Ho Mei M, Kairo Satnam K, Corbel Michael J
Division of Bacteriology, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire, UK.
Hum Vaccin. 2006 Jan-Feb;2(1):29-33. doi: 10.4161/hv.2.1.2523. Epub 2006 Jan 20.
Tuberculin purified protein derivative (PPD) currently can only be standardised by delayed hypersensitivity skin reactions in sensitised guinea pigs. An in vitro dot blot immunoassay was developed for both identity and confirmation of potency estimation of PPD. Polyclonal antibodies (mainly IgG) were generated and immunoreacted with human, bovine and, to lesser extent, avian PPD preparations. Combining size exclusion chromatography (FPLC-SEC) and dot blot immunoassay, the results showed that PPD preparations were mixtures of very heterogeneous tuberculoproteins ranging in size from very large aggregates to very small degraded molecules. All individual fractions of PPD separated by size were immunoreactive, although those of the largest molecular sizes appeared the most immunoreactive in this in vitro dot blot immunoassay. This method is very sensitive and specific to tuberculoproteins and can be an in vitro alternative for the in vivo intradermal skin assay which uses guinea pigs for identity of PPD preparations. Although the capacity of PPD to elicit cell-mediated immune responses on intradermal testing has to be confirmed by in vivo assay, the dot blot immunoassay offers a rapid, sensitive and animal-free alternative to in vivo testing for confirming the identity of PPD preparations with appropriate potencies. This alternative assay would be particularly useful for national regulatory laboratories for confirming the data of manufacturers and thus reducing the use of animals.
结核菌素纯蛋白衍生物（PPD）目前只能通过致敏豚鼠的迟发型超敏皮肤反应来标准化。开发了一种体外斑点印迹免疫测定法，用于PPD的鉴定和效价估计的确认。制备了多克隆抗体（主要是IgG），并使其与人、牛以及程度较低的禽PPD制剂发生免疫反应。结合尺寸排阻色谱法（FPLC-SEC）和斑点印迹免疫测定法，结果表明PPD制剂是非常异质的结核蛋白混合物，其大小范围从非常大的聚集体到非常小的降解分子。通过尺寸分离的PPD的所有单个级分都具有免疫反应性，尽管在这种体外斑点印迹免疫测定法中，分子尺寸最大的那些级分似乎免疫反应性最强。该方法对结核蛋白非常敏感且特异，并且可以作为体内皮内皮肤试验的体外替代方法，体内皮内皮肤试验使用豚鼠来鉴定PPD制剂。尽管PPD在皮内试验中引发细胞介导免疫反应的能力必须通过体内试验来确认，但斑点印迹免疫测定法为体内试验提供了一种快速、灵敏且无动物的替代方法，用于确认具有适当效价的PPD制剂的身份。这种替代测定法对于国家监管实验室确认制造商的数据从而减少动物使用将特别有用。
