建立和验证一种流式细胞微球检测方法以评估针对多种恶性疟原虫重组抗原的抗体。

BACKGROUND

Multiplex cytometric bead assay (CBA) have a number of advantages over ELISA for antibody testing, but little information is available on standardization and validation of antibody CBA to multiple Plasmodium falciparum antigens. The present study was set to determine optimal parameters for multiplex testing of antibodies to P. falciparum antigens, and to compare results of multiplex CBA to ELISA.

METHODS

Antibodies to ten recombinant P. falciparum antigens were measured by CBA and ELISA in samples from 30 individuals from a malaria endemic area of Kenya and compared to known positive and negative control plasma samples. Optimal antigen amounts, monoplex vs multiplex testing, plasma dilution, optimal buffer, number of beads required were assessed for CBA testing, and results from CBA vs. ELISA testing were compared.

RESULTS

Optimal amounts for CBA antibody testing differed according to antigen. Results for monoplex CBA testing correlated strongly with multiplex testing for all antigens (r = 0.88-0.99, P values from <0.0001 - 0.004), and antibodies to variants of the same antigen were accurately distinguished within a multiplex reaction. Plasma dilutions of 1:100 or 1:200 were optimal for all antigens for CBA testing. Plasma diluted in a buffer containing 0.05% sodium azide, 0.5% polyvinylalcohol, and 0.8% polyvinylpyrrolidone had the lowest background activity. CBA median fluorescence intensity (MFI) values with 1,000 antigen-conjugated beads/well did not differ significantly from MFI with 5,000 beads/well. CBA and ELISA results correlated well for all antigens except apical membrane antigen-1 (AMA-1). CBA testing produced a greater range of values in samples from malaria endemic areas and less background reactivity for blank samples than ELISA.

CONCLUSION

With optimization, CBA may be the preferred method of testing for antibodies to P. falciparum antigens, as CBA can test for antibodies to multiple recombinant antigens from a single plasma sample and produces a greater range of values in positive samples and lower background readings for blank samples than ELISA.

背景

与 ELISA 相比，多指标流式细胞术检测（CBA）在抗体检测方面具有许多优势，但关于多重检测疟原虫 falciparum 抗原的抗体的标准化和验证，信息却很少。本研究旨在确定多重检测疟原虫 falciparum 抗原抗体的最佳参数，并比较 CBA 与 ELISA 的检测结果。

方法

采用 CBA 和 ELISA 检测 30 名肯尼亚疟疾流行地区个体的样本中的 10 种重组疟原虫 falciparum 抗原抗体，并与已知的阳性和阴性对照血浆样本进行比较。评估 CBA 检测的最佳抗原量、单指标与多指标检测、血浆稀释度、最佳缓冲液、所需的珠子数量，并比较 CBA 与 ELISA 检测的结果。

结果

CBA 抗体检测的最佳抗原量因抗原而异。对于所有抗原，单指标 CBA 检测结果与多指标检测结果相关性很强（r = 0.88-0.99，P 值均<0.0001-0.004），且在多指标反应中可以准确区分同一抗原的变体抗体。对于所有抗原，CBA 检测的最佳血浆稀释度为 1:100 或 1:200。在含有 0.05%叠氮化钠、0.5%聚乙烯醇和 0.8%聚乙烯吡咯烷酮的缓冲液中稀释的血浆背景活性最低。CBA 检测中，1000 个抗原偶联珠/孔的中位荧光强度（MFI）与 5000 个珠/孔的 MFI 无显著差异。除顶膜抗原-1（AMA-1）外，所有抗原的 CBA 和 ELISA 结果均具有良好的相关性。与 ELISA 相比，CBA 检测在疟疾流行地区的样本中产生了更大范围的数值，且空白样本的背景反应更小。