This study was initiated with the following objectives: (1) to standardize the protein nitrogen unit PNU method for the determination of the concentration of allergenic extracts, (2) to determine the stability of the allergenic extract PNU value throughout the dating period, (3) to determine between laboratory reproducibility for assayed PNU values, and (4) to improve the detection limit for the determination of nitrogen and decrease analysis time. Parameters were optimized for the PNU precipitation procedure for aqueous, freeze-dried, glycerinated and alum precipitated allergenic extracts. The stability study indicated stability for PNU values when the products tested have been stored at a constant 2-8oC. Although the allergens lose their reactivity with time, the PNU value does not change significantly. It is an estimate of the concentration of a freshly prepared allergenic extract. The collaborative study of the optimized PNU precipitation procedure consisted of the analysis of six samples in duplicate by six laboratories using the CBER Kjeldahl methodology. A chemiluminescence method is being explored as a method for nitrogen determination that is more sensitive than the micro-Kjeldahl method and which can detect about 10 micrograms of protein/mL or 1.6 micrograms of nitrogen/mL. Methodology is being studied to determine the protein content in Anthrax Vaccine, Typhoid Vaccine, Cholera Vaccine, Influenzae Virus Vaccine and other vaccines such as Hepatitis B Vaccine and Hepatitis C100-3 Antigen in which protein measurement by the Lowry method would be subjected to interferences by compounds which are present such as SDS, Tris, EDTA, and thiol reagents (DTT and thimerosal). A study of the above methods is underway.
