Antibody antiserum

In addition to conventional staining and absorption controls, the following procedure is recommended. Parallel sections are processed through the above protocol up to step 10. Instead of specific antibody/antiserum, normal serum from the same species is applied. Steps 11-15 are carried out as described above. These control sections must not show the... 

Chemical inhibition with CYP-selective direct-acting and metabolism-dependent inhibitors and antibody inhibition with CYP-selective polyclonal or monoclonal antibodies (antiserum, ascites fluid or purified antibody)... 

Apply anti-Ig antibodies (antiserum dilution 1/23) incubation as in step 2. 

The primary alternative to the instrumental methods summarized above is immunodetection of EA with specific antibodies. Antiserum raised in response to immunization with lysergol (6a) coupled to human serum albumin recognizes several EA (100). Used for competitive enzyme-linked immunosorbent assay (ELISA), this broad-spectrum antiserum allows rapid detection of total EA in many samples concurrently. However, it does not separately quantify the different EA that contribute to the profile, and different EA show different reactivities to the antiserum. Monoclonal antibodies specific for individual EA species, such as ergotamine, also have been raised and provide an effective means for detection and quantification of the particular EA, as well as a valuable tool for physiological studies (101). 

Primary antibody-antiserum and secondary antibody raised in the appropriate animal. The dilution of antibodies is determined empirically in each case. Store at 4°C or -20°C long term. 

Incubate with primary antibody-antiserum at the desired dilution in PBS plus 5% sheep serum for l-2h at room tanperature. 

Bergseid et al. (2000) also reported on the use of SHA-activated chromatography supports for the coupling of boronate-containing affinity ligands. In this case, immobilized RNase A was used to purify anti-RNase antibodies from antiserum samples. RNase A was modified with an NHS-PDBA crosslinker at a molar ratio of 100 1 (crosslinkenprotein), purified to remove excess crosslinker, and then coupled to an SHA-agarose support in 0.1 M sodium bicarbonate, pH 8.0. 

Antiserum A serum containing an antibody or antibodies produced from animals or humans that have survived exposure to an antigen. 

Antiserum (plural antisera) Blood serum containing one or more polyclonal antibodies that are specific for one or more antigens. The antibodies in an antiserum result from previous immunization or exposure to an agent of disease. 

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. 

Mix a fixed volume (fixed concentration ) of antiserum containing the specific antibody with a constant amount of radiolabelled antigen,... 

The tube which contained radioactive dihydromorphine and antiserum but no unlabelled morphine, served as a measure of maximum antibody-bound radioactivity,... 

Immunization and Antibody Production The lypphilized immunogen obtained above is dissolved in normal saline and emulsified with equal volumes of complete Freund s adjuvant into a thick paste. Three New Zealand albino rabbits are immunized with the immunogen-paste through intradermal injections. The process is repeated twice at 2-weeks intervals followed by booster doses at monthly intervals. The antiserum is harvested when the plasma titer value is attained maximum. 

Where more than one antibody in an antiserum can combine simultaneously with an antigen, the sum of the binding strength is defined as the avidity of the antiserum. 

A polyclonal antiserum contains a heterogeneous mix of antibodies directed against an antigen. 

Antiserums produced in this manner are known as polyclonal because they contain many antibodies produced by different lymphocytes, each one responding either to different antigenic determinants of the original antigen or to other immunogenic substances in the injected material. Such a range of antibodies reduces the specificity of the method. 

In addition to their improved specificity, monoclonal antibodies offer other significant advantages over polyclonal antiserums there is an indefinite supply of antibodies with constant characteristics together with relative ease in purification. 

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