained in the standard serum endpoint dilution. For example, the endpoint dilution of standard diphtheria antitoxin (10 IU/mL) is 1:2048, containing 0.0048 IU/mL, while the endpoint dilution of the serum to be tested is 1:8, so its unit 398
is 0.038 IU/ml.
C1.4.5 Result judgment standard
GB15997-1995
a) Agglutinated red blood cells are evenly spread on the bottom of the well to form a thin layer, which is "ten-ten-ten"; b) Agglutinated red blood cells are basically the same as a), but there is a very weak red circle at the bottom of this thin layer, which is "ten-ten-ten"; c) Agglutinated red blood cells form a thin layer at the bottom of the well, and a loose red circle can be seen in the middle, which is "ten-ten"; d) Agglutinated red blood cells form a circle at the bottom of the well, and a small number of red blood cells can be seen around it, which is slightly larger than the control circle, which is "+" e) Blood cells form a tight circle at the bottom of the well to form a dot, which is "-". There are many factors that affect the accuracy and sensitivity of the hemagglutination test, such as the cleanliness of the utensils, the standardization of materials, and the meticulousness of the operation. C2 ELISA for detection of diphtheria toxin antibodies C2.1 Principle
Use diphtheria toxoid coated on a solid phase carrier to capture the corresponding anti-toxin antibodies in the serum to be tested, then use enzyme-labeled anti-human IgG antibody to remove the reaction (antigen-antibody-enzyme-labeled anti-antibody), and use the substrate to make the enzyme color to detect whether the antibody exists. C2.2 Materials
C2.2.1 Antigen
Purified diphtheria toxoid 1110Lf/mL.
C2.2.2 Conjugate
Sheep anti-human IgG enzyme-labeled antibody.
C2.2.3 Carrier
1cm×4cm polystyrene plastic tube with flat bottom and cap. C2.2.4 Substrate
O-phenylenediamine,
C2.2.5 Reference serum
Multiple indirect hemagglutination positive high-titer sera are mixed as positive reference serum, and multiple negative sera are mixed as negative reference serum. C2.2.6 Serum to be tested
Patients and healthy people in diphtheria outbreak areas. C2.2.7 Measuring instrument
721 spectrophotometer.
C2.3 Methods and steps
C2.3.1 Determination of the amount of coated antigen and the optimal dilution of the tested serum. The positive and negative reference serum were used for square titration with the antigen to determine that the concentration of the coated antigen in this test was 20ug/mL and the optimal initial dilution of the tested serum was 1:40.
C2.3.2 Plotting the standard curve of reference serum
Both positive and negative reference serum were diluted from 1:40 to 10240 by double dilution, and the reaction results were measured by 721 spectrophotometer for optical density (OD value, = 492nm). In this test, the average of the three determination results was taken, and the serum dilution was used as the horizontal axis and the corresponding OD value as the vertical axis to draw a curve; then the correlation regression method was used to determine the standard curve corresponding to the OD value and the serum dilution multiple. The highest OD value of positive serum was 0.6, and the lowest was 0.1. Because the boundary OD value of the obvious difference between positive and negative sera was 0.3, it was determined that (OD value greater than or equal to 0.3 was positive, and the corresponding serum dilution was 1:2560, which was one unit. Each sample of blood was tested The serum dilution is 1:40, and the titer is found from the standard curve. After calculation, the P values ​​of the two coefficients are both less than 0.01. C2.3.3 Antigen coating
Add 1.0mL of diluted antigen to each tube, place at 4C overnight, and then wash three times with pH7.2 phosphate buffer (PBS)-Tween 20, leaving it to rest for 3 minutes between each wash.
C2.3.4 Add 1.0mL of the test serum, leave it at room temperature for 2h, and wash three times again, using the same method as before. 399
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C2.3.5 Add 1.0mL of conjugate (1:8000), leave it at room temperature for 2h, and wash as before. C2.3.6 Add 1.0mL of substrate solution (0.04%), leave it at room temperature for 30min, add 2 0.015mL mol/L sulfuric acid. If the reaction is terminated, measure the OD value immediately.
C3 Solid phase radioimmunoassay for diphtheria antibodies
C3.1 Basic principle
Solid phase radioimmunoassay (SPRIA) is a double antibody method that uses purified diphtheria toxins to be adsorbed onto a solid phase carrier, and specific antiserum is added to form an antigen-antibody complex on the carrier. Then, a second antibody labeled with an isotope is added to form an antigen-antibody-antibody complex. The content of the corresponding antibody can be determined by measuring the radioactivity of the labeled second antibody. C3.2 Materials and methods
C3.2.1 Source of serum specimens
The subjects of diphtheria antibody level testing in the population are mostly serum specimens collected from children. C3. 2.2 The purified diphtheria toxoid was further purified, filtered through Sephadex G-200, and its concentration was measured. C3.2.3 Antiserum (standard product)
C3.2.4 Secondary antibody and standardization method
Using horse anti-human IgG (purified) as the secondary antibody, the 125I-horse anti-human IgG was obtained by radioiodination standardization according to the high-concentration standardization method of Greenwood and Hunter. C3.2.5 Solid phase carrier
U-shaped polyvinyl chloride soft plastic plate.
C3.2.6 Buffer and filling solution
a) Buffer: PBST, i.e., 0.01.mol/L pH 7.4 phosphate buffered saline containing 0.05% Tween 20. b) Diluent: PBST-BSA, i.e., adding 0.5% bovine serum albumin to the buffer. c) Filling solution: PBS-BSA, that is, subtract Tween 20 from the diluent. C3.3 Operation method
C3.3.1 Coating antigen, dilute the purified diphtheria toxoid to 5μg/mL with carbonate buffer (pH9.6), add 100μL to each well of the polynitrogen plate, and place in a 4℃ refrigerator overnight.
C3.3.2 Shake off the antigen solution, wash 5 times with PBST, let it stand for about 1min each time, fill each well with filling solution, put the plate in a wet box, incubate at 37'C for 1h, and suck out the filling solution with a water pump. C3.3.3 Add 100uL of serum sample diluted with PBST-BSA 1:20 to each well (do duplicate), incubate at 37℃ for 2h, suck out the liquid in the well, and wash 5 times with PBST.
C3.3.4Add 100uL of 200,000 CPM125I-labeled horse anti-human immunoglobulin (125I-IgG) to each well, keep warm at 37℃ for 2h, remove the liquid and wash with PBST 5 times, remove and wait for drying.
C3.3.5.Cut out each well on the polyvinyl chloride soft plastic plate with scissors, measure the radioactivity, and count each well for 1min. C3.3.6Draw a standard curve on the coordinate paper with the results of the standard samples with different dilutions, and use the average value of the two wells of each specimen to find the corresponding antitoxin IU/mL on the standard curve. 1000.025mL to the third well, and so on, take out 0.025mL from the last well and discard it. Or use a 0.025mL dilution stick to make a series of dilutions starting from the second well. Use a micropipette to draw 0.025mL of 0.5% sensitized blood cells and add them to each well. Do not touch the wall of the well when adding, so as not to be stained with antiserum. Then shake the hook, put it at 37℃ for 2h, take it out, put it in a refrigerator at 2~～10℃, and read the results the next morning. "Ten plus ten" is the end point of the agglutination test. Each serum to be tested can be used as a control of three dilutions of antiserum plus acid blood cells. The dilution method is as described above (i.e. 1:2, 1:4, 1:8) to exclude non-specific agglutination in the serum. C1.4.4 Result calculation
Test serum unit = test serum end point dilution × number of units contained in the standard serum end point dilution. For example, the endpoint dilution of standard diphtheria antitoxin (10 IU/mL) is 1:2048, containing 0.0048 IU/mL, while the endpoint dilution of the serum to be tested is 1:8, so its unit 398
is 0.038 IU/ml.
C1.4.5 Result judgment standard
GB15997-1995
a) Agglutinated red blood cells are evenly spread on the bottom of the well to form a thin layer, which is "ten-ten-ten"; b) Agglutinated red blood cells are basically the same as a), but there is a very weak red circle at the bottom of this thin layer, which is "ten-ten-ten"; c) Agglutinated red blood cells form a thin layer at the bottom of the well, and a loose red circle can be seen in the middle, which is "ten-ten"; d) Agglutinated red blood cells form a circle at the bottom of the well, and a small number of red blood cells can be seen around it, which is slightly larger than the control circle, which is "+" e) Blood cells form a tight circle at the bottom of the well to form a dot, which is "-". There are many factors that affect the accuracy and sensitivity of the hemagglutination test, such as the cleanliness of the utensils, the standardization of materials, and the meticulousness of the operation. C2 ELISA for detection of diphtheria toxin antibodies C2.1 Principle
Use diphtheria toxoid coated on a solid phase carrier to capture the corresponding anti-toxin antibodies in the serum to be tested, then use enzyme-labeled anti-human IgG antibody to remove the reaction (antigen-antibody-enzyme-labeled anti-antibody), and use the substrate to make the enzyme color to detect whether the antibody exists. C2.2 Materials
C2.2.1 Antigen
Purified diphtheria toxoid 1110Lf/mL.
C2.2.2 Conjugate
Sheep anti-human IgG enzyme-labeled antibody.
C2.2.3 Carrier
1cm×4cm polystyrene plastic tube with flat bottom and cap. C2.2.4 Substrate
O-phenylenediamine,
C2.2.5 Reference serum
Multiple indirect hemagglutination positive high-titer sera are mixed as positive reference serum, and multiple negative sera are mixed as negative reference serum. C2.2.6 Serum to be tested
Patients and healthy people in diphtheria outbreak areas. C2.2.7 Measuring instrument
721 spectrophotometer.
C2.3 Methods and steps
C2.3.1 Determination of the amount of coated antigen and the optimal dilution of the tested serum. The positive and negative reference serum were used for square titration with the antigen to determine that the concentration of the coated antigen in this test was 20ug/mL and the optimal initial dilution of the tested serum was 1:40.
C2.3.2 Plotting the standard curve of reference serum
Both positive and negative reference serum were diluted from 1:40 to 10240 by double dilution, and the reaction results were measured by 721 spectrophotometer for optical density (OD value, = 492nm). In this test, the average of the three determination results was taken, and the serum dilution was used as the horizontal axis and the corresponding OD value as the vertical axis to draw a curve; then the correlation regression method was used to determine the standard curve corresponding to the OD value and the serum dilution multiple. The highest OD value of positive serum was 0.6, and the lowest was 0.1. Because the boundary OD value of the obvious difference between positive and negative sera was 0.3, it was determined that (OD value greater than or equal to 0.3 was positive, and the corresponding serum dilution was 1:2560, which was one unit. Each sample of blood was tested The serum dilution is 1:40, and the titer is found from the standard curve. After calculation, the P values ​​of the two coefficients are both less than 0.01. C2.3.3 Antigen coating
Add 1.0mL of diluted antigen to each tube, place at 4C overnight, and then wash three times with pH7.2 phosphate buffer (PBS)-Tween 20, leaving it to rest for 3 minutes between each wash.
C2.3.4 Add 1.0mL of the test serum, leave it at room temperature for 2h, and wash three times again, using the same method as before. 399
GB15997—1995
C2.3.5 Add 1.0mL of conjugate (1:8000), leave it at room temperature for 2h, and wash as before. C2.3.6 Add 1.0mL of substrate solution (0.04%), leave it at room temperature for 30min, add 2 0.015mL mol/L sulfuric acid. If the reaction is terminated, measure the OD value immediately.
C3 Solid phase radioimmunoassay for diphtheria antibodies
C3.1 Basic principle
Solid phase radioimmunoassay (SPRIA) is a double antibody method that uses purified diphtheria toxins to be adsorbed onto a solid phase carrier, and specific antiserum is added to form an antigen-antibody complex on the carrier. Then, a second antibody labeled with an isotope is added to form an antigen-antibody-antibody complex. The content of the corresponding antibody can be determined by measuring the radioactivity of the labeled second antibody. C3.2 Materials and methods
C3.2.1 Source of serum specimens
The subjects of diphtheria antibody level testing in the population are mostly serum specimens collected from children. C3. 2.2 The purified diphtheria toxoid was further purified, filtered through Sephadex G-200, and its concentration was measured. C3.2.3 Antiserum (standard product)
C3.2.4 Secondary antibody and standardization method
Using horse anti-human IgG (purified) as the secondary antibody, the 125I-horse anti-human IgG was obtained by radioiodination standardization according to the high-concentration standardization method of Greenwood and Hunter. C3.2.5 Solid phase carrier
U-shaped polyvinyl chloride soft plastic plate.
C3.2.6 Buffer and filling solution
a) Buffer: PBST, i.e., 0.01.mol/L pH 7.4 phosphate buffered saline containing 0.05% Tween 20. b) Diluent: PBST-BSA, i.e., adding 0.5% bovine serum albumin to the buffer. c) Filling solution: PBS-BSA, that is, subtract Tween 20 from the diluent. C3.3 Operation method
C3.3.1 Coating antigen, dilute the purified diphtheria toxoid to 5μg/mL with carbonate buffer (pH9.6), add 100μL to each well of the polynitrogen plate, and place in a 4℃ refrigerator overnight.
C3.3.2 Shake off the antigen solution, wash 5 times with PBST, let it stand for about 1min each time, fill each well with filling solution, put the plate in a wet box, incubate at 37'C for 1h, and suck out the filling solution with a water pump. C3.3.3 Add 100uL of serum sample diluted with PBST-BSA 1:20 to each well (do duplicate), incubate at 37℃ for 2h, suck out the liquid in the well, and wash 5 times with PBST.
C3.3.4Add 100uL of 200,000 CPM125I-labeled horse anti-human immunoglobulin (125I-IgG) to each well, keep warm at 37℃ for 2h, remove the liquid and wash with PBST 5 times, remove and wait for drying.
C3.3.5.Cut out each well on the polyvinyl chloride soft plastic plate with scissors, measure the radioactivity, and count each well for 1min. C3.3.6Draw a standard curve on the coordinate paper with the results of the standard samples with different dilutions, and use the average value of the two wells of each specimen to find the corresponding antitoxin IU/mL on the standard curve. 1000.025mL to the third well, and so on, take out 0.025mL from the last well and discard it. Or use a 0.025mL dilution stick to make a series of dilutions starting from the second well. Use a micropipette to draw 0.025mL of 0.5% sensitized blood cells and add them to each well. Do not touch the wall of the well when adding, so as not to be stained with antiserum. Then shake the hook, put it at 37℃ for 2h, take it out, put it in a refrigerator at 2~～10℃, and read the results the next morning. "Ten plus ten" is the end point of the agglutination test. Each serum to be tested can be used as a control of three dilutions of antiserum plus acid blood cells. The dilution method is as described above (i.e. 1:2, 1:4, 1:8) to exclude non-specific agglutination in the serum. C1.4.4 Result calculation
Test serum unit = test serum end point dilution × number of units contained in the standard serum end point dilution. For example, the endpoint dilution of standard diphtheria antitoxin (10 IU/mL) is 1:2048, containing 0.0048 IU/mL, while the endpoint dilution of the serum to be tested is 1:8, so its unit 398
is 0.038 IU/ml.
C1.4.5 Result judgment standard
GB15997-1995
a) Agglutinated red blood cells are evenly spread on the bottom of the well to form a thin layer, which is "ten-ten-ten"; b) Agglutinated red blood cells are basically the same as a), but there is a very weak red circle at the bottom of this thin layer, which is "ten-ten-ten"; c) Agglutinated red blood cells form a thin layer at the bottom of the well, and a loose red circle can be seen in the middle, which is "ten-ten"; d) Agglutinated red blood cells form a circle at the bottom of the well, and a small number of red blood cells can be seen around it, which is slightly larger than the control circle, which is "+" e) Blood cells form a tight circle at the bottom of the well to form a dot, which is "-". There are many factors that affect the accuracy and sensitivity of the hemagglutination test, such as the cleanliness of the utensils, the standardization of materials, and the meticulousness of the operation. C2 ELISA for detection of diphtheria toxin antibodies C2.1 Principle
Use diphtheria toxoid coated on a solid phase carrier to capture the corresponding anti-toxin antibodies in the serum to be tested, then use enzyme-labeled anti-human IgG antibody to remove the reaction (antigen-antibody-enzyme-labeled anti-antibody), and use the substrate to make the enzyme color to detect whether the antibody exists. C2.2 Materials
C2.2.1 Antigen
Purified diphtheria toxoid 1110Lf/mL.
C2.2.2 Conjugate
Sheep anti-human IgG enzyme-labeled antibody.
C2.2.3 Carrier
1cm×4cm polystyrene plastic tube with flat bottom and cap. C2.2.4 Substrate
O-phenylenediamine,
C2.2.5 Reference serum
Multiple indirect hemagglutination positive high-titer sera are mixed as positive reference serum, and multiple negative sera are mixed as negative reference serum. C2.2.6 Serum to be tested
Patients and healthy people in diphtheria outbreak areas. C2.2.7 Measuring instrument
721 spectrophotometer.
C2.3 Methods and steps
C2.3.1 Determination of the amount of coated antigen and the optimal dilution of the tested serum. The positive and negative reference serum were used for square titration with the antigen to determine that the concentration of the coated antigen in this test was 20ug/mL and the optimal initial dilution of the tested serum was 1:40.
C2.3.2 Plotting the standard curve of reference serum
Both positive and negative reference serum were diluted from 1:40 to 10240 by double dilution, and the reaction results were measured by 721 spectrophotometer for optical density (OD value, = 492nm). In this test, the average of the three determination results was taken, and the serum dilution was used as the horizontal axis and the corresponding OD value as the vertical axis to draw a curve; then the correlation regression method was used to determine the standard curve corresponding to the OD value and the serum dilution multiple. The highest OD value of positive serum was 0.6, and the lowest was 0.1. Because the boundary OD value of the obvious difference between positive and negative sera was 0.3, it was determined that (OD value greater than or equal to 0.3 was positive, and the corresponding serum dilution was 1:2560, which was one unit. Each sample of blood was tested The serum dilution is 1:40, and the titer is found from the standard curve. After calculation, the P values ​​of the two coefficients are both less than 0.01. C2.3.3 Antigen coating
Add 1.0mL of diluted antigen to each tube, place at 4C overnight, and then wash three times with pH7.2 phosphate buffer (PBS)-Tween 20, leaving it to rest for 3 minutes between each wash.
C2.3.4 Add 1.0mL of the test serum, leave it at room temperature for 2h, and wash three times again, using the same method as before. 399
GB15997—1995
C2.3.5 Add 1.0mL of conjugate (1:8000), leave it at room temperature for 2h, and wash as before. C2.3.6 Add 1.0mL of substrate solution (0.04%), leave it at room temperature for 30min, add 2 0.015mL mol/L sulfuric acid. If the reaction is terminated, measure the OD value immediately.
C3 Solid phase radioimmunoassay for diphtheria antibodies
C3.1 Basic principle
Solid phase radioimmunoassay (SPRIA) is a double antibody method that uses purified diphtheria toxins to be adsorbed onto a solid phase carrier, and specific antiserum is added to form an antigen-antibody complex on the carrier. Then, a second antibody labeled with an isotope is added to form an antigen-antibody-antibody complex. The content of the corresponding antibody can be determined by measuring the radioactivity of the labeled second antibody. C3.2 Materials and methods
C3.2.1 Source of serum specimens
The subjects of diphtheria antibody level testing in the population are mostly serum specimens collected from children. C3. 2.2 The purified diphtheria toxoid was further purified, filtered through Sephadex G-200, and its concentration was measured. C3.2.3 Antiserum (standard product)
C3.2.4 Secondary antibody and standardization method
Using horse anti-human IgG (purified) as the secondary antibody, the 125I-horse anti-human IgG was obtained by radioiodination standardization according to the high-concentration standardization method of Greenwood and Hunter. C3.2.5 Solid phase carrier
U-shaped polyvinyl chloride soft plastic plate.
C3.2.6 Buffer and filling solution
a) Buffer: PBST, i.e., 0.01.mol/L pH 7.4 phosphate buffered saline containing 0.05% Tween 20. b) Diluent: PBST-BSA, i.e., adding 0.5% bovine serum albumin to the buffer. c) Filling solution: PBS-BSA, that is, subtract Tween 20 from the diluent. C3.3 Operation method
C3.3.1 Coating antigen, dilute the purified diphtheria toxoid to 5μg/mL with carbonate buffer (pH9.6), add 100μL to each well of the polynitrogen plate, and place in a 4℃ refrigerator overnight.
C3.3.2 Shake off the antigen solution, wash 5 times with PBST, let it stand for about 1min each time, fill each well with filling solution, put the plate in a wet box, incubate at 37'C for 1h, and suck out the filling solution with a water pump. C3.3.3 Add 100uL of serum sample diluted with PBST-BSA 1:20 to each well (do duplicate), incubate at 37℃ for 2h, suck out the liquid in the well, and wash 5 times with PBST.
C3.3.4Add 100uL of 200,000 CPM125I-labeled horse anti-human immunoglobulin (125I-IgG) to each well, keep warm at 37℃ for 2h, remove the liquid and wash with PBST 5 times, remove and wait for drying.
C3.3.5.Cut out each well on the polyvinyl chloride soft plastic plate with scissors, measure the radioactivity, and count each well for 1min. C3.3.6Draw a standard curve on the coordinate paper with the results of the standard samples with different dilutions, and use the average value of the two wells of each specimen to find the corresponding antitoxin IU/mL on the standard curve. 1005 Result judgment standard
GB15997-1995
a) Agglutinated red blood cells evenly spread on the bottom of the well to form a thin layer, which is "ten-ten-ten"; b) Agglutinated red blood cells are basically the same as a), but there is a very weak red circle at the bottom of this thin layer, which is "ten-ten-ten"; c) Agglutinated red blood cells form a thin layer at the bottom of the well, and a loose red circle can be seen in the middle, which is "ten-ten"; d) Agglutinated red blood cells form a circle at the bottom of the well, and a small number of red blood cells can be seen around it, which is slightly larger than the control circle, which is "+" e) Blood cells form a tight circle at the bottom of the well to form a dot, which is "-". There are many factors that affect the accuracy and sensitivity of the hemagglutination test, such as the cleanliness of the equipment, the standardization of materials, and the meticulousness of the operation. C2 ELISA for detection of diphtheria toxin antibodies C2.1 Principle
Use diphtheria toxoid coated on a solid phase carrier to capture the corresponding anti-toxin antibodies in the serum to be tested, then use enzyme-labeled anti-human IgG antibody to remove the reaction (antigen-antibody-enzyme-labeled anti-antibody), and use the substrate to make the enzyme color to detect whether the antibody exists. C2.2 Materials
C2.2.1 Antigen
Purified diphtheria toxoid 1110Lf/mL.
C2.2.2 Conjugate
Sheep anti-human IgG enzyme-labeled antibody.
C2.2.3 Carrier
1cm×4cm polystyrene plastic tube with flat bottom and cap. C2.2.4 Substrate
O-phenylenediamine,
C2.2.5 Reference serum
Multiple indirect hemagglutination positive high-titer sera are mixed as positive reference serum, and multiple negative sera are mixed as negative reference serum. C2.2.6 Serum to be tested
Patients and healthy people in diphtheria outbreak areas. C2.2.7 Measuring instrument
721 spectrophotometer.
C2.3 Methods and steps
C2.3.1 Determination of the amount of coated antigen and the optimal dilution of the tested serum. The positive and negative reference serum were used for square titration with the antigen to determine that the concentration of the coated antigen in this test was 20ug/mL and the optimal initial dilution of the tested serum was 1:40.
C2.3.2 Plotting the standard curve of reference serum
Both positive and negative reference serum were diluted from 1:40 to 10240 by double dilution, and the reaction results were measured by 721 spectrophotometer for optical density (OD value, = 492nm). In this test, the average of the three determination results was taken, and the serum dilution was used as the horizontal axis and the corresponding OD value as the vertical axis to draw a curve; then the correlation regression method was used to determine the standard curve corresponding to the OD value and the serum dilution multiple. The highest OD value of positive serum was 0.6, and the lowest was 0.1. Because the boundary OD value of the obvious difference between positive and negative sera was 0.3, it was determined that (OD value greater than or equal to 0.3 was positive, and the corresponding serum dilution was 1:2560, which was one unit. Each sample of blood was tested The serum dilution is 1:40, and the titer is found from the standard curve. After calculation, the P values ​​of the two coefficients are both less than 0.01. C2.3.3 Antigen coating
Add 1.0mL of diluted antigen to each tube, place at 4C overnight, and then wash three times with pH7.2 phosphate buffer (PBS)-Tween 20, leaving it to rest for 3 minutes between each wash.
C2.3.4 Add 1.0mL of the test serum, leave it at room temperature for 2h, and wash three times again, using the same method as before. 399
GB15997—1995
C2.3.5 Add 1.0mL of conjugate (1:8000), leave it at room temperature for 2h, and wash as before. C2.3.6 Add 1.0mL of substrate solution (0.04%), leave it at room temperature for 30min, add 2 0.015mL mol/L sulfuric acid. If the reaction is terminated, measure the OD value immediately.
C3 Solid phase radioimmunoassay for diphtheria antibodies
C3.1 Basic principle
Solid phase radioimmunoassay (SPRIA) is a double antibody method that uses purified diphtheria toxins to be adsorbed onto a solid phase carrier, and specific antiserum is added to form an antigen-antibody complex on the carrier. Then, a second antibody labeled with an isotope is added to form an antigen-antibody-antibody complex. The content of the corresponding antibody can be determined by measuring the radioactivity of the labeled second antibody. C3.2 Materials and methods
C3.2.1 Source of serum specimens
The subjects of diphtheria antibody level testing in the population are mostly serum specimens collected from children. C3. 2.2 The purified diphtheria toxoid was further purified, filtered through Sephadex G-200, and its concentration was measured. C3.2.3 Antiserum (standard product)
C3.2.4 Secondary antibody and standardization method
Using horse anti-human IgG (purified) as the secondary antibody, the 125I-horse anti-human IgG was obtained by radioiodination standardization according to the high-concentration standardization method of Greenwood and Hunter. C3.2.5 Solid phase carrier
U-shaped polyvinyl chloride soft plastic plate.
C3.2.6 Buffer and filling solutionbzxZ.net
a) Buffer: PBST, i.e., 0.01.mol/L pH 7.4 phosphate buffered saline containing 0.05% Tween 20. b) Diluent: PBST-BSA, i.e., adding 0.5% bovine serum albumin to the buffer. c) Filling solution: PBS-BSA, that is, subtract Tween 20 from the diluent. C3.3 Operation method
C3.3.1 Coating antigen, dilute the purified diphtheria toxoid to 5μg/mL with carbonate buffer (pH9.6), add 100μL to each well of the polynitrogen plate, and place in a 4℃ refrigerator overnight.
C3.3.2 Shake off the antigen solution, wash 5 times with PBST, let it stand for about 1min each time, fill each well with filling solution, put the plate in a wet box, incubate at 37'C for 1h, and suck out the filling solution with a water pump. C3.3.3 Add 100uL of serum sample diluted with PBST-BSA 1:20 to each well (do duplicate), incubate at 37℃ for 2h, suck out the liquid in the well, and wash 5 times with PBST.
C3.3.4Add 100uL of 200,000 CPM125I-labeled horse anti-human immunoglobulin (125I-IgG) to each well, keep warm at 37℃ for 2h, remove the liquid and wash with PBST 5 times, remove and wait for drying.
C3.3.5.Cut out each well on the polyvinyl chloride soft plastic plate with scissors, measure the radioactivity, and count each well for 1min. C3.3.6Draw a standard curve on the coordinate paper with the results of the standard samples with different dilutions, and use the average value of the two wells of each specimen to find the corresponding antitoxin IU/mL on the standard curve. 1005 Result judgment standard
GB15997-1995
a) Agglutinated red blood cells evenly spread on the bottom of the well to form a thin layer, which is "ten-ten-ten"; b) Agglutinated red blood cells are basically the same as a), but there is a very weak red circle at the bottom of this thin layer, which is "ten-ten-ten"; c) Agglutinated red blood cells form a thin layer at the bottom of the well, and a loose red circle can be seen in the middle, which is "ten-ten"; d) Agglutinated red blood cells form a circle at the bottom of the well, and a small number of red blood cells can be seen around it, which is slightly larger than the control circle, which is "+" e) Blood cells form a tight circle at the bottom of the well to form a dot, which is "-". There are many factors that affect the accuracy and sensitivity of the hemagglutination test, such as the cleanliness of the equipment, the standardization of materials, and the meticulousness of the operation. C2 ELISA for detection of diphtheria toxin antibodies C2.1 Principle
Use diphtheria toxoid coated on a solid phase carrier to capture the corresponding anti-toxin antibodies in the serum to be tested, then use enzyme-labeled anti-human IgG antibody to remove the reaction (antigen-antibody-enzyme-labeled anti-antibody), and use the substrate to make the enzyme color to detect whether the antibody exists. C2.2 Materials
C2.2.1 Antigen
Purified diphtheria toxoid 1110Lf/mL.
C2.2.2 Conjugate
Sheep anti-human IgG enzyme-labeled antibody.
C2.2.3 Carrier
1cm×4cm polystyrene plastic tube with flat bottom and cap. C2.2.4 Substrate
O-phenylenediamine,
C2.2.5 Reference serum
Multiple indirect hemagglutination positive high-titer sera are mixed as positive reference serum, and multiple negative sera are mixed as negative reference serum. C2.2.6 Serum to be tested
Patients and healthy people in diphtheria outbreak areas. C2.2.7 Measuring instrument
721 spectrophotometer.
C2.3 Methods and steps
C2.3.1 Determination of the amount of coated antigen and the optimal dilution of the tested serum. The positive and negative reference serum were used for square titration with the antigen to determine that the concentration of the coated antigen in this test was 20ug/mL and the optimal initial dilution of the tested serum was 1:40.
C2.3.2 Plotting the standard curve of reference serum
Both positive and negative reference serum were diluted from 1:40 to 10240 by double dilution, and the reaction results were measured by 721 spectrophotometer for optical density (OD value, = 492nm). In this test, the average of the three determination results was taken, and the serum dilution was used as the horizontal axis and the corresponding OD value as the vertical axis to draw a curve; then the correlation regression method was used to determine the standard curve corresponding to the OD value and the serum dilution multiple. The highest OD value of positive serum was 0.6, and the lowest was 0.1. Because the boundary OD value of the obvious difference between positive and negative sera was 0.3, it was determined that (OD value greater than or equal to 0.3 was positive, and the corresponding serum dilution was 1:2560, which was one unit. Each sample of blood was tested The serum dilution is 1:40, and the titer is found from the standard curve. After calculation, the P values ​​of the two coefficients are both less than 0.01. C2.3.3 Antigen coating
Add 1.0mL of diluted antigen to each tube, place at 4C overnight, and then wash three times with pH7.2 phosphate buffer (PBS)-Tween 20, leaving it to rest for 3 minutes between each wash.
C2.3.4 Add 1.0mL of the test serum, leave it at room temperature for 2h, and wash three times again, using the same method as before. 399
GB15997—1995
C2.3.5 Add 1.0mL of conjugate (1:8000), leave it at room temperature for 2h, and wash as before. C2.3.6 Add 1.0mL of substrate solution (0.04%), leave it at room temperature for 30min, add 2 0.015mL mol/L sulfuric acid. If the reaction is terminated, measure the OD value immediately.
C3 Solid phase radioimmunoassay for diphtheria antibodies
C3.1 Basic principle
Solid phase radioimmunoassay (SPRIA) is a double antibody method that uses purified diphtheria toxins to be adsorbed onto a solid phase carrier, and specific antiserum is added to form an antigen-antibody complex on the carrier. Then, a second antibody labeled with an isotope is added to form an antigen-antibody-antibody complex. The content of the corresponding antibody can be determined by measuring the radioactivity of the labeled second antibody. C3.2 Materials and methods
C3.2.1 Source of serum specimens
The subjects of diphtheria antibody level testing in the population are mostly serum specimens collected from children. C3. 2.2 The purified diphtheria toxoid was further purified, filtered through Sephadex G-200, and its concentration was measured. C3.2.3 Antiserum (standard product)
C3.2.4 Secondary antibody and standardization method
Using horse anti-human IgG (purified) as the secondary antibody, the 125I-horse anti-human IgG was obtained by radioiodination standardization according to the high-concentration standardization method of Greenwood and Hunter. C3.2.5 Solid phase carrier
U-shaped polyvinyl chloride soft plastic plate.
C3.2.6 Buffer and filling solution
a) Buffer: PBST, i.e., 0.01.mol/L pH 7.4 phosphate buffered saline containing 0.05% Tween 20. b) Diluent: PBST-BSA, i.e., adding 0.5% bovine serum albumin to the buffer. c) Filling solution: PBS-BSA, that is, subtract Tween 20 from the diluent. C3.3 Operation method
C3.3.1 Coating antigen, dilute the purified diphtheria toxoid to 5μg/mL with carbonate buffer (pH9.6), add 100μL to each well of the polynitrogen plate, and place in a 4℃ refrigerator overnight.
C3.3.2 Shake off the antigen solution, wash 5 times with PBST, let it stand for about 1min each time, fill each well with filling solution, put the plate in a wet box, incubate at 37'C for 1h, and suck out the filling solution with a water pump. C3.3.3 Add 100uL of serum sample diluted with PBST-BSA 1:20 to each well (do duplicate), incubate at 37℃ for 2h, suck out the liquid in the well, and wash 5 times with PBST.
C3.3.4Add 100uL of 200,000 CPM125I-labeled horse anti-human immunoglobulin (125I-IgG) to each well, keep warm at 37℃ for 2h, remove the liquid and wash with PBST 5 times, remove and wait for drying.
C3.3.5.Cut out each well on the polyvinyl chloride soft plastic plate with scissors, measure the radioactivity, and count each well for 1min. C3.3.6Draw a standard curve on the coordinate paper with the results of the standard samples with different dilutions, and use the average value of the two wells of each specimen to find the corresponding antitoxin IU/mL on the standard curve. 1003 is positive, and the corresponding serum dilution is 1:2560, which is one unit. The dilution of each test serum is 1:40, and the titer is found from the standard curve. After calculation, the P values ​​of both coefficients are less than 0.01. C2.3.3 Antigen coating
Add 1.0mL of diluted antigen to each tube, place at 4C overnight, and then wash three times with pH7.2 phosphate buffer (PBS)-Tween 20, and let it stand for 3 minutes between two times.
C2.3.4 Add 1.0mL of the test serum, place at room temperature for 2h, and wash three times again, the method is the same as before. 399
GB15997—1995
C2.3.5 Add 1.0mL of conjugate (1:8000), place at room temperature for 2h, and wash as before. C2.3.6 Add 1.0 mL of substrate solution (0.04%), keep at room temperature for 30 minutes, add 0.015 mL of 2 mol/L sulfuric acid, and measure the OD value immediately if the reaction is terminated.
C3 Solid phase radioimmunoassay for diphtheria antibodies
C3.1 Basic principle
Solid phase radioimmunoassay (SPRIA) is a double antibody method, in which purified diphtheria toxoid is adsorbed onto a solid phase carrier, and specific antiserum is added to form an antigen-antibody complex on the carrier. Then, a second antibody labeled with an isotope is added to form an antigen-antibody-antibody complex. The content of the corresponding antibody can be determined by measuring the radioactivity of the labeled second antibody. C3.2 Materials and methods
C3.2.1 Source of serum samples
The subjects of diphtheria antibody level detection in the population are mostly serum samples collected from children. C3.2.2 The purified diphtheria toxoid is further purified, filtered through Sephadex G-200, and its concentration is measured. C3.2.3 Antiserum (standard)
C3.2.4 Secondary antibody and standardization method
Use horse anti-human IgG (purified) as the secondary antibody and obtain 125I-horse anti-human IgG by radioiodination standardization according to the high-concentration standardization method of Greenwood and Hunter. C3.2.5 Solid phase carrier
U-shaped polyvinyl chloride soft plastic plate.
C3.2.6 Buffer and filling solution
a) Buffer: PBST, i.e. 0.01.mol/L pH 7.4 phosphate buffered saline containing 0.05% Tween 20. b) Diluent: PBST-BSA, i.e. 0.5% bovine serum albumin is added to the buffer. c) Filling solution: PBS-BSA, i.e. Tween 20 is subtracted from the diluent. C3.3 Operation method
C3.3.1 Coating antigen, dilute the purified diphtheria toxoid to 5μg/mL with carbonate buffer (pH9.6), add 100μL to each well of the polynitrogen plate, and place in a 4℃ refrigerator overnight.
C3.3.2 Shake off the antigen solution, wash 5 times with PBST, let stand for about 1min each time, fill each well with filling liquid, put the plate in a wet box, incubate at 37'C for 1h, and suck out the filling liquid with a water pump. C3.3.3 Add 100uL of serum sample diluted with PBST-BSA1:20 to each well (do duplicate), incubate at 37℃ for 2h, suck out the liquid in the well, and wash 5 times with PBST.
C3.3.4Add 100uL of 200,000 CPM125I-labeled horse anti-human immunoglobulin (125I-IgG) to each well, keep warm at 37℃ for 2h, remove the liquid and wash with PBST 5 times, remove and wait for drying.
C3.3.5.Cut out each well on the polyvinyl chloride soft plastic plate with scissors, measure the radioactivity, and count each well for 1min. C3.3.6Draw a standard curve on the coordinate paper with the results of the standard samples with different dilutions, and use the average value of the two wells of each specimen to find the corresponding antitoxin IU/mL on the standard curve. 1003 is positive, and the corresponding serum dilution is 1:2560, which is one unit. The dilution of each test serum is 1:40, and the titer is found from the standard curve. After calculation, the P values ​​of both coefficients are less than 0.01. C2.3.3 Antigen coating
Add 1.0mL of diluted antigen to each tube, place at 4C overnight, and then wash three times with pH7.2 phosphate buffer (PBS)-Tween 20, and let it stand for 3 minutes between two times.
C2.3.4 Add 1.0mL of the test serum, place at room temperature for 2h, and wash three times again, the method is the same as before. 399
GB15997—1995
C2.3.5 Add 1.0mL of conjugate (1:8000), place at room temperature for 2h, and wash as before. C2.3.6 Add 1.0 mL of substrate solution (0.04%), keep at room temperature for 30 minutes, add 0.015 mL of 2 mol/L sulfuric acid, and measure the OD value immediately if the reaction is terminated.
C3 Solid phase radioimmunoassay for diphtheria antibodies
C3.1 Basic principle
Solid phase radioimmunoassay (SPRIA) is a double antibody method, in which purified diphtheria toxoid is adsorbed onto a solid phase carrier, and specific antiserum is added to form an antigen-antibody complex on the carrier. Then, a second antibody labeled with an isotope is added to form an antigen-antibody-antibody complex. The content of the corresponding antibody can be determined by measuring the radioactivity of the labeled second antibody. C3.2 Materials and methods
C3.2.1 Source of serum samples
The subjects of diphtheria antibody level detection in the population are mostly serum samples collected from children. C3.2.2 The purified diphtheria toxoid is further purified, filtered through Sephadex G-200, and its concentration is measured. C3.2.3 Antiserum (standard)
C3.2.4 Secondary antibody and standardization method
Use horse anti-human IgG (purified) as the secondary antibody and obtain 125I-horse anti-human IgG by radioiodination standardization according to the high-concentration standardization method of Greenwood and Hunter. C3.2.5 Solid phase carrier
U-shaped polyvinyl chloride soft plastic plate.
C3.2.6 Buffer and filling solution
a) Buffer: PBST, i.e. 0.01.mol/L pH 7.4 phosphate buffered saline containing 0.05% Tween 20. b) Diluent: PBST-BSA, i.e. 0.5% bovine serum albumin is added to the buffer. c) Filling solution: PBS-BSA, i.e. Tween 20 is subtracted from the diluent. C3.3 Operation method
C3.3.1 Coating antigen, dilute the purified diphtheria toxoid to 5μg/mL with carbonate buffer (pH9.6), add 100μL to each well of the polynitrogen plate, and place in a 4℃ refrigerator overnight.
C3.3.2 Shake off the antigen solution, wash 5 times with PBST, let stand for about 1min each time, fill each well with filling liquid, put the plate in a wet box, incubate at 37'C for 1h, and suck out the filling liquid with a water pump. C3.3.3 Add 100uL of serum sample diluted with PBST-BSA1:20 to each well (do duplicate), incubate at 37℃ for 2h, suck out the liquid in the well, and wash 5 times with PBST.
C3.3.4Add 100uL of 200,000 CPM125I-labeled horse anti-human immunoglobulin (125I-IgG) to each well, keep warm at 37℃ for 2h, remove the liquid and wash with PBST 5 times, remove and wait for drying.
C3.3.5.Cut out each well on the polyvinyl chloride soft plastic plate with scissors, measure the radioactivity, and count each well for 1min. C3.3.6Draw a standard curve on the coordinate paper with the results of the standard samples with different dilutions, and use the average value of the two wells of each specimen to find the corresponding antitoxin IU/mL on the standard curve. 100
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