Practical applications of monoclonal antibodies

2.14 Practical applications of monoclonal antibodies

The production, selection and maintenance of hybridoma clones synthesizing antibody of a required specificity is so time consuming. Is it the best way? For example, the monospecific anti- body is a distinct disadvantage in the production of antibodies for class-specific precipitation of immunoglobulins or screening of recombinant DNA expression libraries. Monoclonal antibodies bind to only one determinant per molecule when used with non-polymeric antigens, thus pre- cluding the formation of a matrix for precipitation.

The availability of monoclonal antibodies compared with conventional antisera has greatly improved existing technology and has been fundamental for the generation of new techniques in that: (a) monospecific, hybridoma-derived antibodies can be used to estimate degree of structural homo-

logy between antigens. For example, with influenza virus, antibodies against chemically defined antigens have been used to investigate strains of virus for the presence of identical or closely related antigens;

(b) within solid-phase binding assays, these antibodies can be used at very low concentrations.

These assays are essentially affinity independent and detect the very low-affinity non-specific protein–protein interactions often found with conventional sera. As the degree of non- specific binding shown by an unrelated monoclonal antibody is much less than would be found with a normal control serum or unrelated antiserum the results can be interpreted with greater confidence; and

(c) the production of hybridoma-derived antibody is highly reproducible. Thus, whenever a new

batch of antibody is produced from the same cloned cell line, it will have the same specificity. In general the use of monospecific antibody is limited to research and diagnostic applications.

One obvious example is the definition of cell-surface markers for the investigation of specialized or abnormal cell function. This has given powerful diagnostic, and in some cases prognostic, tools for the study of tumours. Moreover, such techniques should continue to yield new informa- tion on the development and control of the immune system.

Potential applications of monospecific antibodies include: (a) clinical diagnostic testing; and (b) the production of therapeutic antisera with suitable human plasmacytoma parent line.

Monospecific antibodies produced by mouse fusion hybrids have many immediate applica- tions in the clinical laboratory as diagnostic or immunoassay reagents. (a) Tissue typing. Current typing techniques rely on antisera derived from multiparous women or

from patients who have received multiple blood transfusions. They are of low titre and often possess several specificities. A research programme has been established in the USA for the production of typing reagents using hybridoma techniques, although it seems likely that this approach could be overtaken by the use of gene probes.

(b) Blood group serology. A range of standard reagents are available through the National Blood Service.

2.14PRACTICAL APPLICATIONS OF MONOCLONAL ANTIBODIES

(c) Immunoassay of hormones. Fusions can be performed with spleen cells from mice immun- ized with relatively impure antigen. The need for antigen purification is circumvented by the selection of appropriate cell lines from cloned populations. This, and the monospecificity of the antibody thus obtained, has greatly enhanced the range and sensitivity of immuno- assay techniques. For example, an improved pregnancy test has been introduced using hybridoma-derived antibody for the immunoenzymatic detection of human chorionic gonadotrophin (hCG). These tests are now sensitive, rapid, accurate and unequivocal in their interpretation. In one variant, the enzyme substrate develops a minus sign in the event of

a negative test; the anti-hCG antibody is arranged so that it then adds an additional bar, to create a plus sign, in the event of a positive test. Monoclonal antibody technology has transformed this test from a highly specialized laboratory technique to a ‘home-based’ immunoassay.

(d) Immunodiagnosis of infectious disease. Sensitive immunoassays using monoclonal antibodies allow the diagnosis of infectious disease by the detection of microbial antigen rather than antibody. This is of much greater clinical value as it is a direct measure of the current state of the patient. Antibody detection has the disadvantage that it is practically impossible to dis- tinguish between a past or present infection using a single blood sample. In addition these reagents avoid the diagnostic imprecision due to cross-reactivity between serologically related organisms. Attempts to produce human monoclonal antibodies have met with only limited success.

Major limitations are that: (a) peripheral blood is the only readily available source of lymphocytes (which must be activated in vitro as deliberate immunization in vivo would be unethical); and (b) there are really no human tumour parents equivalent to the MOPC 21 sublines used in the murine system. Attempts have been made to circumvent these deficiencies, e.g. EBV lines used both alone and fused with B-cell tumours, but no generally applicable technique has emerged.

Recombinant DNA techniques have helped to overcome the problem of antigenicity of murine antibodies destined for clinical use. The solution has been fusion of the DNA sequences encoding murine variable regions with the sequence encoding human constant regions, thus producing a minimally antigenic molecule. Although there is the problem of an anti-idiotypic response, there appears to be a sufficiently large time window in which to achieve therapeutic potential. Recombinant techniques may be applied to the production of novel hybrid molecules in which the combining site of antibody is used to target a secondary effector function; for example, an enzyme active site.