Antibodies preparation method

Figure 13.5 Outline of the production strategy of CEA-SCAN. The antibody-producing hybridoma cell line was originally obtained by standard methods of hybridoma generation. Spleen-derived murine B-lymphocytes were fused with murine myeloma calls. The resulting stable hybridomas were screened for the production of anti-CEA monoclonals. The clone chosen produces an IgG anti-CEA antibody. Note that the finished product outlined above is not radiolabelled. The freeze-dried antibody preparation (which has a shelf life of 2 years at 2-8 °C) is reconstituted immediately prior to its medical use. The reconstituting solution contains 99mTc, and is formulated to facilitate direct conjugation of the radiolabel to the antibody fragment...

Ascites production, however, suffers from a number of drawbacks. It is costly, and the product is contaminated by significant levels of various mouse proteins, rendering subsequent downstream processing more complex. As a result, monoclonal antibody production by standard animal cell culture techniques has become the method of choice for the production of pharmaceutical-grade monoclonal antibody preparations. 

Notes Chiral preparations include the proline-catalyzed reactions2 and recently an aldolase antibody 38C2 method has been reported.3 See also 4... 

Stemberger, L. A (1970) The unlabelled antibody-enzyme method of histochemistry Preparation and properties of soluble anigen-antibody complex (horseradish peroxidase-antihorseradish peroxidase) and its use m identification of spirochetes. J Histochem Cytochem 18,315—333... 

An even more indirect method which does not require the use of a fluorescence microscope is to use enzyme linked antibodies, e.g. the peroxidase anti-peroxidase (PAP) method of Stemberger et al. (1970). Here, after the antigen has been reacted with a rabbit antibody preparation this is conjugated to sheep anti-rabbit IgG which in turn is conjugated to a complex of rabbit anti-horse-radish peroxidase and horseradish peroxidase. This then reacts with diaminobenzidine and hydrogen peroxide when a brown colour indicates the presence of the antigen. 

Gel electrophoresis yielded a broad band and indicated several protein components, Fig. (15B). It was necessary to establish whether the preparation contained several types of antibodies or only anti-lactose antibodies. The preparation was subjected to electrofocusing [39], Duplicate gels were made by electrofocusing. One gel was stained for protein and showed the presence of multiproteins, Fig. (15C). The unstained gel was examined by the coupled method and yielded a precipitin which was opposite each band in the stained gel, Fig. (15D-F). These results showed that the purified antibody preparation consisted of 13 protein species each with antibody activity directed at lactose-containing antigen. 

Affinity Absorption A method of separation by affinity chromatography. It may be used, for example, to remove unwanted antibodies from an antibody preparation. The preparation is passed through a column matrix containing antigens against which the unwanted antibodies are directed. Thus, the unwanted antibodies remain bound to the column. The antibody solution leaving the column contains only the desired antibodies, purified by affinity absorption. 

In immunoblotting procedures, a mixture of antigens is first separated by gel electrophoresis and subsequently transferred from the gel to a membrane, where it is detected by antibody. Comparative levels of reactivity with antibody can be assessed by incubation with serially diluted antibody preparations. A comprehensive review of the technique is available.44 Because the test is done with proteins that are at least partially degraded, relatively distant relationships can be detected.43 46 The method has been used to calculate SDI values for proteins of potyviruses.47... 

The molecular size and molecular homogeneity of antibodies may be determined by a sucrose density-gradient ultracentrifugation method.31 32 Samples of 0.2 mL of 0.4% solutions of each antibody preparation are placed carefully on top of separate sucrose density-gradient tubes prepared from 5,10,20,30, and 40% sucrose. The tubes are centrifuged in a swinging... 

On gel electrophoresis, the purified antibody preparations yield single bands when the gel is stained for proteins. However, on gel isoelectrofocus-ing, differences in the results maybe noted, as shown in Fig. 15. Several protein isomers were present in each antibody preparation, with 7 isomers being detected in the anti-BSA antibodies (B) and 11 isomers in the anti-fucose antibodies (F). The coupled electrofocusing-agar diffusion method showed that each isomer of the anti-fucose set possessed the same antibody activity with the antigen, a-L-fucosyl-BSA. 

The most often used procedures for evaluating the specificity of antibody preparations are double diffusion methods developed by Ouchterlony and immunoelectrophoretic methods developed by Grabor and Williams. For a discussion of variations of these techniques as well as other useful procedures the reader is referred elsewhere (7-9). 

If the labeled antibody preparation proves to be satisfactory by the above rapid test it is usual to store it prior to its evaluation in an assay. It has been found that a very satisfactory and convenient method of storage is to bind the labeled antibody back onto immunoadsorbent (the appropriate amount required may be judged from the immunoadsorbent dilution curve). The rebound antibody is then frozen in aliquots that contain enough radioactivity to produce a single assay. Since one iodination nearly always provides more label than will be used in subsequent assays, before the preparation of more labeled antibody it is advisable to err on... 

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