Laboratory techniques used for Immunological laboratory methods
Dr. Tatiana Jones, MD, PhD NCC
How to Make Serial Dilutions? Interpretation can be made differently depending on the nature of test.
For example, if we need to figure out in what sample the concentration of the antibody or antigen is higher, we will go by TITER, which is the lowest serial dilution (let’s say that it is 1:32 in the picture on the left) that gives us positive result. This mean that even diluted 32 times sample is still capable of reacting.
The other scenario when we are interpreting quantitative assays, such as ELISA. In this case we need to match results of our samples to known concentrations of STANDARD and MULTIPLY be our dilution factor. What is Antibody Titer?
An antibody titer is a measurement of how much antibody an organism has produced that recognizes a particular antigen. Titer is expressed as the inverse of the greatest dilution that still gives a positive result. ELISA is a common means of determining antibody titers. How to Determine Antibody Titer? Where we can use Indirect Coombs test detects the presence of anti-Rh antibodies in blood serum. A patient might be reported to have an "indirect Antibody Titer? Coombs titer" of 16. This means that the patient's serum gives a positive indirect Coombs test at any dilution down to 1/16 (1 part serum to 15 parts diluent). At greater dilutions the indirect Coombs test is negative. If a few weeks later the same patient had an indirect Coombs titer of 32 (1/32 dilution which is 1 part serum to 31 parts diluent), this would mean that more anti-Rh antibody was made, since it took a greater dilution to eradicate the positive test.
Many traditional serological tests such as Rheumatoid Factor, hemagglutination or complement fixation employ this principle. Such tests can typically be read visually, which makes them fast and cost-effective in a "low-tech" environment. The interpretation of serological titers is guided by reference values that are specific for the antigen or antibody in question; a titer of 1:32 may be below the cut-off for one test but above for another. How Does it Work? Examples of Interpretation Phases of Antibody Response Antigen : Antibody Ratio
Ratio Ag : Ab affects agglutination reactions, both the first and second stages. In double immunodiffusion, if antigen to be detected, a known reagent antibody is placed in the center well and the unknown samples are placed in the surrounding well. If antibody is to be detected, unknown antigen is placed in the center. After each of the samples and reagents have been added to the appropriate wells, diffusion occurs and both antigen and antibody diffuse radially from wells toward each other, thereby establishing a concentration gradient. A line of precipitation forms at the zone of equivalence. https://www.youtube.com/watch?v=4rX_q6Tndx8 and https://www.youtube.com/watch?v=oYnXeAPieN0 videos. Ouchterlony Double Immunodiffusion
Ouchterlony double immunodiffusion (also known as agar gel immunodiffusion or passive double immunodiffusion) is an immunological technique used in the detection, identification and quantification of antibodies and antigens, such as immunoglobulins and extractable nuclear antigens. In radial immunodiffusion, an antigen sample is placed in a well and allowed to diffuse into agar containing a suitable dilution of an antibody. The antigen diffuses in all directions from the well, and accordingly the region of equivalence is established and a ring of precipitation (precipitin ring) forms around the well. The area of the precipitation ring is proportional to the concentration of antigen. The diameter of the area of precipitation (including the well diameter) is measured to determine the concentration of antigen. Enzyme-Linked Immunosorbent Assay ELISA Activity (The ELISA Method)
Antigen Detection Antibody
Capture Antibody
Enzyme
Positive test Negative test
Substrate “Sandwich” ELISA
1. Cover tray with Capture Ab (primary Ab) – 8-24 hrs. 2. Wash. 3. Block to prevent non-specific binding – 2-48 hrs. 4. Wash. 5. Place Standard and Samples – 1-2 hrs. 6. Wash. 7. Place Detection Ab (secondary Ab) – 1-2 hrs. 8. Wash. 9. Place Enzyme – 30 min – 1 hr. 10. Wash. 11. Place Substrate. 12. Read results using plate reader.
Immunoelectrophoresis is a gel electrophoretic technique which uses both electrophoresis and double diffusion. The samples that contain the proteins (the antigen) are added to the wells on the gel plate. The samples could be a mixture and contain serum from healthy as well as from infected persons. The antigen mixture is first electrophoresed to separate its components by charge. This creates line of the electric field, into which a purified known antibody is added. The antibody molecules diffuse into the gel. When an antigen is encountered by an antibody, formation of a visual precipitate occurs. Precipitin arcs form at the zone of equivalence between the antigen and specific antibody. The pattern of precipitation can reveal antigenic differences between the normal serum and the serum from an affected person. Immunoelectrophoresis is used in clinical laboratories to detect the presence or absence of proteins in the serum, especially changes concerned with immunoglobulins. Change in the immunoglobulin profile can be the result of immunodeficiencies, chronic bacterial or viral infections, and https://www.youtube.com/watch?v=52opF5Cc1-k video. infections of a fetus. Rheumatoid Factor (RF). Rheumatoid arthritis (RA) is a chronic autoimmune disease affecting primarily joints. The autoimmune antibody generated in patients with RA belongs to group of IgM class immunoglobulins that interacts with self-IgG molecules, thus patients with RA have “anti- antibodies”. Rheumatoid factor (RF) is detected in 60-80% of cases of diagnosed RA. RF is an IgM class anti-antibody, which in-vitro, is detected by its ability to agglutinate latex particles (or red blood cells) coated with human IgG. RF in patient sample, if present, will attach to the IgG coating the latex particles. Agglutination of the latex particles is a positive result indicating the presence of RF. Latex or Erythrocyte agglutination.
Two-fold dilutions of samples of different influenza viruses (A – H) were prepared, mixed with chicken red blood cells, and added to the wells of a 96-well plate. After 30 minutes the wells were photographed. Sample A causes hemagglutination up to the 1:256 dilution; therefore the HA titer of this virus stock is 256. The sample in row B and C contain no detectable virus, while that in row D has an HA titer of 512, E - 1024. Antibody titration. An antibody titer is a measurement of how much antibody an organism has produced that recognizes a particular antigen, expressed as the inverse of the greatest dilution that still gives a positive result. ELISA, RF, Rh Factor are a common Make serial dilutions of a sample to be tested for antibody and then add a fixed number of red means of determining blood cells or bacteria or other particulate antibody titers. antigen and determine the maximum dilution, which gives agglutination. The maximum dilution that gives visible agglutination is called the titer.