Total Protein and Total and Specific Antibody in Nasal Secretions (BCS)
Pre-challenge nasal washings for assay of total and specific immunoglobulins and total protein were shaken with glass beads, centrifuged to sediment the mucus, and stored at -20°C. Prior to assay, specimens were tested for the presence of blood by the Haemastix test (Miles Laboratories, Ltd.) and excluded if they gave more than a trace reaction.
Enzyme-linked immunosorbant assay (ELISA) was used to measure total IgA and IgG in nasal lavage specimens. Conjugates, substrate (p-nitrophenol phosphate dissolved in 10% diethanolamine buffer [1 mg/ml]), washing methods, and measurement of optical density (OD) were the same as those described for the detection of antibody in serum (see Total Serum Antibody and Specific Antibody in Serum, Coronaviruses and Respiratory Syncytial Virus). ELISA plates were coated overnight with rabbit anti-human α chain IgA serum (Hoechst UK Ltd) or goat anti-human IgG (Sigma) at about 5 µg/ml protein coating buffer. Nasal washings at a dilution of 1:4000 for IgA assay or 1:500 for IgG assay were added, and the plates incubated for 6 hours at 4°C. A standard curve of doubling dilutions of human IgG or colostrum IgA (Sigma) diluted in PBS-T was set up on each plate.
Specific Antibody to Coronaviruses and to Respiratory Syncytial Virus1
For measurement of specific IgA in nasal washings, specimens were diluted 1:20 in 5% control antigen in PBS-T and added to wells coated with virus or control antigen in the same experiment in which total IgA was assayed (see Total Antibody above). The low ODs obtained against control antigen were subtracted from those obtained in virus-coated wells, and the concentrations read off from the IgA standard curve.
Specific Antibody to Rhinoviruses2
The assay used to measure specific antibody to rhinovirus in nasal secretions is the same as that used to measure antibody in serum (see Specific Antibody in Serum, Rhinoviruses). The only procedural difference is that nasal wash samples were tested in duplicate 2-fold serial dilutions rather than duplicate 10-fold serial dilutions.
Total protein in nasal washings was measured using the Lowry method3. Briefly, a complex-forming reagent was prepared by mixing the following stock solutions in the proportion of 100:1:1, respectively: 2% Na2CO3 in distilled water; 1% CuSO4•5H2O in distilled water; and 2% sodium potassium tartrate in distilled water. 0.1 ml of 2N NaOH was added to 0.1 ml of extracted sample and the resulting solution hydrolyzed at 100°C for 10 minutes. After cooling at room temperature, 1ml of complex-forming reagent was added, and the solution left at room temperature for 10 minutes. After adding 0.1 ml of Folin reagent with a vortex mixer, the mixture was left at room temperature for 30-60 minutes to allow color to develop. Samples were read in a spectrophotometer and compared to a standard curve.
1 Callow, K. A. (1985). Effect of specific humoral immunity and some non-specific factors on resistance of volunteers to respiratory coronavirus infection. Journal of Hygiene, 95, 173-189.
2 Barklay, W. S. & Al-Nakib, W. (1987). An ELISA for the detection of rhinovirus specific antibody in serum and nasal secretion. Journal of Virological Methods, 15, 53-64.
3 Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry, 193, 265-275.