Reference sera with neutralizing antibody (NA) titers to HIV-1 were prepared in our laboratory for the World Health Organization (WHO). According to the WHO guidelines for the preparation of reference reagents, a number of procedures and tests are required to assure the quality of the samples. There are 2500-3000 freeze-dried ampules of two different sera ready for labelling and distribution. Uniformity of fill of the ampules was achieved for both lots with a standard of deviation of < or = 0.012 ml and a coefficient of variation of < or = 0.024. Moisture content was analyzed using the Gravimetric or Loss on Drying method and was within the appropriate range and residual oxygen was not present since the ampules were sealed under vacuum and tested with a high frequency vacuum testing coil to confirm that a vacuum was present. The samples were assessed for microbial contamination using fluid thioglycollate media and soybean casein digest media and no growth was observed in either. A short term stability study was conducted with 5 samples of each at 56 C, 35 C, 27 C, and 4 C. The results indicated that lyophilized samples placed at 56 C for up to 3 weeks were stable, however at 5 weeks these samples became gelled upon reconstitution. At 35 C, 27 C, and 4 C, samples were stable with no loss in titer for a period of 8 weeks, 5 months, and 7 months, respectively. An infectivity assay was done with no detectable virus present at the end of four weeks as determined on a HIV-p24 core profile Elisa test kit. An international collaborative study was designed using ten coded samples and six laboratories participated from the U.S. and Europe with each using their own in-house method for determining NA titers. Mean titers were calculated for each of the coded samples which included the reference sera in duplicate for the HIVMN, HIVIIIB, HIVRF, and HIVSF2 strains of HIV. Currently, unitage is being decided upon for each of the two samples so that labels can be prepared and the samples can be distributed to WHO centers.
