PRINCIPLE AND PRODUCTION OF ANTI HUMAN GLOBULIN REAGENT
Normal Human red blood cells, in presence of antibody directed towards the antigen they possess, may fail to agglutinate and become sensitized. This may be due to the particular nature of the antigen and antibody involved. Anti human globulin reagent would react with red cell sensitized with gamma globulin or components of human complement involved and cause agglutination of the red blood cells.
PRODUCTION OF AHG (POLYSPECIFIC AHG)
POLYCLONAL ANTI HUMAN GLOBULIN
Polyclonal antibodies are a mixture of antibodies from different plasma cell-clones and the resulting polyclonal antibodies recognize different antigenic epitopes.
Polyclonal antihumanglobulin is prepared in rabbits, although when large volumes of antibody are required, sheep or goats may be used. In contrast with the early production methods, in which a crude globulin Fraction of serum was used as the immunogen, modern production commences with the purification of the immunogen from a large pool of normal sera.
Conventional polyspecific antiglobulin reagents are produced by immunizing one colony of rabbits with human immunoglobulin (lgG) antigen and another colony with human C3 antigen. Because of the heterogeneity of lgG molecules, the use of serum from many donors to prepare the pooled lgG antigen to immunize the rabbits and the pooling of anti-lgG from many immunized rabbits are essential in producing reagents for routine use that are capable of detecting the many different lgG antibodies. This is an advantage of using anti-lgG of polyclonal origin for antiglobulin serm. (Issitt, 1997).
Both colonies of animals are hyperimmunized to produce high-titer, high-avidity lgG antibodies. Blood specimens are drawn from the immunized animals, and if the antibody potency and specificity meet predetermined
specifications, the animals are bled for a production batch of reagent. Separate blends of the anti-lgG and anticomplement antibodies are made, and each pool is then absorbed with A, B, and O cells to remove heterospecific antibodies. The total antibodies content of each pool is determined and the potency of the pools is analyzed to calculate the optimum antibody dilution for use.
Block titrations for anti-lgG pools are performed by reacting dilutions of each antibody against cells sensitized with different amount of lgG. This is a critical step in the manufacturing process because excess antibody, especially with anti-lgG, may lead to prozoning and hence, false-negative test results.
Because it is not possible to coat cells measured amounts of complement, the potency, of anti-C3 pools is measured using at least two examples each of a C3b-and C3d- coated cell. Both anti-C3d (C3c) and anti-C3d are present in the polyclonal anti-C3 pool. The level of anti-C3d is particularly critical yet detecting clinically significant amounts of red blood cell-bound C3d. additionally, if the dilution of the anti-C3 pool is determined on the basis of the anti-C3d present, the level of anti-C3b (3c) varies.
The inability to determine the potency of anto-C3b and anti-C3d individually is one of the difficulties with polyclonal reagents that can be avoided with monoclonal product (Issitt, 1997). Once the required performance characteristics of the trial blend are obtained a production blend of the separate anti-lgG and anticomplement pools is made.
Monoclonal Anti human globulin Production
Monoclonal antibodies are derived from one clone of plasma cells and recognize a single epitope.
The monoclonal antibodies technique devised by Kohler and Milstein (Kohler et al., 1975), has been used to produce AHG and has proved particularly useful in producing high-titer- antibodies with well defined specificities to lgG and to the fragments of C3. (Lachman, 1983, Holt 1985).
Monoclonal antibody production begins with the immunization of laboratory animals, usually mice, with purified human globulin. After a suitable immune response, mouse spleen cells containing antibody-secreting lymphocytes are fused with myeloma cells. The resulting “hybridomas” are screened for antibodies with the required specificity and affinity.
The antibodies-secreting clones may then be propagated in tissue culture or by inoculation into mice, in which case the antibody is collected as ascites. Because the clonal line produces a single antibody, there is no need for absorption to remove heterspecific antibodies.
All antibody molecules produced by a clone of hybridoma cells are identical in terms of antibody structure and antigen specificity. This has advantage and disadvantages in Anti human globulin production. Once an antibody-secreting clone of cells has been established, antibody with the same specificity and reaction characteristics will be available indefinitely. This allows the production of a consistently pure and uncontaminated Anti Human Globulin reagent.
The disadvantage is that all antibodies produced by a clone of cells recognize single epitope present on an antigen. For antigens composed of multiple epitopes such as lgG, several different monoclonal antibodies reacting with different epitopes may need to be blended, or monoclonal antibodies specificity for an epitope on all variants of a particular antigen may need to be selected to ensure that all different expressions of the antigen are detected.
Monoclonal antibodies to human complement components anti-C3b and anti-C3d may be blended with polyclonal anti-lgG from rabbits to achieve potent reagents that give fewer false positive reactions as a result of anti-complement. The anti-lgG component produced by exposing mice to red blood cells coated with lgG. The resulting anti-lgG reacts with the C43 region of the gamma chain of lgG subclass 1, 2 and 3. The antibody does not react with human antibodies of subclass lgG4, but these are not considered to be clinically significant. Blending the monoclonal anti-lgG with a monoclonal anti-C3b and monoclonal anti-C3d results in a polyspecific antihumanglobulin reagents.
Monospecific Anti human globulin Production
Monospecific AHG is prepared by a production process similar to that described by poly specific AHG; however, it contains only one antibody specificity. Monospecific anti-lgG is usually of polyclonal origin; however, monoclonal anti-lgG has been prepared effectively by hybridoma technology.
Monospecific anti-complement reagents are often a blend of monoclonal anti-C3b and monoclonal anti-C3d.
Polyspecific antihuman globulin may be manufactured by combining polyclonal anti-lgG with either polyclonal or monoclonal anticomplement components.
A monoclonal blend may be manufactured by blending monoclonal anti-C3b, monoclonal anti-C3d, and monoclonal anti-lgG. Monospecific globulin reagents can be manufactured by conventional or hybridoma technology.