Generalov Sergey V, Erokhin Pavel S, Kuznetsov Oleg S, Abramova Elena G, Zhulidov Ivan M, Osina Natalya A
Russian Research Anti-Plague Institute "Microbe", Saratov, Russia.
Saratov State Vavilov Agrarian University, Saratov, Russia.
Avicenna J Med Biotechnol. 2021 Jul-Sep;13(3):136-142. doi: 10.18502/ajmb.v13i3.6362.
Mouse neutralization test is widely used to determine the level of anti-rabies antibodies, but it is labor-intensive and time consuming. Alternative methods for determining the neutralizing activity of anti-rabies sera and immunoglobulin in cell cultures are also known. Methods such as FAVN and RFFIT involve the use of fluorescent diagnostics. Determination of Cytopathic Effect (CPE) is often complicated due to features of rabies virus replication in cells. Atomic Force Microscopy (AFM) is able to detect the interaction of the virus with the cell at an early stage. Therefore, in this study, a method has been developed for determining the specific activity of anti-rabies sera and immunoglobulin using AFM of cell cultures.
The method is based on the preliminary interaction of rabies virus with samples of rabies sera or immunoglobulin drug, adding the specified reaction mixture to cell culture (Vero or BHK-21), and then measuring the surface roughness of the cells using AFM. AFM was carried out in the intermittent contact mode by the mismatch method in the semi-contact mode. The results were compared with the values obtained in the mouse neutralization test. The consistency of the results obtained by both methods was evaluated by Bland-Altman method.
The increment in the surface roughness of the cells is a consequence of the damaging effect of the virus, which is weakened as a result of its neutralization by rabies antibodies. A dilution allowing 50% suppression of the increase in the surface roughness of cells was selected as the titer of rabies sera or immunoglobulin. In this case, the recommended range for determining the antibody titer is from 1:100 to 1:3000.
For the first time, a new methodological approach in virology and pharmaceutical research is presented in this study. The use of the proposed methodological technique will reduce the time from 21 to 2 days to obtain results in comparison with the mouse neutralization test; also, fewer laboratory animals are required in this approach which is in agreement with 3 R Principle.
小鼠中和试验广泛用于测定抗狂犬病抗体水平，但该试验劳动强度大且耗时。已知在细胞培养中测定抗狂犬病血清和免疫球蛋白中和活性的替代方法。如快速荧光灶抑制试验（FAVN）和快速荧光焦点抑制试验（RFFIT）等方法涉及使用荧光诊断。由于狂犬病病毒在细胞中的复制特性，细胞病变效应（CPE）的测定往往很复杂。原子力显微镜（AFM）能够在早期检测病毒与细胞的相互作用。因此，在本研究中，开发了一种利用细胞培养的原子力显微镜测定抗狂犬病血清和免疫球蛋白比活性的方法。
该方法基于狂犬病病毒与狂犬病血清样本或免疫球蛋白药物的初步相互作用，将特定反应混合物加入细胞培养物（Vero或BHK - 21）中，然后使用原子力显微镜测量细胞的表面粗糙度。原子力显微镜通过半接触模式下的失配法以间歇接触模式进行。将结果与小鼠中和试验中获得的值进行比较。通过Bland - Altman方法评估两种方法所得结果的一致性。
细胞表面粗糙度的增加是病毒破坏作用的结果，而这种破坏作用会因狂犬病抗体的中和作用而减弱。选择能抑制细胞表面粗糙度增加50%的稀释度作为狂犬病血清或免疫球蛋白的效价。在这种情况下，测定抗体效价的推荐范围是1:100至1:3000。
本研究首次在病毒学和药物研究中提出了一种新的方法学途径。与小鼠中和试验相比采用所提出的方法技术将使获得结果的时间从21天缩短至2天；此外，该方法所需实验动物较少，符合3R原则。
