sensors Article Lateral Flow Serodiagnosis in the Double-Antigen Sandwich Format: Theoretical Consideration and Confirmation of Advantages Dmitriy V. Sotnikov *, Anatoly V. Zherdev and Boris B. Dzantiev A.N. Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Leninsky Prospect 33, 119071 Moscow, Russia; [email protected] (A.V.Z.); [email protected] (B.B.D.) * Correspondence: [email protected]; Tel.: +7-495-9543142 Abstract: Determination of the presence in the blood of antibodies specific to the causative agent of a particular disease (serodiagnosis) is an effective approach in medical analytical chemistry. Sero- diagnostics performed in the lateral flow immunoassay format (immunochromatography) meet the modern requirements for point-of-care testing and are supported by existing technologies of large-scale diagnostic tests production, thus increasing the amount of attention in a tense epidemio- logical situation. For traditional lateral flow serodiagnostics formats, a large number of nonspecific immunoglobulins in the sample significantly reduces the degree of detectable binding. To overcome these limitations, an assay based on the formation of immobilized antigen-specific antibody-labeled antigen complexes detection was proposed. However, the requirements for its implementation, providing maximum sensitivity, have not been established. This article describes the mathematical model for the above assay. The influence of the ratio of reagent concentrations on the analysis results is considered. It is noted that the formation of specific antibody complexes with several labeled antigens is the main limiting factor in reducing the detection limit, and methods are proposed to minimize this factor. Recommendations for the choice of the assay conditions, following from the analysis of the model, are confirmed experimentally. Citation: Sotnikov, D.V.; Zherdev, A.V.; Keywords: point-of-care assay; membrane tests; immunochromatography; lateral flow immunoassay; Dzantiev, B.B. Lateral Flow Serodiagnosis immune response; detection of antibodies; antigen-antibody reactions; mathematical modelling; in the Double-Antigen Sandwich assay sensitivity; limit of detection Format: Theoretical Consideration and Confirmation of Advantages. Sensors 2021, 21, 39. https://dx.doi.org/10.3390/ s21010039 1. Introduction Received: 19 November 2020 The presence of antibodies specific to the causative agent of a certain disease in Accepted: 16 December 2020 the blood (serodiagnosis) indicates the contact between the organism and the pathogen, Published: 23 December 2020 and, for many diseases, it is considered an effective diagnostic parameter [1–3]. The ad- vantages of such diagnostics in comparison with the detection and identification of the Publisher’s Note: MDPI stays neu- pathogen itself consist in the relatively simple sampling, the reproducible results that do not tral with regard to jurisdictional claims depend on the choice of the sampling site or that slowly change for the patient over time, in published maps and institutional and the excluded need to grow the pathogen until it reaches the detectable concentration. affiliations. Serodiagnostics is actively used for many widespread and socially significant infections, including tuberculosis, human immunodeficiency virus, and more [4–8]. Particular interest in this diagnostic method has arisen this year due to the coronavirus pandemic [9–11]. Copyright: © 2020 by the authors. Li- Serodiagnostics can be implemented in different formats; however, today two of censee MDPI, Basel, Switzerland. This them dominate in mass practice—microplate enzyme immunoassay and immunochromato- article is an open access article distributed graphic analysis—which correspond to their leading place in relation to immunodiagnostics under the terms and conditions of the in general [12–14]. At the same time, recent years have seen the accelerated development Creative Commons Attribution (CC BY) of point-of-care diagnostic tools, which make it possible to carry out methodically simple license (https://creativecommons.org/ testing directly at the sampling site and quickly obtain results [15–18]. This requirement is licenses/by/4.0/). fully met by immunochromatographic analysis (lateral flow immunoassay). All reagents Sensors 2021, 21, 39. https://dx.doi.org/10.3390/s21010039 https://www.mdpi.com/journal/sensors Sensors 2021, 21, x FOR PEER REVIEW 2 of 16 Sensors 2021, 21, 39 2 of 16 simple testing directly at the sampling site and quickly obtain results [15–18]. This require- ment is fully met by immunochromatographic analysis (lateral flow immunoassay). All requiredreagents forrequired the assay for arethe pre-applied assay are pre-applie to the testd strip to the (a compositetest strip (a of composite several membranes). of several Contactmembranes). of the Contact sample withof the this sample test strip with initiates this test the strip movement initiates of the liquid movement along the of poresliquid ofalong the membranesthe pores of andthe membranes analytical reactions and analytical with the reactions applied with reagents, the applied including reagents, reagents in- containingcluding reagents a colored containing label. Most a colored often, label. nanodispersed Most often, gold nanodispersed particles are gold used particles as such are a labelused dueas such to their a label intense due color,to their ease intense of preparation, color, ease and of preparation, modification and [19– 21modification]. As a result [19– of these21]. As processes, a result of labeled these coloredprocesses, immune labeled complexes colored immune are formed complexes in certain are zones formed of the in testcer- strip.tain zones A visual of the assessment test strip. of A the visual presence assessmen or absencet of the of presence coloration or of absence these zones of coloration allows us of tothese make zones conclusions allows us about to make the testconclu resultssions [22 about,23]. the test results [22,23]. ForFor serodiagnostics,serodiagnostics, several several formats formats are are possible, possible, differing differing in in the the location location and and labeling label- ofing immunoreagents. of immunoreagents. The The most most known known format format consists consists of immobilizing of immobilizing an antigen an antigen (specific (spe- forcific a for given a given pathogen) pathogen) in the in the analytical analytical zone zone and and labeling labeling an an immunoglobulin-binding immunoglobulin-binding reagent-anti-speciesreagent-anti-species antibodies,antibodies, bacterialbacterial immunoglobulin-bindingimmunoglobulin-binding proteinsproteins (protein(protein A,A, proteinprotein G),G), etc.etc. [[24–29].24–29]. TheThe presencepresence ofof specificspecific antibodiesantibodies toto thethe pathogenpathogen ininthe the sample sample leadsleads toto thethe formationformation ofof aa detectabledetectable complexcomplex ofof immobilizedimmobilized antigenantigen molecules,molecules, specificspecific antibodiesantibodies andand labeledlabeled immunoglobulin-bindingimmunoglobulin-binding proteinprotein (Figure(Figure1 A).1A). When When carrying carrying out out suchsuch serodiagnostics,serodiagnostics, a a reliable reliable difference difference in resultsin results is achieved is achieved for samplesfor samples containing containing and notand containing not containing specific specific antibodies. antibodies. However, However, the intensitythe intensity of the of the recorded recorded color color is often is of- low,ten low, which which prevents prevents a correct a correct assessment assessment of the of results the results obtained. obtained. The reason The reason for this for effect this iseffect that is the that labeled the labeled immunoglobulin-binding immunoglobulin-binding protein protein binds tobinds all immunoglobulins to all immunoglobulins in the testedin the bloodtested orblood serum, or serum, of which of onlywhich a fewonly percent a few percent are specific are specific to this pathogen to this pathogen [30,31]. The[30,31]. reaction The reaction of immunoglobulin-binding of immunoglobulin-bin proteinsding proteins with nonspecificwith nonspecific immunoglobulins immunoglob- reducesulins reduces the proportion the proportion of the of label the thatlabel is that capable is capable of participating of participating in the in formation the formation of a detectable complex of antigen-specific antibody-immunoglobulin-binding protein label of a detectable complex of antigen-specific antibody-immunoglobulin-binding protein la- when the fluid passes through the analytical zone. bel when the fluid passes through the analytical zone. FigureFigure 1. 1. TheThe three three serodiagnostic serodiagnostic immunochromatography immunochromatography formats: formats: (A) with labeled (A) with immuno- labeled immunoglobulin-bindingglobulin-binding protein and protein immobilized and immobilized antigen in antigen the analytical in the analytical zone; (B) zone; with (labeledB) with antigen labeled antigenand immobilized and immobilized immunoglobulin-binding immunoglobulin-binding protein proteinin the analytical in the analytical zone; (C zone;) with ( Clabeled) with labeledantigen antigenand immobilized and immobilized antigen antigenin the analytical in the analytical zone (see zone additional (see additional comments comments in the paper). in the paper). Sensors 2021, 21, 39 3 of 16 To overcome this limitation, an alternative immunochromatographic serodiagnos- tics format