Department of Pathobiology, College of Veterinary Medicine, Auburn University, Auburn, AL, USA.
Parasit Vectors. 2023 Dec 19;16(1):463. doi: 10.1186/s13071-023-06083-7.
The dissociation of antigen-antibody complexes has been utilized to enhance the accuracy of serological tests for infectious diseases, including Dirofilaria immitis. Currently, the antigen detected by available tests is primarily a glycoprotein found in the reproductive tract of female worms. However, this antigen can become inaccessible when bound to excessive circulating antibodies, leading to reduced test sensitivity and false-negative results. Acid and heat treatments of the sera or plasma have been established as reliable methods for inducing immune complex dissociation (ICD). Previous antigen testing for heartworm infection in dogs and cats has demonstrated that these treatments improve the diagnostic sensitivity without compromising specificity. This study aims to evaluate the performance of four distinct ICD methods in the detection of D. immitis antigen.
We utilized twofold serial dilutions of a well-characterized plasma (ranging from 1:2 to 1:4096) obtained from a D. immitis-infected dog to simulate the diverse antigen levels encountered in real-life infected dogs. The presence of antigen in the diluted samples, both without treatment and treated with four ICD protocols, was assessed in triplicate visually using DiroCHEK by observing color changes. OD values were also obtained using the microplate reader SpectraMax® i Series-Spectramax Id3. A Factorial ANOVA test was conducted to compare the OD values between samples with and without treatments.
The highest dilution at which color changes were observed was 1:128 for untreated samples and for samples subjected to acid treatments in ICD-3 and the hybrid ICD-4 protocol. In contrast, both heat treatment protocols (ICD-1 and ICD-2) exhibited color changes at a 512-fold dilution. The OD values in samples subjected to heat treatment were significantly higher than those in untreated samples, up to dilutions of 512-fold. Although OD values tended to be higher in samples subjected to acid treatment and the hybrid protocol compared to untreated samples up to a 128-fold dilution, this difference was not significant as the samples underwent further dilution.
Our findings affirm that heat treatments, rather than acid treatment, efficiently enhance the detection of D. immitis antigen by liberating the sequestered antigen from the immune complexes.
抗原抗体复合物的解离已被用于提高传染病血清学检测的准确性，包括犬心丝虫。目前，可用检测方法检测的抗原主要是存在于雌性虫生殖道中的糖蛋白。然而，当与过多的循环抗体结合时，这种抗原变得难以获得，导致检测灵敏度降低和假阴性结果。血清或血浆的酸和热处理已被确立为诱导免疫复合物解离（ICD）的可靠方法。先前对犬和猫心丝虫感染的抗原检测表明，这些处理方法在不影响特异性的情况下提高了诊断灵敏度。本研究旨在评估四种不同 ICD 方法在检测犬心丝虫抗原中的性能。
我们使用一种经过充分表征的犬心丝虫感染犬血浆（从 1:2 到 1:4096 进行两倍连续稀释）来模拟现实生活中感染犬中遇到的不同抗原水平。使用 DiroCHEK 通过观察颜色变化，对未经处理和用四种 ICD 方案处理的稀释样本中的抗原进行三次重复目视检测。还使用微孔板读数仪 SpectraMax® i 系列-Spectramax Id3 获得 OD 值。使用析因方差分析比较处理前后样本的 OD 值。
未经处理的样本和 ICD-3 中酸处理以及混合 ICD-4 方案的样本观察到颜色变化的最高稀释度为 1:128。相比之下，两种热处理方案（ICD-1 和 ICD-2）在 512 倍稀释时均发生颜色变化。经热处理的样本的 OD 值明显高于未经处理的样本，最高可达 512 倍稀释。尽管与未经处理的样本相比，经酸处理和混合方案处理的样本的 OD 值在 128 倍稀释时更高，但随着样本进一步稀释，这种差异并不显著。
我们的研究结果证实，与酸处理相比，热处理通过将被隔离的抗原从免疫复合物中释放出来，更有效地增强了犬心丝虫抗原的检测。
