Malaria Branch, Division of Parasitic Diseases and Malaria, Centers for Disease Control and Prevention, Atlanta, GA, 30329, USA.
IHRC, Inc, Atlanta, GA, 30346, USA.
Malar J. 2021 Oct 17;20(1):405. doi: 10.1186/s12936-021-03946-1.
Despite the widespread use of histidine-rich protein 2 (HRP2)-based rapid diagnostic tests (RDTs), purified native HRP2 antigen is not standardly used in research applications or assessment of RDTs used in the field.
This report describes the purification of native HRP2 (nHRP2) from the HB3 Plasmodium falciparum culture strain. As this culture strain lacks pfhrp3 from its genome, it is an excellent source of HRP2 protein only and does not produce the closely-related HRP3. The nHRP2 protein was isolated from culture supernatant, infected red blood cells (iRBCs), and whole parasite lysate using nickel-metal chelate chromatography. Biochemical characterization of nHRP2 from HB3 culture was conducted by SDS-PAGE and western blotting, and nHRP2 was assayed by RDT, ELISA, and bead-based immunoassay.
Purified nHRP2 was identified by SDS-PAGE and western blot as a - 60 kDa protein that bound anti-HRP-2 monoclonal antibodies. Mouse anti-HRP2 monoclonal antibody was found to produce high optical density readings between dilutions of 1:100 and 1:3,200 by ELISA with assay signal observed up to a 1:200,000 dilution. nHRP2 yield from HB3 culture by bead-based immunoassay revealed that both culture supernatant and iRBC lysate were practical sources of large quantities of this antigen, producing a total yield of 292.4 µg of nHRP2 from two pooled culture preparations. Assessment of nHRP2 recognition by RDTs revealed that Carestart Pf HRP2 and HRP2/pLDH RDTs detected purified nHRP2 when applied at concentrations between 20.6 and 2060 ng/mL, performing within a log-fold dilution of commercially-available recombinant HRP2. The band intensity observed for the nHRP2 dilutions was equivalent to that observed for P. falciparum culture strain dilutions of 3D7 and US06 F Nigeria XII between 12.5 and 1000 parasites/µL.
Purified nHRP2 could be a valuable reagent for laboratory applications as well as assessment of new and existing RDTs prior to their use in clinical settings. These results establish that it is possible to extract microgram quantities of the native HRP2 antigen from HB3 culture and that this purified protein is well recognized by existing monoclonal antibody lines and RDTs.
尽管广泛使用富含组氨酸蛋白 2（HRP2）的快速诊断检测（RDT），但在研究应用或评估现场使用的 RDT 时，并未标准使用纯化的天然 HRP2 抗原。
本报告描述了从 HB3 恶性疟原虫培养株中纯化天然 HRP2（nHRP2）。由于该培养株基因组中缺乏 pfhrp3，因此它是 HRP2 蛋白的绝佳来源，而不会产生密切相关的 HRP3。使用镍金属螯合物色谱法从培养上清液、感染的红细胞（iRBC）和全寄生虫裂解物中分离 nHRP2 蛋白。通过 SDS-PAGE 和 Western blot 对 HB3 培养物的 nHRP2 进行生化特征分析，并通过 RDT、ELISA 和基于珠的免疫测定法测定 nHRP2。
通过 SDS-PAGE 和 Western blot 鉴定纯化的 nHRP2 为 -60 kDa 蛋白，可与抗 HRP-2 单克隆抗体结合。通过 ELISA 发现，抗 HRP2 单克隆抗体在 1:100 至 1:3200 的稀释度下产生高光密度读数，在 1:200000 的稀释度下观察到检测信号。基于珠的免疫测定法显示，HB3 培养物的 nHRP2 产量表明，培养上清液和 iRBC 裂解物均是大量该抗原的实用来源，从两个混合培养物中总共产生了 292.4 µg nHRP2。评估 RDT 对 nHRP2 的识别情况表明，Carestart Pf HRP2 和 HRP2/pLDH RDT 在 20.6 至 2060 ng/mL 的浓度下检测到纯化的 nHRP2，在商业可获得的重组 HRP2 的对数稀释范围内进行检测。nHRP2 稀释度观察到的条带强度与 3D7 和 US06 F Nigeria XII 恶性疟原虫培养株稀释度在 12.5 至 1000 个寄生虫/µL 之间观察到的强度相当。
纯化的 nHRP2 可作为实验室应用的有价值试剂，也可在将新的和现有的 RDT 用于临床环境之前对其进行评估。这些结果表明，从 HB3 培养物中提取微克级天然 HRP2 抗原是可行的，并且该纯化蛋白可被现有的单克隆抗体系和 RDT 很好地识别。
