Using a recombinant protein antigen for antibody testing shows a sum of antibody responses to multiple different immune epitopes existing in the protein antigen. In contrast, the antibody testing to an immunogenic peptide epitope reflects a singular antibody response to the individual peptide epitope. Therefore, using a panel of peptide epitopes provides an advantage for profiling multiple singular antibody responses with potential to estimate recent malaria exposure in human infections. However, transitioning from malaria immune epitope peptide-based ELISA to an all peptide bead-based multiplex Luminex assay presents some challenges including variation in the ability of different peptides to bind beads. The aim of this study was to develop a peptide coupling method while demonstrating the utility of these peptide epitopes from multiple stage antigens of Plasmodium falciparum for measuring antibodies.

Successful coupling of peptide epitopes to beads followed three steps: 1) development of a peptide tag appended to the C-terminus of each peptide epitope consisting of beta-alanine–lysine (x 4)--cysteine, 2) bead modification with a high concentration of adipic acid dihydrazide, and 3) use of the peptide epitope as a blocker in place of the traditional choice, bovine serum albumin (BSA). This new method was used to couple 12 peptide epitopes from multiple stage specific antigens of P. falciparum, 1 Anopheles mosquito salivary gland peptide, and 1 Epstein-Barr virus peptide as an assay control. The new method was applied to testing of IgG in pooled samples from 30 individuals with previously repeated malaria exposure in western Kenya and IgM and IgG in samples from 37 U.S. travelers with recent exposure to malaria.

The new peptide-bead coupling method and subsequent multiplex Luminex assay showed reliable detection of IgG to all 14 peptides in Kenyan samples. Among 37 samples from U.S. travelers recently diagnosed with malaria, IgM and IgG to the peptide epitopes were detected with high sensitivity and variation. Overall, the U.S. travelers had a much lower positivity rates of IgM than IgG to different peptide epitopes, ranging from a high of 62.2% positive for one epitope to a low of only 5.4% positive for another epitope. In contrast, the travelers had IgG positive rates from 97.3% to 91.9% to various peptide epitopes. Based on the different distribution in IgM and IgG positivity to overall number of peptide epitopes and to the number of pre-erythrocytic, erythrocytic, gametocytic, and salivary stage epitopes at the individual level, four distinct patterns of IgM and IgG responses among the 37 samples from US travelers were observed. Independent peptide-bead coupling and antibody level readout between two different instruments also showed comparable results.

Overall, this new coupling method resolves the peptide-bead coupling challenge, is reproducible, and can be applied to any other immunogenic peptide epitopes. The resulting all peptide bead-based multiplex Luminex assay can be expanded to include other peptide epitopes of P. falciparum, different malaria species, or other diseases for surveillance, either in US travelers or endemic areas.

使用重组蛋白质抗原进行抗体测试显示了对蛋白质抗原中存在的多个不同免疫表位的抗体反应的总和。相比之下，针对免疫原性肽表位的抗体测试反映了对单个肽表位的单一抗体反应。因此，使用一组肽表位为分析多个单一抗体反应提供了一个优势，有可能估计人类感染中最近的疟疾暴露。然而，从基于疟疾免疫表位肽的 ELISA 过渡到基于全肽微珠的多重 Luminex 检测存在一些挑战，包括不同肽结合微珠的能力存在差异。用于测量抗体的恶性疟原虫。

肽表位与珠子的成功偶联遵循三个步骤：1) 开发附加到每个肽表位 C 端的肽标签，由 β-丙氨酸-赖氨酸 (x 4)-半胱氨酸组成，2) 具有高己二酸二酰肼的浓度，以及 3) 使用肽表位作为阻断剂代替传统选择的牛血清白蛋白 (BSA)。这种新方法用于偶联来自恶性疟原虫、1按蚊的多阶段特异性抗原的 12 个肽表位。蚊子唾液腺肽和 1 种爱泼斯坦-巴尔病毒肽作为检测对照。新方法用于测试来自肯尼亚西部先前反复接触疟疾的 30 名个体的合并样本中的 IgG，以及来自最近接触过疟疾的 37 名美国旅行者的样本中的 IgM 和 IgG。

新的肽-珠偶联方法和随后的多重 Luminex 检测显示了对肯尼亚样品中所有 14 种肽的 IgG 的可靠检测。在最近诊断出患有疟疾的美国旅行者的 37 个样本中，检测到肽表位的 IgM 和 IgG，具有高灵敏度和变异性。总体而言，美国旅行者对不同肽表位的 IgM 阳性率比 IgG 低得多，从一个表位的阳性率高达 62.2% 到另一个表位的阳性率仅为 5.4%。相比之下，旅行者对各种肽表位的 IgG 阳性率为 97.3% 至 91.9%。基于 IgM 和 IgG 阳性对肽表位总数和个体水平的前红细胞、红细胞、配子细胞和唾液阶段表位数量的不同分布，在来自美国旅行者的 37 个样本中观察到四种不同的 IgM 和 IgG 反应模式。两种不同仪器之间的独立肽-珠偶联和抗体水平读数也显示出类似的结果。

总体而言，这种新的偶联方法解决了肽-珠偶联挑战，具有可重复性，并且可以应用于任何其他免疫原性肽表位。由此产生的所有基于肽珠的多重 Luminex 检测可以扩展到包括恶性疟原虫的其他肽表位、不同的疟疾物种或其他用于监测的疾病，无论是在美国旅行者还是在流行地区。