Antibody soluble protein preparation

The fixation conditions used to prepare cells for antibody application are assumed to preserve the distributions of the protein(s) being examined (2). Soluble proteins can be redistributed into inappropriate locations and... 

Mucosal and systemic antibody responses were measured following oral (OR) and SQ immunization by a nanoparticulate-formulated protein. For oral immunizations, C57BL/6 female mice, 6-8 weeks old, were used in groups of 10 animals. Animals were immunized by gavage using a dose volume of 500 pL. Experimental formulations were prepared so each dose contained a certain load of protein (Table 4). Formulations tested included soluble protein, protein in the nanoparticulate form, protein mixed with empty nanoparticles and protein formulated with CRL-1005 non-ionic block copolymer. The immunization protocol included two immunizations one month apart. Blood, feces and saliva were collected from immunized mice on study days 21,28,49 and 56 and stored at -70 °C until tested. 

The fixation conditions used to prepare cells for antibody application are assumed to preserve the distributions of the protein(s) being examined (2). Soluble proteins can be redistributed into inappropriate locations and can be differentially extracted from native locations during the permeabilization and fixation of the cells before antibody application (3,4)- Further, no cell aggregation or alteration of the intracellular antigenicity should occur in the permeabilization/fixation treatment. The fixation/stain methodology, with and without permeabilization, can be accomplished in various ways depending on the exact site of the organelle or cell constituent to be stained. 

A protein sedimenting at 22S is the major soluble protein of the mitotic apparatus as isolated by Kane s hexylene glycol method (see Kane, 1967, and Stephens, 1967, for detailed characterization and earlier literature). This protein, as prepared, does not bind colchicine (Borisy and Taylor, 1967b). A definitive study with ferritin-labeled antibody shows that the protein is not a microtubule component. It is present throughout the egg, and in the mitotic apparatus (after isolation) it is located in the amorphous matrix between the microtubules (Bibring and Baxandall, 1969). Its mitotic role, if any, is unknown. 

Like many other antibodies, the activity of antibody 14D9 is sufficient for preparative application, yet it remains modest when compared to that of enzymes. The protein is relatively difficult to produce, although a recombinant format as a fusion vdth the NusA protein was found to provide the antibody in soluble form with good activity [20]. It should be mentioned that aldolase catalytic antibodies operating by an enamine mechanism, obtained by the principle of reactive immunization mentioned above [15], represent another example of enantioselective antibodies, which have proven to be preparatively useful in organic synthesis [21]. One such aldolase antibody, antibody 38C2, is commercially available and provides a useful alternative to natural aldolases to prepare a variety of enantiomerically pure aldol products, which are otherwise difficult to prepare, allovdng applications in natural product synthesis [22]. 

Serum sickness. This occurs when there is an excess of anhgen to antibody, resulting in the formation of soluble complexes. These may circulate and cause systemic reactions or be widely deposited in the kidneys, joints and skin. A rise in temperature, swollen lymph nodes, a generalized urticarial rash and painful swollen joints occur. The rcpeated administration of foreign serum (e.g. antidiphtheria serum or antitetanus serum prepared in horses) can lead to this condition due to antibodies being produced to the horse protein material. 

NG14. This mutant is capable of elaborating 15 FP units/mL with a productivity of 45 units/L/hr. The final concentration of soluble extracellular protein is 21.2 mg/mL. Samples of this enzyme preparation were sent to G. Pettersson at the University of Uppsala, Sweden, for quantification of each of the enzymes in the cellulase complex using purified antibodies to the individual enzymes. The quantitative antigen-antibody reaction showed that 600 mg/g of this enzyme preparation was one enzyme, cellobiohydrolase. This represents a yield of 13 g/L of cellobio-hydrolase, which is a 100-fold increase over the amount of cellobiohydrolase obtained with strain QM 9414 (130 mg/L) the best previously existing cellulase mutant (G. Pettersson, personal communication). 

Antibody prepared against SSI effectively neutralized souble SSI activity, but had no effect on either the activities of GBSSI or GBSSII, or even on SSII. Alternatively, antibody prepared against the starch granule-bound proteins effectively inhibited GBSSI activity, but had very little effect on the soluble starch synthases.173... 

In 1956 our laboratory, in collaboration with Beiser and Lieberman, became interested in preparing steroid-protein conjugates that were to be used to elicit antisteroid antibodies. An examination of the literature at that time showed that the azo coupling techniques of Landsteiner were still dominant. Like him, we chose to use the serum albumins because they were inexpensive and likely to yield soluble conjugates. However, an examination of the amino acid content of bovine serum albumin (BSA) (Fig. 1) convinced us that substitution by such relatively complex haptens as steriods should be attempted by reaction with the more plentiful c-amino groups of the lysine residues rather than by an azo coupling reaction with tyrosine, tryptophan, and imidazole residues. This meant forma-... 

---