Leave the clotted blood at 4oC overnight to allow absorption of cold agglutinins on the red cells. Centrifuge the container and separate the serum in a second sterile container.
If plasmapheresis is carried out, add one ml of 1 M CaCl2.6 H2O for every 100 ml of plasma to be defibrinated. Incubate at 37oC for 15 to 30 min. allowing clot to form and retract. Separate the clear serum.
To prevent bacterial contamination, use sterile procedures, add bacteriostatic sodium azide to achieve final concentration of 0.1% (0.5 ml of 20% sodium azide for every 100 ml of plasma) and store at 4oC.
This human serum is used to immunize animals to prepare antisera. For this, rabbit, goat, sheep, horse, donkey and chicken are in use. Rabbits being small and easy to handle are preferred, though goat gives ten times more antiserum.
The inoculated animals produce antisera against human’s serum. These antisera require standardising procedures such as absorption to remove unwanted antibodies and dilution to adjust prozone.
Some of the antisera can also be prepared from the plants. Lectins are the saline extracts made from seeds that have ability to agglutinate the red cells. They are highly specific for known red cell antigens and hence are useful as grouping reagents.
The various lectins available with their specific activity are as follows:
Dolichos Biflorus – agglutinates A1 cell, Ulex europeus is anti-H, Vicia Graminea and Bauhinia purpura – anti – N, Iberis amara – anti – M, Arachis hypogea anti – T and Saliva sclaera anti – T. The extracts are easy to make but the seeds may be difficult to obtain.
1. Grind seeds until course.
2. Add 3-4 times their volume of saline.
3. Incubate at room temperature for 4-12 hours stirring occasionally.
4. Centrifuge supernatant for 1 minute and filter.
5. Determine the activity.
Dolichos extracts agglutinates A1 and A1B cells but not the A2B or B cells. If all cells agglutinate add enough saline to dilute so that specific agglutination is achieved. The strength of agglutination of seeds extract is in following order: A2, B, A1, A1B.
Store the extracts at 4oC. The shelf life is 1 year at least.
Monoclonal and Polyclonal Antibody:
The antibody produced has a single specificity is called monoclonal antibody. The antibodies pooled from several immunized individuals having heterogeneous character are called as polyclonal antibody.
The monoclonal reagents are commonly in use for ABO, Rho (D) and anti-human globulin tests.
The monoclonal antibody technique devised by Kohler and Milsten has proved useful in producing high-titre and specific antibodies.
Laboratory animals, usually mice are immunized for the production of monoclonal antibody. After suitable immune response, mouse spleen cells containing antibody- secreting lymphocytes are fused to neoplastic plasma cells of infinite reproductive capacity from a mouse (i.e. myeloma cells).
The resulting hybridomas are screened for antibody with the required specificity and affinity. The antibody-secreting clones may then be propagated in tissue culture or by inoculation into mice, in which case the antibody molecules produced by a clone of hybridoma cells are identical in terms of antibody structure and antigen specificity.
Once one antibody-secreting clone of cells has been established, antibody with same specificity and reaction characteristics will be available for sure. These will be available indefinitely.