Antiserum preparation

The neutral FDPase and SDPase activities, which were present in the crude spinach extracts, were precipitated at lower ammonium sulfate concentration and could thus be separated from the specific alkaline FDPase. These activities appeared to be associated with the chloroplast fraction and did not require the presence of a divalent cation for activity. In crude extracts only the alkaline FDPase activity was inhibited by antiserum prepared by immunizing rabbits with the purified alkaline FDPase. The neutral FDPase was also active with ribulose diphosphate (RuDP) (98). 

Two key steps involved in the traditional methods are antiserum preparation and the agglutination tests. Sera for O determination are produced by immunization of rabbits with cultures that have been heated at 100°C for 2 h. Broth cultures or agar plate suspensions heated at 100°C for 1 h are used as antigens for typing. With these two procedures, bacterial agglutination is a very simple and sensitive method for qualitative O antigen determination. 

This kit is very similar to the method described before with the exception that a highly specific antibody for Apidra (guinea pig anti-HMR 1964 antiserum prepared by Linco) is used. The antibody meets the specificity requirements (cross-reactivity e.g. to human insulin < 0.001 %) and the sensitivity requirements (< 0.2 ng/ml). 

Proteins from the black kidney-bean were fractionated.726 Analysis showed species that had both hemagglutinating and mitogenic properties these agglutinins proved to be glycoproteins.726 Rabbit antiserum, prepared against proteins from black kidney-beans, cross-reacted with the water-soluble proteins from white and red kidney-beans (although each variety of bean gave very different immunoelectrophoretic patterns).726,727... 

Methanococcus voltae contains a membrane-bound vanadate-sensitive ATPase [48] that is inhibited by diethylstilbestrol, an inhibitor of eukaryotic P-type ATPases. The purified enzyme is composed of a single subunit (Mr 74 000), forms a covalent acyl-phosphate enzyme intermediate, and is not inhibited by nitrate or bafilomycin [49]. No such ATPase activity has been reported in other archaea. The presence of a second ATPase in M. voltae has been inferred since membranes react with antiserum prepared against the 3 subunit from the V-type ATPase of S. acidocaldarius [50]. Two peptides are detected whose Mr values (51 000 and 65 000) correspond to the masses for the two laigest subunits of the S. acidocaldarius ATPase [51]. There is evidence that ATP synthesis in the M. voltae enzyme is due to the operation of a sodium-translocating ATPase [50]. The relationship of the putative V-like ATPase to the sodium-translocating ATPase has not been established. 

Various preparations of antisera to atropine have been reported. A racemic hemisuccinate ester was prepared and conjugated to bovine serum albumin by the carbodiimide technique. Antisera formed to the original immunogen selectively bound the R isomer (34), but a later antiserum prepared by this approach was reported to bind both R and S forms with "equal efficiency" (35). R, S-atropine was treated with diazotized p-amino-benzoic acid, and the resulting compound (which was not further characterized) was used for conjugation to bovine serum albumin by means of a carbodiimide-mediated reaction. Antisera resulting from use of this material were quite selective for the R isomer, with a cross-reaction of only about 2% for the S isomer (36). Virtanen et al. followed this procedure with S-atropine. Their antiserum bound equally to S- and R,S-atropine, as measured by displacement of tritium-labeled R,S-atropine (37). In another study (31), both racemic atropine and the S isomer were coupled to human serum albumin by the technique of Wurtzburger et al. (36), Antisera were obtained that were selective for both the R and S isomers (33). 

IgG in antiserum prepared as described above is purified by affinity chromatography. To prepare the affinity column, conjugate the antigen (component I or II) to Affi-Gel 15 (Bio-Rad Laboratories) according to the manufacturer s Instruction Manual. 

Antiserum Preparation. Anti-clomazone antisera were prepared by Pel-Freeze Biologicals, Rogers, AR. Three New Zealand white rabbits were inoculated in several intradermal sites with a total of 1 mg BSA conjugate (1 1 emulsification in Freund s complete adjuvant) at 0, 2 and 4 weeks. Boosters (1 mg intradermal and 0.5 mg intravenous) were given at 4 to 6 week intervals, with the animals bled 10 to 14 days later. 

Itrformation on DNA adduct structure is needed for the preparation of specific antibodies, when highly specific antibodies are prepared. Therefore not appUcable for unknown compounds, unless whole damaged DNA is irsed fo" antiserum preparation. 

The presence of cross-reacting material to antiserum prepared against normal NADP-GDH (189). 

Figure 5. Pea epicotyl segments 72 hours after inoculation with conldial suspension of F. solan f. sp. pisi in water (control), water containing IgG from preimmune rabbit serum (serum), water containing IgG from antiserum prepared against cutlnase (antiserum) and water containing 10 VM dllsopropylfluorophosphate (DFP).

Enzyme Isolation and Antiserum Preparation. Periplasmic CA was prepared from air-grown Chlamydomonas reinhardtii (137 mt" ") by p-methylaminobenzenesulfonamide affinity chromatography as described by Yang et al, (6). Antiserum against the isolated enzyme was prepared in rabbits as we described previously (7). Chemically deglycosylated CA was prepared by trifluoromethanesulfonic acid hydrolysis (8,9). 

The reference strain taxon field is used to record the generic name and specific epithet of the reference strain. This is essential, if the names are known, to avoid confusion. If the names are not known, distinctive operational names or symbols must be assigned to all reference strains. For example, an antiserum prepared against a Salmonella strain might be included in a number of tests developed for the genus Escherichia. Although components of earlier coded data might exist in both the Escherichia and the Salmonella sections of the RSC, the reference strain taxon would be Salmonella, not Escherichia. Computer searches of the reference strain number and the reference strain taxon fields would determine if the RSC contains any information about specifically numbered or named reference strains. 

Two lipases, i.e. LPL and H-TGL, in this postheparin plasma were separately measured using the antiserum prepared against H-TGL(3). Plasma samples from 12 age-matched control subjects were used for the lipoprotein analysis. Normal values of postheparin plasma lipase activities were those for 13 normo-lipidemic male subjects reported elesewhere(3). The serum concentration of apolipoprotein A-I, A-IE and apolipoprotein B were measured by a immunodiffusion technique using specific anti-serum. 

Fig. 4. Immunodiffusion test of rabbit antiserum prepared against cellulase CIV with purified enzymes. In center well 10 ul of undiluted antiserum was placed and the outer wells contained each 10 jjl of 3-glucosidase 31 (1.5 x 10 unit) and 3II x 10 unit),...

As defined by Oudin, idiotypy applies to determinants recognized by an antiserum prepared in the same species, and the term, individually specific antigenic determinant, has been utilized in reference to determinants defined by an antiserum prepared in a heterologous species, such as a rabbit antiserum directed to a purified human antibody or... 

Fig. 11.4. Inhibition of binding of labeled FCab ) fiagments of anti-p-azobenzoate antibodies from rabbit AZl to antiidiotype antiserum prepared in a recipient rabbit. The antibenzoate antibodies were isolated from sera taken approximately 8 months after the start of immunization of rabbit AZL Competitors are unlabeled specifically purified antibenzoate antibodies prepared from the sera of rabbit AZl at various times after the start of immunization the approximate number of months is indicated by the numeral on each curve. Reprinted, by permission, from MacDonald and Nisonoff (41).

Inhibition by antiserum. An intracellular localization for cathepsin D different from that of the other lysosomal acid hydrolases in rat TDL led us to explore some of the biochemical properties of this enzyme. As illustrated in Figure 3, an antiserum prepared in rabbits against rat liver soluble lysosomal enzymes effectively inhibited rat liver cathepsin D, although it did not inhibit the cathepsin D of rat TDL. In this case the incubations were carried out at pH 5 instead of pH 3.6 to avoid dissociation of the antigen-antibody complex. Both rat liver and rat TDL cathepsin D, however, have identical pH activity curves toward denatured bovine hemoglobin as substrate. 

In two cases of Lesch .Nyhan syndrome with complete and partial deficiency of HG-PRT activity the presence of enzyme protein could be demonstrated using an antiserum prepared against the enzyme from normal erythrocytes The immunoreactive proteins in hemolysates of both normal and sick patients showed a complete identity ... 

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