Antiserum specificity

Sensitivity defines the degree to which an assay can distinguish one compound from another of the same nature and an immunoassay is a function of the particular antibody molecules contained in the antiserum. Specificity of the antiserum is a function of the particular antigen used to immunize the animal. Affinity usually measures how strongly bound is the antigen to the antibody. Titer refers to the concentration level of, in the context of the usage, antibody contained in the obtained serum. 

Antiserum specific for the biologically more active X-pentazocine has been produced in rabbits using an -pentazocine-2 -carboxymethyl ether-bovine serum albumin conjugate. A low 0.08 cross-reactivity was found for [Pg.391]

Although raised against THC, the assay antiserum is not specific for that compound, but binds several cannabinoids to varying degrees. An example of the unpredictable nature of antiserum specificity is demon-... 

Fig. 2. Typical densitometnc scan. Calf thymus DNA, methylated in vitro with 5 mM of methylnitrosourea, was denatured in 50 mM sodium hydroxide and blotted onto nitrocellulose. After incubation with a rabbit antiserum specific for O -methyldeoiQrguanosine (NPZ-193-1,1 8,000 see ref. 4) and alkaline phosphatase conjugated goat-antirabbit IgG, the blot was developed with 5-bromo-4-chloro-3-indolylphosphate toluidine salt and nitroblue tetrazolium chloride in diethanolamine buffer, as described in the Methods section, and scanned by reflectance densitometry at 530 nm. Slots contain 283, 94.7, 31.3, 10.4, 3.4, 1.1, and 0 fmol of 0 -methyl-deoxyguanosine, corresponding to 181-0.7 pmol/mol of deoxyguanosine.

The procedure should be performed at 4°. Overnight, stir 20 ml of rabbit antiserum specific for human IgE with an equal volume of Sepharose-IgE. Pour into a chromatography column and elute unbound protein with bicarbonate-buffered saline. Desorb the y-globulin fraction of anti-IgE serum with 0.1 M acetic acid it is now suitable for labeling with I. 

The assay can be conveniently done in glass or plastic petri plates (60 mm) containing about 4 ml of 0.8% agarose in complement buffer. Guinea pig complement is readily available commercially and generally does not require absorption. Lymphoid cells are prepared by teasing spleens or lymph nodes in cold tissue culture medium buffered with Hepes. If the indirect plaque assay is to be done, an antiserum specific for IgG of the antibody-secreting cells is also required. 

The extremely low levels of vitamin ID and its metabolites in biological systems make it very difficult to assay these products by traditional methods. Calcium-binding protein is not found in the intestinal mucosa of vitamin D-deficient animals. It is synthesized only in response to the presence of a material with vitamin D activity. Thus, using antiserum specific to intestinal calcium-binding protein, a radioimmunodiffiision assay (98) conducted on homogenates of intestinal mucosa of chicks fed the test material for 7—10 d allows assay of the material for vitamin D activity down to 1—200 lU. The... 

MDP stimulated human monocytes to release lymphocyte-activating factor. Similarly, when purified mouse macrophages were exposed to MDP, they were found to produce a supernatant factor which restored macrophage-deficient lymph-node cell cultures.35 Moreover, in the presence of MDP, cultured murine spleen cells were reported to release a soluble mediator which stimulated antibody responses of other cultured spleen cells. 3- production of this factor could be blocked by an antiserum specific for mouse macrophages. 3. Finally, MDP was also capable of inducing the formation of colony-stimulating factor in cultures of mouse macrophages. ... 

Identification may be helped by changing the precipitating capacity of the antiserum. If the latter is absorbed with pure fractions of plasma proteins until no precipitation lines are formed in the Ouchterlony test, the pattern of ImEl will lack the corresponding lines. If the unknown antigen mixture contains fractions of tissues or tumors, the antiserum has to be absorbed first with pooled lyophilized human plasma. Another technique absorbs the antiserum specifically with a considerable excess of the unknown antigen a control ImEl shows the effect of absorption, thereby revealing the active antigen. Heremans (HI) has used this... 

We perceived the need for sensitive assays that do not rely on the use of radioisotopes or extensive analytical methodology and that could accurately detect protein-bound acetaminophen in biological fluids in the presence of unbound acetaminophen. To this end, we recently developed sensitive avidin biotin-amplified ELISA (A-B ELISA) and particle concentration fluorescence immunoassays (PCFIA) which use antiserum specific for the major acetaminophen-protein adduct associated with toxicity (13-16). These assays are new tools to study the relation between formation of the 3-Cys-A protein adduct and acetaminophen-induced toxicity. In this report we review how these assays were developed, validated in laboratory animals, and used to quantify 3-Cys-A protein adduct formation in human acetaminophen overdose patients. 

Previous reports described an ELISA specific for 3-Cys-A protein adducts that does not rely on radiolabeled samples (1 ) characterization of the relevant epitope (H) and quantification of 3-Cys-A adducts in liver and serum as a function of hepatotoxicity (9). This higlily characterized antiserum specific for the acetaminophen-protein adduct, 3-Cys-A, was used to correlate the immunohistochemical localization of 3-Cys-A protein adducts with the development of cell injury, in microwave-fixed liver. 

Control experiments using an antiserum specific for D1 confirmed that no full-length D1 polypeptide was present in membranes of TD35 (data not shown). However, the presence of a truncated form of D1 produced by the remnant of psbA3 in TD35 cannot yet be discounted. 

This partially purified preparation of P subunit catalyzed a measurable rate of ATP hydrolysis. The ATPase activity depended on the presence of Mg but not Ca + and was detectable in the absence of known activators of ATPase in isolated CFi. The activity was destroyed by heating the protein and was inhibited (Fig. 2) by an antiserum specific... 

Fig. 4. MxA forms complexes with viral target structures and cellular membranes. (A) Colocalization of MxA/N protein complexes with smooth ER membranes. VA3 cells were infected with LACV for 16 h, fixed as described above, and stained with monoclonal anti-MxA antibody (a, green), goat anti-Syntaxinl7 antibody (b, red), and a rabbit antiserum specific for the viral N protein (c, blue). The primary antibodies were detected with fiuorophore (Cy2, Cy3, and Cy5)-conjugated secondary antibodies, respectively. The pictures were recorded using a Leica TCS confocal laser scanning microscope. Bar = 8 /rm. (B) VA3 cells were infected with LACV or left uninfected. Cells were lysed in the presence or absence of GTP-yS and subjected to immunoprecipitation using the N-specific antiserum. Bound MxA was detected by Western blotting using the monoclonal anti-MxA antibody M143 (from Kochs et al., 2002, 2002).

Antiserum specific for a subclass is generally obtained by immunization with a myeloma protein or its Fc fragment and absorption of the antiserum with myeloma proteins of the other subclasses. Primates... 

That the 19 S protein of the chicken is indeed homologous to mammalian IgM is shown by the fairly strong immunological cross-reaction observed between the chicken and human proteins the tests were carried out with antiserum specific for human /u. chains (18). No crossreaction could be detected between human IgG and the chicken immunoglobulin of comparable molecular weight. 

A third, and most definitive method for observing persistence is the analysis of amino acid sequence the idiotype of a molecule must be defined by sequences in the variable portions of its H and L chains. Amino acid sequence analysis has been used to observe the persistence of molecules of related or identical structure during repeated immunizations with type VIII pneumococcal vaccine (71). A rabbit received three 1-month courses of immunization with a rest period of 1 month after each course. Antibodies isolated after successive courses are referred to as Ab-1, Ab-2, and Ab-3. Specifically purified Ab-2 was homogeneous by the criterion of electrophoresis. Antiidiotypic antiserum specific for this purified antibody also reacted, although not as strongly, with Ab-1 and Ab-3. Light chains of Ab-2 were found to have a unique sequence for the first N-terminal 21 residues. This contrasts with nonspecific L chains of the same allotype (b4) which have multiple residues at many... 

Pursuing similar types of experiments with reovirus-infected L cells, Skup et al. studied the ability of CBP to restore translation of capped reovirus mRNAs. They found that CBP could partially restore activity to lysates from infected cells (Zarbl and Millward, 1983). Zarbl and Millward, however, point out that inactivation of CBP does not explain why uncapped reovirus mRNAs can be translated in infected cells because, unlike other uncapped mRNAs which can be translated in lysates from uninfected cells, uncapped reovirus mRNAs cannot be translated under these conditions (Skup and Millward, 1980a, >). Therefore, they propose that an additional factor, presumably viral, is necessary for the translation of reovirus uncapped mRNAs. In order to determine whether this is the case, Skup and Millward have examined the effect of antireovirus antibodies on the translation of capped and uncapped reovirus mRNAs in vitro. The antiserum specifically inhibits the translation of uncapped viral... 

---