Protein antiserum

Antiserum - protein A agarose (immobilized antibody for boronate chromatography)... 

Figure 9. Anti-PbTx antiserum inhibition of [ H]PbTx-3 binding to its receptor site in rat brain membrane preparations. Labeled toxin (0.5 nM in 1 ml PBS) was incubated with rat brain membranes (125 fig total protein) and increasing amounts of anti-PbTx antiserum (- -) or pre-immune serum (- -) for 1 hr at 4 C. Membrane-bound radioactivity was then measured in a centrifugation assay as previously described (8),...

The PemB cellular localisation was determined both in E. chrysanthenu and in an E. coli recombinant strain by Western blot of the cell fractions with a PemB-antiserum. No PemB was detected in the culture supernatant and only trace amounts were found in the soluble cell fractions - periplasm and cytoplasm (Figure 2). PemB was found mostly in the total membrane fraction from which it could be completely extracted by Triton X-100/Mg2+ and partially extracted by Sarkosyl (Figure 2). This behaviour is typical of inner membrane proteins, but since some exceptions have been noticed it does not positively indicate the PemB localisation (15). We performed cell membrane fractionation in sucrose density gradient centrifugation both by sedimentation and flotation, using several markers of inner and outer membrane vesicles. PemB was found in the outer membrane vesicles (data not shown). 

Figure 5. Proteins extracted from different E. chrysanthemi strains by Triton X-100 were analysed by immunoblotting with PemB-antiserum.

Markwell, M.A.K. (1982) A new solid-state reagent to iodinate proteins conditions for the efficient labeling of antiserum. Anal. Biochem. 125, 427-432. 

Nunoi and co-workers (1988) fractionated neutrophil cytoplasm by Mono Q anion-exchange chromatography and obtained three fractions (NCF-1, -2 and -3) that were active in the assembly of the oxidase. Independently, Volpp and colleagues (Volpp, Nauseef Clark, 1988) prepared antiserum from cytosolic factors that eluted from a GTP-affinity column, and this antiserum (Bl) recognised cytoplasmic factors of relative molecular masses 47 kDa and 66 kDa. It was later shown by this group that these cytosolic factors translocated to the plasma membrane during activation. NCF-1 was shown to contain the 47-kDa protein and NCF-2 the 66-kDa protein. Analysis of the defect in the cytosol of autosomal recessive CGD patients revealed that most of these (88%) lacked the 47-kDa protein (p41 -phox), whereas the remainder lacked the 66-kDa protein (p66-phox). Both of these components have now been cloned and recombinant proteins expressed. Interestingly, in the cell-free system, recombinant p47-phox and p66-phox can restore oxidase activity of the cytosol from autosomal recessive CGD patients who lack these components. 

Figure 11.15 Immunoelectrophoresis of human serum proteins. The proteins are separated electrophoretically from wells cut in a suitable gel. After electrophoresis, a trough is cut in the gel parallel to the direction of migration and filled with an antiserum. The components are allowed to diffuse for 24-48 hours for precipitation lines to develop. Human serum contains many proteins, among which the immunoglobulins can be identified.

The PAP method was pioneered by Sternberger in 1979 (1). The method uses an immunological sandwich amplification and the enzyme peroxidase to effect a signal. The unique feature of this procedure is the enzyme/antibody solution, the PAP immune complex. The horseradish peroxidase enzyme, itself an immunogenic protein, is used to inoculate a given species, and a polyclonal immune response is generated against the enzyme. This antiserum is harvested and placed in solution with the enzyme so that immune complexes form that... 

Thanks are expressed to Dr. K.-E. Eriksson (STFI, Stockholm, Sweden) for the gift of the enzyme extract and inoculation of the wood samples to Professor R. Guinet (Institut Pasteur, Lyon Lentilly, France) for the preparation of the IgG directed against the crude protein extracted and to Dr. E. Odier (INA P.G., France) for providing the anti-ligninase antiserum. 

In addition Choi et al. utilized FlTC-labeled methamphetamine for competitive immunoassay of methamphetamine in urine (19). Instead of purified antibody or antibody fragment, antiserum was used. An aminobutyl derivative of metamphetamine was conjugated with proteins and used as an immunogen to produce antibodies for the assay. The free FITC-labeled tracer was well separated from the antibody-bound fraction, and the detection limit for the CE assay was lower than that for ELISA. 

Rinse a protein A column (4-5 ml bed volume 15 x 22.5 and 15 x 28.5 mm, respectively) with 40 - 50 ml of Soln. A, flow rate 1 ml/min. Dilute 2 - 5 ml of rabbit antiserum with the same volume of Soln. A and apply with the same flow rate to the column. After sample... 

An indirect competitive ELISA has been also developed for the determination of streptomycin and dihydrosticptomyciri in milk (24). Prior to the analysis, the milk sample was skimmed and treated with oxalic acid. The antiserum was raised in rabbits using streptomycin linked to a bacterial protein as the antigen. To perform the test, microtiter plates were coated with streptomycin, and antiserum and milk samples were mixed to be added in the wells where they were incubated for 1 h. Depending on the amount of residues in the sample, more or less antibody remained available for binding to the streptomycin coat. A pig antirabbit antibody-enzyme conjugate was subsequently added and incubated for 90 min. Using a suitable substrate, streptomycin and dihydrostreptomycin could be detected down to 1.6 ppb, whereas quantification could be made possible up to 100 ppb when samples were used undiluted. 

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