In vivo induction and detection of antibodies
In vivo induction and detection of antibodies
Detection of Antibody Production in Vivo by ELISA by J.E. Colligan et al, Translated by Xuitao Cao et al. This experiment is from the "Compendium of Immunology Laboratory Guide".
Operation method
In vivo induction and detection of antibodies Move Basic Scheme 1 In vivo induction of antibodies by protein or polysaccharide antigens Materials Human peripheral blood Multivalent pneumococcal vaccines (e.g., Pneumovax 23, Merck or Pnu-Immune 23, Lederle) Hypodermic syringe and alcohol swabs 1 . Collect normal human peripheral blood (Appendix 3G), hemoconcentrate, centrifuge the serum, and store at 20 to 70 degrees Celsius. 2 . Inject 0.5 ml of the toxoid mixture into the muscle of the triceps or thigh muscle using a hypodermic syringe, taking care to avoid blood vessels. The injection should be monitored closely for 24 hours to prevent the occurrence of allergic reactions (although rare), and the situation should be dealt with promptly. 3 . Or subcutaneous injection of 0.5 ml of polyvalent pneumococcal vaccine. The injection should be monitored closely and treated promptly (allergic reactions are more likely to occur in previously vaccinated persons). 4. Blood should be collected at weeks 1, 2 and 3 after immunization. If blood is collected only once, it can be collected in the second week after immunization. For IgM detection, blood should be collected on the 3rd to 7th day after immunization. After separating the serum, store the serum at 20 to 70 degrees Celsius. 5. Determine the amount of antibody in the sample by the ELISA method (see Basic Plan 2). Carbonate buffer, P H 9.6 or PBS (see related manuals) Antigens: diphtheria toxoid, tetanus toxoid, pneumococcal type I or II polysaccharide or protein-coupled type III Pneumococcal polysaccharide or protein-coupled type III polysaccharide (see supplemental regimen) NaN3, recommended Tween/PBS: PBS with 0 -0.5 % (VTV) Tween 20 (1 month storage at 400 C) PBS with 1 % (VAO fetalcalfserum (FCS, 56°C, Ih inactivated) or PBS with 0.1% (m/V) BSA is recommended. Serum standards and sera to be tested, avoid repeated freezing and thawing. FCS/Tween/PBS: Tween/PBS with 0.1% FCS (56°C, Ih inactivated). alkaline phosphatase (AP)-labeled secondary antibodies: K and light chains specific to human antibodies, and anti-human IgG, Ig A, and IgE antibodies (Tago) Substrate, freshly prepared (approx. 6mL per plate) 3mol/L N a O H Tetanus toxoid and diphtheria toxoid standards 9 6-well flat-bottomed culture plates and lids (Costar) Semi- or fully automated plate washer (Dynatech) Enzyme couplers 1 . Take a 96-well plate, set aside the first column as a blank, and add 150 M1 Carbonate Buffer (for toxoid antigens) or PBS (for polysaccharide antigens) at intervals in each of the remaining columns (e.g., columns 2, 4, 6, 8, 10). 2 . Dilute diphtheria toxoid and tetanus toxoid to 0.22Lf U /m l and 0.56LfU /m l, respectively, in carbonate buffer; dilute I/I/I pneumococcal polysaccharide and protein-coupled type III pneumococcal polysaccharide to 0 -025m g /m l and 1~3/^g/m l, respectively, in PBS. Add 150^1 of each diluted antigen to the remaining columns (e.g., columns 3, 5, 7, 9, 11) and incubate at 4°C overnight. Incubate overnight at 4°C. If 0.2% NaN3 is added, incubate overnight at 4°C. The plates can be stored for 4 weeks if 0.02% NAN3 is added. 3. Wash the plates three times with a semi-automatic or fully automatic plate washer, adding 200 Ml Tween/PBS each time, and washing the plates at intervals of 1 to 2 min. Finally, pat dry the residual washings. 4 . Alternate procedure: To reduce non-specific binding, add 100~150/xl of PBS containing 1 % FCS or 0.1 % BSA. Incubate at room temperature for Ih (or overnight at 4°C) and wash the plates (this step is not necessary for detection of diphtheria toxoid and tetanus toxoid antigens). 5 . Make a standard curve by serial dilution of the standard serum 3 times. The method is as follows: In the first row of wells in columns 2 to 3, add 150ul of the highest concentration of standard (including antigen-coated and antigen-uncoated wells). Add 100ul of FCS/Tween/PBS to the remaining wells in columns 2 to 3. Pipette 50M1 of serum from each of the two wells in the first row into the corresponding two wells in the second row, and then dilute the serum 3-fold. After gently blowing and mixing, perform serial 3-fold dilutions of serum in the remaining wells of rows 2 to 3 in the same manner as above. Finally, the serum was pipetted from each of the two wells in the last row. 6 . Dilute pairs of sera to be tested (pre- and post-immunization) and add to the 96-well plate as described above. Add 150ul of diluted serum to the top two wells of each of the two columns (including the antigen-coated column and the antigen-uncoated column) (usually 1:40, but higher dilutions can be used for post-immunization sera). 2 Add IOOul of FCS/Tween/PBS to the remaining wells in the vertical column. Make a 3-fold serial dilution of the serum to be tested as in step 5. 7 . Incubate at room temperature for 2 h or at 4°C overnight. Wash the plate 3 times with Tween/PBS and pat dry the residual wash solution. 8 . Dilute the AP-labeled secondary antibody with FCS/Tween/P B S to the appropriate concentration. Add IOOul of antibody to each well using a multichannel pipette. The first column is a blank control and only FCS/Tween/PBS is added. Incubate at room temperature for 2 h or at 4°C overnight. 9. Wash the plates three times with Tween/PBS. Wash the plate three times with Tween/PBS and pat dry the residual washings. Add IOOmI freshly prepared substrate solution to each well and develop the color at room temperature. The absorbance value was detected by ELISA, and the color development was terminated by adding lOOjul of 3mol/L N aOH to each well when the absorbance value of A 4q5 in the well with the highest concentration of the standard reached 1.0. 10 . Enter the data into the computer for processing and analysis. When analyzing the results, the absorbance value of the sample wells should be subtracted from the absorbance value of the blank control wells. 11 . Compare the absorbance value of the sample to be tested with the absorbance value of the standard. Specify the antibody potency of the undiluted standard as 1000 antibody units and calculate the antibody units of the sample. It is desirable to convert the standards to international standard values against the international standards for diphtheria toxoid and tetanus toxoid and then calculate the sample values. Pneumococcal polysaccharides type III (and other types of pneumococcal polysaccharides such as 6A, 9, 14, and 19) need to be coupled with proteins in order to be stably adsorbed on the surface of the solid phase and to obtain reproducible experimental results. This method can also be used for the coupling of type I and II pneumococcal polysaccharides to proteins, in which case the dose of polysaccharide antigen should be reduced by a factor of 5 to 10. Indicator Fluid Cyanuric chloride crystals 0.1% ( m A O poly(lysine) (relative molecular mass 30,000-70,000; Sigma) Pneumococcal polysaccharide type III PBS 1. Take 3 test tubes and label them A, B and C. Take three test tubes and label them as A, B and C. Add 0.5 ml of indicator solution - to tube A. Add 0.5 ml of indicator solution- to tube A, 0.5 m g of cyanuric chloride crystals to tube B and 0.1 ml of 0.1% (m/V) polylysine to tube C. 2 . Dilute pneumococcal polysaccharide antigen to l m g /m l with water. Add 0.1 m l of diluted antigen to tube A and mix for IOs (the solution is pink in color). Transfer the liquid from tube A to tube B and mix the IOs (the solution becomes colorless when the p H drops to 8.0~8.2). Then transfer the liquid from tube B to tube C. Mix well. After homogenization, the solution was allowed to set at 4 °C for 2 h. The solution was allowed to set at 4 °C for 3 h. 3 . Dilute pneumococcal polysaccharide antigen with PBS to 2-5 ug/ml (100 Mg of coupled antigen is obtained). For more product details, please visit Aladdin Scientific website.
Toxoid mixture: 25Lf U/ml diphtheria toxoid (Connaught Laboratories or State Laboratoriy Institute of Massachusetts) mixed with IOLf U/ml tetanus toxoid (State Laboratoriy Institute of Massachusetts). State Laboratoriy Institute of Massachusetts)
Finally, 50M1 of diluted serum was aspirated from each of the two wells in the last row (the final volume of all wells at this point was lOOul).