Quantification of bacterial lipopolysaccharides by the purpald assay: Measuring formaldehyde generated from 2-keto-3-deoxyoctonate and heptose at the inner core by periodate oxidation
Quantification of bacterial lipopolysaccharides by the purpald assay: Measuring formaldehyde generated from 2-keto-3-deoxyoctonate and heptose at the inner core by periodate oxidation
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DOI：
10.1006/abio.1998.2961
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发表时间：
1999-02-01
影响因子：
2.9
通讯作者：
Tsai, CM
中科院分区：
文献类型：
--
作者：
Lee, CH;Tsai, CM
我们采用了Purpald分析法（M，S。Quesenberry和Y. C.李，阿纳尔。234，50-55，1996）来定量室温下96孔微量滴定板中溶液中的脂多糖（LPS）含量。该方法利用高碘酸盐氧化LPS分子的2-酮基-3-脱氧辛酸酯（Kdo）和L-（或D-）甘油-D-甘露-庚糖中未取代的末端邻位二醇基团以释放甲醛。甲醛在550 nm（或530-570 nm）处通过与purpald试剂反应，然后用NaIO 4氧化来定量。Purpald测定法的灵敏度与Kdo测定法测定LPS的灵敏度相当。然而，Purpald测定法比Kdo测定法优越上级，因为：（i）在测定过程中不需要样品的酸水解和煮沸;因此，它可以在室温下直接用微量滴定板进行大量样品的测定，（二）Purpald测定法可以检测来自各种细菌的许多类型的LPS，因为LPS含有Kdo和庚糖，其在LPS中具有未取代的末端邻位二醇。其结构，而Kdo测定不能检测来自某些细菌的LPS（例如，流感嗜血杆菌、百日咳杆菌和霍乱弧菌），这是由于在Kdo的C-4和C-5位置的取代。
We have adapted the purpald assay (M, S. Quesenberry and Y. C. Lee, Anal. Biochem. 234, 50-55, 1996) to quantify lipopolysaccharide (LPS) content in solution in 96-well microtiter plates at room temperature. This method employs the oxidation of unsubstituted terminal vicinal glycol groups in 2-keto-3-deoxyoctonate (Kdo) and L-(or D-)glycero-D-manno-heptose of LPS molecules by periodate to release formaldehyde. The formaldehyde is quantified at 550 nm (or 530-570 nm) by reacting with purpald reagent followed by oxidation with NaIO4. The sensitivity of the purpald assay is comparable to that of the Kdo assay for LPS determination. However, the purpald assay is superior to the Kdo assay because: (i) No acid hydrolysis of the samples and no boiling in the assay process are required; thus, it can be directly carried out with microtiter plates for a large number of samples at room temperature, (ii) The purpald assay can detect many types of LPS from various bacteria since LPS contains Kdo and heptose which possess unsubstituted terminal vicinal glycol in its structure, while the Kdo assay cannot detect LPS from certain bacteria (e.g., Haemophilus influenzae, Bordetella pertussis, and Vibrio cholerae) due to substitution at the C-4 and C-5 positions of Kdo.