Rh antigens are widely spaced on the red blood cell surface, and so the IgG anti-Rh antibodies do not bind in the correct conformation to fix complement and so do not cause lysis of red blood cells in vitro. Furthermore, for reasons that are not fully understood, antibodies against Rh antigens do not agglutinate red blood cells, unlike antibodies against the ABO blood-group antigens. Thus, detecting anti-Rh antibodies was difficult until anti-human immunoglobulin antibodies were developed. With these, maternal IgG antibodies bound to the fetal red blood cells can be detected after washing the cells to remove unbound immunoglobulin that is present in the fetal serum. Adding anti-human immunoglobulin antibodies against the washed fetal red blood cells agglutinates any cells to which maternal antibodies are bound. This is the direct Coombs test (Fig. A.8), so called because it directly detects antibody bound to the surface of the fetal red blood cells. An indirect Coombs test is used to detect nonagglutinating anti-Rh antibody in maternal serum: the serum is first incubated with Rh-positive red blood cells, which bind the anti-Rh antibody, after which the antibody-coated cells are washed to remove unbound immunoglobulin and are then agglutinated with antiimmunoglobulin antibody (see Fig. A.8). The indirect Coombs test allows Rh incompatibilities that might lead to hemolytic disease of the newborn to be detected, and this knowledge allows the disease to be prevented (see Section 15-10). The Coombs test is also commonly used to detect antibodies against drugs that bind to red blood cells and cause hemolytic anemia.
A-7 Monoclonal antibodies.
The antibodies generated in a natural immune response or after immunization in the laboratory are a mixture of molecules of different specificities and affinities. Some of this heterogeneity results from the production of antibodies that bind to different epitopes on the immunizing antigen, but even antibodies directed at a single antigenic determinant such as a hapten can be markedly heterogeneous, as shown by isoelectric focusing. In this technique, proteins are separated on the basis of their isoelectric point, the pH at which their net charge is zero. By electrophoresing proteins in a pH gradient for long enough, each molecule migrates along the pH gradient until it reaches the pH at which it is neutral and is thus concentrated (focused) at that point. When antiserum containing anti-hapten antibodies is treated in this way and then transferred to a solid support such as nitrocellulose paper, the anti-hapten antibodies can be detected by their ability to bind labeled hapten. The binding of antibodies of various isoelectric points to the hapten shows that even antibodies that bind the same antigenic determinant can be heterogeneous.
Antisera are valuable for many biological purposes but they have certain inherent disadvantages that relate to the heterogeneity of the antibodies they contain. First, each antiserum is different from all other antisera, even if raised in a genetically identical animal by using the identical preparation of antigen and the same immunization protocol. Second, antisera can be produced in only limited volumes, and thus it is impossible to use the identical serological reagent in a long or complex series of experiments or clinical tests. Finally, even antibodies purified by affinity chromatography (see Section A-3) can include minor populations of antibodies that give unexpected cross-reactions, which confound the analysis of experiments. To avoid these problems, and to harness the full potential of antibodies, it was necessary to develop a way of making an unlimited supply of antibody molecules of homogeneous structure and known specificity. This has been achieved through the production of monoclonal antibodies from cultures of hybrid antibody-forming cells or, more recently, by genetic engineering.