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首页> 外文期刊>Vaccine >Physiochemical and functional characterization of antigen proteins eluted from aluminum hydroxide adjuvant
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Physiochemical and functional characterization of antigen proteins eluted from aluminum hydroxide adjuvant

机译：从氢氧化铝佐剂洗脱的抗原蛋白的理化和功能表征

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We have characterized protein antigens after quantitative dissociation from aluminum hydroxide adjuvant. Bovine serum albumin (BSA) and a multi-antigen vaccine for Group A Streptococcus (GrAS Vaccine) were formulated on aluminum hydroxide, stored for & or =10 days then eluted with a 48-h treatment at 4 degrees C with 0.85% H(3)PO(4) plus 4M guanidine HCl (GnHCl). BSA is recovered from adjuvant at 92+/-2%. GrAS antigens are equally recovered from GrAS Vaccine (95+/-11% of total protein expected using multiple lots stored for up to 12 months). Recovery after elution is similar when determined by RP-HPLC, SEC-HPLC, UV absorbance, or Bradford methods. Eluted antigens are structurally and functionally intact as judged relative to both treated and untreated antigen controls by SDS-PAGE, RP-HPLC, SEC-HPLC, and after desalting by circular dichroism, bis-ANS binding, and antigenicity determined by ELISA. When formulated and stored for a few weeks, BSA has more dimer (31+/-5%) relative to the elution control (9% dimer) as detected by SEC-HPLC, suggesting that BSA microaggregation is promoted on aluminum. Antigens eluted from very aged GrAS Vaccine (&12 months) show marked changes by RP-HPLC. Structural changes in the antigens under elution conditions were evaluated using bis-ANS, a fluorescent probe of protein structure. Binding of bis-ANS increases fluorescence approximately 100-fold and is significantly diminished with increasing GnHCl concentrations indicating a progressive denaturing of the proteins. At 4M GnHCl (with or without 0.85% H(3)PO(4)) the GrAS antigens are fully denatured and BSA is partially denatured. Interestingly, the addition of 0.85% H(3)PO(4) increases bis-ANS binding on GrAS antigens and reduces the denaturing of GrAS antigens and BSA by chaotropes. Desalting or diluting the eluted antigens results in renaturing of the proteins as judged by bis-ANS fluorescence, circular dichroism and antigenicity testing. The elution method provides a novel approach for high recovery and characterization of GrAS Vaccine antigens and may be applicable to the study of many aluminum hydroxide-bound vaccines.
机译：从氢氧化铝佐剂定量解离后，我们已经表征了蛋白质抗原。将牛血清白蛋白（BSA）和用于A组链球菌的多抗原疫苗（GrAS疫苗）配制在氢氧化铝上，并储存于＞ 200℃。或= 10天，然后在4摄氏度下用0.85％H（3）PO（4）加4M盐酸胍（GnHCl）进行48小时处理洗脱。从佐剂中回收的BSA的含量为92 +/- 2％。从GrAS疫苗中平均回收GrAS抗原（使用最多可存储12个月的多批样品，预期总蛋白的95 +/- 11％）。通过RP-HPLC，SEC-HPLC，UV吸光度或Bradford方法测定时，洗脱后的回收率相似。相对于通过SDS-PAGE，RP-HPLC，SEC-HPLC进行处理和未处理的抗原对照，以及通过圆二色性，bis-ANS结合脱盐和通过ELISA确定的抗原性判断，洗脱的抗原在结构和功能上都是完整的。当配制并保存数周时，相对于洗脱对照（9％二聚体），BSA具有更多的二聚体（31 +/- 5％），这是通过SEC-HPLC检测到的，表明BSA微聚集在铝上得到了促进。从非常老的GrAS疫苗（＞ 12个月）洗脱的抗原通过RP-HPLC显示出明显的变化。使用bis-ANS（一种蛋白质结构的荧光探针）评估了洗脱条件下抗原的结构变化。 bis-ANS的结合使荧光增加了约100倍，并且随着GnHCl浓度的增加而显着降低，表明蛋白质逐渐变性。在4M GnHCl（有或没有0.85％H（3）PO（4）时），GrAS抗原被完全变性，BSA被部分变性。有趣的是，添加0.85％H（3）PO（4）增加了对GrAS抗原的bis-ANS结合，并减少了离液剂对GrAS抗原和BSA的变性。通过bis-ANS荧光，圆二色性和抗原性测试判断，对洗脱的抗原进行脱盐或稀释可导致蛋白质复性。洗脱方法为GrAS疫苗抗原的高回收率和鉴定提供了一种新颖的方法，可用于研究许多氢氧化铝结合疫苗。

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