Recombinant antibodies spleen

Preparation of recombinant antibodies from immune rodent spleens and the design of their humanization by CDR grafting... 

The quality of mRNA is important in generating recombinant antibodies. It is particularly important to ensure minimal ribosomal RNA contamination when cDNA is random primed for Ubraiy construction to reduce the proportion of clones which represent non-mRNA species. The spleen mRNA of immunized mice is presmnably derived mainly from plasma cells, as the level of Ig mRNA in these cells is up to 1000-fold greater than in resting B cells. For spleens, extraction of total RNA followed ly mRNA isolation on oligo(dT)-cellulose is recommended in order to effectively process the large number of cells present in a spleen. Cytoplasmic RNA Extraction Kit (see Protocol 2) followed by mRNA Purification Kit is hi y recommended. 

It is preferable to use NS-0 as a fusion partner, it does not secrete a constitutive antibody but has the ability to produce monoclonal antibodies once fused to an immune spleen cell. NS-1 has a constitutive antibody, which may cause interference in screening assays for recombinant hybridoma cells. 

The —50 kDa band (48-53 kDa) is identified as dysbindin-1 A in our WESTERNS, because it runs close to the molecular mass of our histidine-tagged recombinant mouse dysbindin-1 A. Its identity is confirmed by the fact that it is recognized by antibodies we have recently generated to amino acid sequences in the CTR of human dysbindin-lA, but not found in dysbindin-lB, -2, or -3. The —50 kDa band is the most consistently observed dysbindin-1 band across tissues. We find it in all tissues examined to date the adrenal gland, heart, kidney, liver, lung, spleen, skeletal muscle, testes, spinal cord, cerebellum, striatum, hippocampus, and cerebral cortex (e.g., Figure 2.2-12a and c). In mouse and human synaptosomes, the —50 kDa isoform is heavily concentrated in the PSD fraction with a much lesser amount in the presynaptic membrane and no detectable amount in the synaptic vesicle fraction (Talbot et al., in preparation). 

Table 1 shows some animal cell lines that are commonly used in various applications. Creating a stable, permanent cell line is the first critical step in producing recombinant proteins for therapeutic and diagnostic applications. Hybridoma, commonly used in the production of MAh, are generated by fusing antibody-producing spleen cells, which have a limited life... 

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