Neutralizing assays

Despite a considerable decrease in its inhibitory activity in the solid-phase inhibition assays (with an IC50 of 300 nM, corresponding to a 50-fold increase compared with 160), an equivalent decrease in performance of the so-called DAISY (161) in the more challenging SLT-I and SLT-II verocytotoxicity-neutralization assays was not... 

For other plant-derived antibodies, stability was shown to be similar to mammalian counterparts. For instance, a humanized anti-herpes simplex virus monoclonal antibody (IgGl) was expressed in soybean and showed stability in human semen and cervical mucus over 24 h similar to the antibody obtained from mammalian cell culture. In addition, the plant-derived and mammalian antibodies were tested in a standard neutralization assay with no apparent differences in their ability to neutralize HSV-2. As glycans may play a role in immune exclusion mechanisms in mucus, the diffusion of these monoclonal antibodies in human cerival mucus was tested. No differences were found in terms of the prevention of vaginal HSV-2 transmission in a mouse model, i.e. the plant-derived antibody provided efficient protection against a vaginal inoculum of HSV-2 [58]. This shows that glycosylation differences do not necessarily affect efficacy. 

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. 

In general, the evaluation of the immunogenicity of impurities is restricted to binding antibodies. Only in the case where the biological activity of the impurity is known and its inactivation is biologically relevant, a neutralization assay may be necessary. 

Palytoxin hemolysis neutralization assay 0.3 pg cell palytoxin equivalents (isolated from Rangiputa)... 

Bignami, G.S. 1993. A rapid and sensitive hemolysis neutralization assay for palytoxin. Toxicon 3, 817-820. 

However, the presence of cytotoxicity in the extract could be due to other toxins or bacterial products, it is necessary to perform a neutralization assay with specific antisera to confirm that the cytopathic effect is due to the production of Shiga toxins. Moreover, in absence of neutralization assay, VGA lacks specificity (Rahn et al. 1996). Therefore, the Vero Cells assay have been largely supplanted by immunologically-based and DNA-based method for the detection of STEC, but is still used for confirmation of Shiga toxin production from pure cultures. 

If available, a potency assay developed for the batch release of the biological is a good starting point because it can often be adapted for use as a Nab bioassay. For example, a neutralization assay for GM-CSF can be a modification of the proliferation assay used for potency assessment for GM-CSF. This approach is advantageous, as optimization of cell line maintenance and culture conditions had already been performed. However, this assay needs to be tailored for use as a neutralization assay, as it may not be adequately sensitive for the purpose in its original format. Therefore, the performance of the potency assay in the presence of test sample (appropriate species serum) and response to the therapeutic to yield a sufficiently sensitive assay requires validation. Ideally, the cell line should yield a functional end point upon treatment with the therapeutic protein, the assay should be simple to perform, and the biological end point should be tolerant of the test sample matrix and perform adequately over a range of concentrations. 

Neutralizing Assays. The detection of neutralizing antibodies is performed in assays that can show inhibition of the biological activity of the therapeutic protein. In general this will be a bioassay. The therapeutic protein is incubated with the serum to be tested, and the mixture is tested in the bioassay. If the antibodies in the serum have neutralizing capacities, the biological activity of the mixture is reduced, as compared with the pure therapeutic protein. 

Briggs, L., et al., Detection of palytoxin using a haemolysis neutralization assay, in Proceedings, 10th Marine Biotoxin Science Workshop, Wellington, New Zealand, New Zealand Food Safety Authority, 91, 1998. 

Tryptic soy broth (TSB) is suggested for use in neutralization assay and for preparing the E. coli inocula to be distributed into the ground hamburger. 

The affinity matrices were (0.5 ml bed volume) equilibrated with PBS-BT buffer and 0.5 ml of antiserum fractionated on each column. The unbound material was washed with 20.0 ml loading buffer (PBS-BT) followed by a 0.5 M NaCl to remove nonspecific binding. Specific antibody fraction was eluted in the same buffer adjusted to pH 2.0 and the fraction (2.0 ml each) were immediately neutralized. Assayed 100 tds of each fraction with either [3H]NAD (33,400 cpm = 0.90 ng) or 3H-5 AMP (33, 610 cpm = 0.83 ng) in PBS-BT buffer. 

---