Antiserum titer

Antiserum Production The immunogen, carboxymethylmorphine-bovine-serum-albumin, is emulsified with equal volume of complete Freund s adjuvant. Initial immunization doses are injected into the New Zealand albino rabbits and later on this followed up with booster injections after a period of 6 weeks. The antiserum titer is determined with each booster dose injection and is duly harvested when the titre value is maximum. This is diluted suitably and employed in the radioimmunoassay. ... 

Check the antiserum titer Repeat the booster injection, and proceed with the protocol if the titer is not satisfactory. 

Monitoring of the antiserum titer before killing rabbits is important to obtain specific antisera. It is recommended that the titer of a small volume of serum bled from ears is monitored periodically by ELISA (Follow the same procedure described in section 3.7. using 2 p.g/ml Dig-OVA as a coating conjugate). Collecting antisera should be carried out when the titer reaches the stationary phase. 

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. 

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. 

Production of antiserum with high titer and specility is done by trial and error, especially because each immunized animal gives antisera with different characteristics therefore, several groups of animals should be immunized with different antigen preparations. 

Pipet 100 pi of the antibody or antiserum dilution (for titer determination a serial dilution in PBS), blank (buffer without antiserum) and, if available, controls into the respective wells and incubate on a shaker at RT for 30 min. Remove the antibody dilution and wash three times with 250 pi of Soln. B each. 

Another booster should be given when the antibody titer begins to decrease if it is necessary to obtain a very large volume of antiserum (see Note 7)... 

Figure 7. Specific binding of Sendai virus to GQlb ganglioside and its inhibition by antiserum as demonstrated with (a) the VCS method and (b) hemadsorption (see text). A virus in fourfold serial dilutions in buffer B in lower-titer antiserum, 1% C in the same serum, 10% D in higher titer antiserum, 1% this serum, 10%.

The specificity of an antibody titer refers to the degree of cross-reactivity one sees with the antiserum used. By using different tagged haptens one can vary the specificity of the resulting assay however, there is an intrinsic specificity of the antiserum which is difficult (although possible) to improve. The specificity of an antiserum is usually established by compe-... 

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. 

Titer In immunohistochemistry, the highest dilution of an antiserum, which results in optimal specific staining with the least amount of background. 

An important issue in the neutralization assay is the amount of therapeutic protein product to add. This dose should be based on the dose-response curve of the protein in the assay. The reduction should be in the linear part of the curve and be significant enough to be reliably testable in the assay. The titer of the neutralization is often expressed in units. The highest dilution of the serum with a significant inhibition is defined to contain one neutralizing unit. Sometimes the activity is expressed in arbitrary units using an animal antiserum as reference. Further characterization of the antibodies may include evaluation of Ig isotype and affinity. Although alternative methods may be employed, the BIAcore assay has become the standard for these types of analyses. 

A blank control (TO) containing no anti-insulin antibodies is used to determine the non-specific binding (NSB). Three titer controls (T1-T3) containing low, medium and high levels of anti-insulin antibodies (guinea pig anti-pore insulin antiserum, e.g. Scant-ibodies Laboratory Inc. T 531 B, Part. 3 AK 025 in buffer solution) are measured in each run. 

Since the titer of antiserum changes from animal to animal and within the same animal with time, it is advisable to use a single preparation for each set of experiments. When this is not possible each new preparation must be completely standardized. In the case of the secondary antibody preparation, only its ability to precipitate the primary antibodies must be quantitated. Primary antibody preparations, however, must be titrated for their ability to serve as both antibodies and antigens. 

The dilution of the primary antibody is dependent on the nature (i.e., monoclonal or polyclonal) and the titer of the antiserum, and should be determined empirically for each individual antiserum. The dilution usually falls between 1 500-1 1000 however, when testing a new antibody preparation, dilutions between 1 50-1 10,000 should be tried. Antibody dilutions can be stored frozen (-20°C) and reused approx 10 times, depending on antibody. 

It must be emphasized, however, that the m or determinant of the sensidvity and quality of Western blots is the titer of the primary antiserum used. 

The repeated booster doses that are usually required for the best antiserum should not be given too frequently. It has been shown that no further rise in titer results from a second injection given before the response to the first is reaching its peak. At least 4 weeks should pass between injections of Freund s emulsions. After the first booster, or sometimes after the second, antibody response may be quite prolonged and many people believe that a rest of 3-6 months is desirable before the next injection if antiserum of the highest avidity is required the evidence in favor of this approach is not strong, but in general terms there is little doubt that pa-... 

Individual animals immunized with the same substance can produce antibodies that may differ in affinities, titer, and specificities. " Such differences are appeu-ent with antibodies studied by the more classical physical chemical procedures. However, even among anti-enzyme sera harvested from individual rabbits that had been immunized with -lactamase, some were found to neutralize the activity of the enzyme, others to stimulate its activity, and still others were stimulatory and then inhibitory at higher concentrations." In radioimmunoassay, each antiserum from an individual animal must be characterized separately to select those that have the proper affinities and specificities. The production of monoclonal antibodies by hybridoma technology can yield molecules with defined specificities and affinities. As this technology becomes affordable for the average research laboratory, problems associated with antibody heterogeneity are due to diminish. 

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