Recombinant HLA-Ⅰ molecules/antigenic peptide complexes (pHLA complexes) are applied in the research of human T cell-specific immune responses. The preparation of pHLA complex is based on genetic engineering and protein in vitro dilution and folding-refolding technology. In an in vitro refolding system, recombinant HLA-Ⅰ molecules correctly fold and bind with antigenic peptides to form complexes. In this study, ultrafiltration-high performance liquid chromatography (ultrafiltration-HPLC) was used for quantitative determination of the antigenic peptides in recombinant pHLA complexes, especially for those in a small amount of prepared products. By adding the recombinant HLA-Ⅰ molecules and antigenic peptides into the refolding buffer, the heavy chain (HC) and light chain (β2m) of recombinant HLA-Ⅰ molecules were refolded and bond with the VYF antigenic peptide containing anchor residues to form a pHLA complex. The unbound free antigenic peptide VYF was removed by ultrafiltration to retain the complex. Finally, the pHLA complex was treated by acid to destroy its interaction, thus releasing the antigenic peptide. The results showed that the prepared recombinant pHLA complex was recognized by HLA-Ⅰ molecule specific antibody W6/32, which indicated that the recombinant HLA-Ⅰ class molecule had correct folding and was identified as pHLA complex. The antigen peptide VYF contained in the pHLA complex was also detected by ultrafiltration-HPLC, so it is feasible to apply ultrafiltration-HPLC for determination of pHLA complex. Compared with Western blotting, the concentration of antigenic peptides detected by ultrafiltration-HPLC was 0-9 μg/mL. The binding conditions can be optimized according to the amount of antigenic peptides bound in the complex in order to improve the folding efficiency of HLA-Ⅰ molecules and promote the binding of HLA-Ⅰ molecules to antigenic peptides. The production rate of pHLA complexes in the refolding system can also be calculated according to the content of antigenic peptides bound by pHLA complexes. Therefore, ultrafiltration-HPLC in this study can be used for the quality control of the preparation process of pHLA complexes, and may facilitate the research of T cell-specific immunity, artificial antigen-presenting cells, and development of specific tetramer probe applications.
Amino Acid Sequence ; Antigens ; Chromatography, High Pressure Liquid ; Humans ; Peptides/chemistry* ; Ultrafiltration

Background Air pollutants PM_2.5 and its adsorbed metal elements are important factors affecting public health. Objective To explore the distribution characteristics and sources of metal elements in atmospheric PM_2.5 in Lanzhou from 2019 to 2020, and to assess the health risks of metal elements to different groups of residents through inhalation. Methods From January 2019 to December 2020 in two districts of Lanzhou City (Chengguan District and Xigu District), regular PM_2.5 and metal elements [antimony (Sb), aluminum (Al), arsenic (As), beryllium (Be), cadmium (Cd), chromium (Cr), mercury (Hg), lead (Pb), manganese (Mn), nickel (Ni), selenium (Se), and thallium (Tl)] were regularly monitored, and their concentrations were described by the median (M) and 25^th and 75^th percentiles (P₂₅, P₇₅) as not following a normal distribution (because the detection rates of the five elements Be, Cr, Hg, Ni, and Se were less than 70%, the five elements were not included in subsequent analysis), and then compared with the secondary concentration limits in the Ambient Air Quality Standards (GB 3095-2012). The differences between the medians of the two groups were compared by the Mann-Whitney U rank sum test, and the differences among the medians of multiple groups were compared by the Kruskal-Wallis H rank sum test; the enrichment factor (EF) method and principal component analysis were used to evaluate the pollution degree of the metals and their sources; the health risks of five non-carcinogenic metals (Sb, Al, Pb, Mn, and Tl) and two carcinogenic metals (As and Cd) in PM_2.5 were evaluated by hazard index (HI) and hazard quotient (HQ) using the incremental lifetime cancer risk (LCR) model and the non-carcinogenic risk assessment model, respectively. Results The PM_2.5 concentrations [M (P₂₅, P₇₅)] in Lanzhou City were 38.50 (26.00, 65.00) and 41.00 (29.00, 63.10) μg·m⁻³ in 2019 and 2020, respectively, and the difference was not statistically significant (Z=−0.989, P > 0.05). The average levels of the metal elements from high to low were: Al > Pb > Mn > As > Cd > Sb > Tl, and the annual average concentration of each metal element in 2019 was higher than that in 2020 (P<0.05). The M ( P₂₅, P₇₅) of PM_2.5 concentrations in Chengguan and Xigu districts were 52.98 (17.00, 61.00) and 55.40 (17.00, 67.00) μg·m⁻³, respectively, with no statistically significant differences (P<0.05); the concentrations of Sb and Al in Chengguan District were lower than those in Xigu District (P<0.05), and the concentrations of other metal elements were not different between the two areas (P>0.05). There were seasonal differences in the concentrations of PM_2.5 and seven metal elements in Lanzhou City (except P_Al=0.007, the other P_s < 0.001). The results of the enrichment factor method showed that the EF values of the six metals (Sb, Al, As, Cd, Pb and Tl) were all greater than 1. Among them, except As, the EF values of other metal elements were all greater than 10, and the EF values of Al and Cd were both greater than 100. The results of principal component analysis showed that the variance contributions of the three principal components were 45.61%, 24.22%, and 14.42%, and the cumulative contribution reached 84.25%. The principal component 1 included Pb, As, Cd, and Sb, the principal component 2 included Al and Mn, and the principal component 3 contained Tl. The non-carcinogenic risks of the five metals were, in descending order, Al > Mn > Pb > Tl > Sb, among which the HQ values of the remaining four metals were less than 1 for adults and children, except the HQ value of Al for adults, which was greater than 1. The ILC values of carcinogenic metal As for adult males, adult females, and children were 2.68×10⁻⁵, 2.51×10⁻⁵, and 1.45×10⁻⁵, respectively; the ILC values of carcinogenic metal Cd for adult males, adult females, and children were 1.53×10⁻⁶, 1.43×10⁻⁶, and 8.26×10⁻⁷, respectively. Conclusion There is pollution of atmospheric PM_2.5 and its adsorbed metal elements in Lanzhou. As and Cd elements may pose potential carcinogenic risks to the residents.

Background Polycyclic aromatic hydrocarbons (PAHs) are one of the most widely distributed and harmful organic pollutants in the atmosphere. Objective To investigate the distribution characteristics and composition sources of PAHs in the atmosphere of two districts of Lanzhou from 2019 to 2020 and evaluate the health risks of PAHs via inhalation to different populations. Methods The PAHs concentrations in two urban areas (Chengguan District and Xigu District) of Lanzhou City from January 2019 to December 2020 were regularly monitored. Mann-Whitney U test was used to compare differences in target pollutant concentrations between the two areas. Diagnostic ratio method and principal component analysis were adopted for source identification. Incremental lifetime cancer risk (ILCR) model was applied to evaluate the health risks of PAHs. Results The M (P₂₅, P₇₅) PAHs concentrations in Chengguan District and Xigu District were 24.04 (14.59, 41.81) ng·m⁻³ and 25.97 (18.59, 42.56) ng·m⁻³, respectively, with no significant difference (Z=−0.970, P>0.05). As to seasonal distribution, most PAHs monomer concentrations in Chengguan District were higher than those in Xigu District in summer, and the concentrations of benzo[a]anthracene and benzo[g,h,i]perylene in Chengguan District were also higher than those in Xigu District in spring and autumn (P<0.05), but there were no significant differences in PAHs monomer concentrations between the two urban areas in winter (P>0.05). Ring number of PAHs exhibited seasonal fluctuations. In winter and spring, the highest proportions in Chengguan District and Xigu District were both 4-ring PAHs (37.32%-41.73%, 35.20%-39.66%), and in summer and autumn, the highest proportions were both 2- and 3-ring PAHs (39.38%-49.54%, 47.17%-51.23%). The results of diagnostic ratio method revealed mixed atmospheric PAHs sources in the two urban areas, including fossil fuel, coal, and biomass combustion. The results of principal component analysis showed that the cumulative contribution rate of the four principal component factors reached 79.54%. Principal component 1 included phenanthrene, anthracene, fluoranthene, pyrene, chrysene, benzo[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, and benzo[a]pyrene; principal component 2 included acenaphthene and dibenzo[a,h]anthracene; principal component 3 was fluorene; principal component 4 was naphthalene. The results of health risk assessment showed that the ILCR values of adult males, adult females, and children in Chengguan District were 2.30×10⁻⁶, 2.16×10⁻⁶, and 1.73×10⁻⁶, respectively; and those in Xigu District were 1.58×10⁻⁶, 1.48×10⁻⁶, and 1.19×10⁻⁶, respectively; all were greater than 10⁻⁶. Conclusion PAHs pollution exists in the atmosphere of the two urban areas of Lanzhou City, mainly comes from mixed sources of fossil fuels, coal, and biomass burning, and may pose potential carcinogenic risks to the population.