Monoclonal antiserum

Figure 3. Combined, direct and ELISA product, photometric monitoring of fractions of the SE-HPLC separation of an extract of a viral protein mixture. SE-HPLC of a Triton X-lOOextract of Newcastle disease virus strain LaSota. The extract was prepared as described in ref. 12. The TSK 4000 SW column was eluted with 50 mM sodium phosphate, pH 6.5, containing 0.1% SDS. The flow-rate was 1 ml/min and the absorbance was monitored at 280 nm. Fractions (1 ml) were analysed with an ELISA (see Table 7). Shaded areas, which represent absorbance at 492 nm, indicate immunological activity. A positive polyclonal antiserum was used as well as a monoclonal antiserum directed against the haemagglutinin-neuraminidase protein (Courtesy of Dr Y.Nagai, Nagoya University School of Medicine, Nagoya, Japan).

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. 

To reduce unspecific binding, mix a 200-pl aliquot of the clear supernatant with 2 pi pre-immune serum or unspecific antibody and a further 200 pi aliquot with 50 pi precipitation aid. Rock at 0 °C for 1 h and spin at 1000 x g. Transfer the supernatant into a fresh container and fill it up to 1000 pi with Soln. A. Add 0.5 - 5 pi of the specific antiserum and monoclonal antibody, respectively, and incubate on ice for 1 h. Prepare a second sample containing pre-immune serum instead of antiserum. 

Variations in Methods, The various immunochemical methods can differ in a number of ways. For example, the analytical reagent may be crude antiserum, monoclonal antibodies, isolated immunoglobulin fractions, etc. The conditions under which the method is run, detection of the antigen—antibody complex, and the techniques used to increase sensitivity or specificity of the reaction all maybe varied. 

The primary antibody can be in the form of a polyclonal antiserum or a monoclonal antibody produced either in a culture supernatant or in an ascitic fluid The concentration of the antibody in an ascitic fluid will be an order of magnitude greater than that in the culture supernatant, but the latter will be free of other immunoglobulins. 

Fig. 1. Double label immunohistochemistry on rai liver. An acetone-fixed rat liver section was incubated with a polyclonal antiserum raised in rabbit to a hepa-tocyte cell surface protein (courtesy of Dr. S. Stamatoglou) and a mouse monoclonal antibody against a bile duct specific cytokeratin (courtesy of Dr. E. B. Lane). The hepatocyte protein was localized by use of a secondary peroxidase-conjugated antibody resulting in a red/brown product (thin arrow). The bile duct cytokeratin was identified by using an alkaline phosphatase-conjugated secondary antibody giving a blue color (thick arrow).

Antibody this should be the pure IgG fraction or, better, affinity-purified antibody from an antiserum or pure monoclonal antibody (see Chapter 7). 

Personal experience has shown that, although monoclonal antibodies can be used in ISEM, better results are more often obtained by using crude antiserum. Others have had the same experience (4). In addition antisera preserved in glycerol can be used successfully for detecting viruses by IEM (8). 

The demonstration that MTs from a wide variety of fish species are recognized by an antiserum raised against one piscine MT has enabled the development of immunotechniques based on ELISA143 and radioimmunoassay (RIA) procedures144 for the quantification of these compounds. A competitive solid-phase assay based on dissociation-enhanced lanthanide fluoroimmuno-detection (DELFI A) of anti-MT monoclonal antibody bound to a solid phase has been reported.145 An electrochemical determination of MTs by square wave cathodic stripping voltammetry has also been developed and optimized.146... 

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)... 

Optimum antibody titer may be defined as the highest dilution of an antiserum (or monoclonal antibody) that results in maximum specific staining with the least amount of background under specific test conditions. This highest dilution is determined primarily by the absolute amount of specific antibodies present. 

Dissociation of primary antibody during washing or incubation with link antibodies A feature of low affinity antibodies Polyclonal primary antiserum Attempt staining at low dilutions. Monoclonal primary antibody Replace with higher affinity antibody of identical specificity. Re-optimize incubation times for washing buffer and link antibody. 5-6... 

Although monoclonal antibodies (MAbs) are preferable to polyclonal antiserum for many types of immunological investigations,50,51 they are not suitable for analyzing quantitatively the degree of antigenic similarity... 

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