Method for detecting antibody concentration and avidity for infectious diseases measured by evanescent field-based biosensor using continuous gradient of ligand density

Abstract

The core of the present invention concerns the invention of a method and device for plug-and-play of concentration (potency) and avidity parameters utilizing a continuous gradient of ligand density applied to a biosensor surface. Previous studies have investigated the humoral antibody response and strength of binding to specific SARS-CoV-2 proteins. Surface plasmon resonance imaging (SPRi) was used to measure the binding strength of IgG, IgM, and IgA to the receptor binding domain and nucleocapsid (RBD / NCP) of SARS-CoV-2 in the serum of 119 COVID-19 patients. Interestingly, in contrast to the antibody titer, the avidity (increasing off-rate) decreased with increasing severity. The present invention measures concentration and affinity / activity parameters in a plug-and-play manner in a single test. Ligand density is a key parameter that has a large impact on the value of the off-rate. A 10-fold mismatch is easily obtained. Not only the off-rate but also the on-rate at the immobilized ligand density needs to be measured, but in that case the effective concentration also needs to be known. The core of the invention is the application of a continuous gradient to the sensor surface to determine antibody titer and avidity, providing a constant but low R max Measure the avidity parameter with a high R value max The goal of the dual gradient method is to measure the concentration of antibodies at a single value. In this way, antibodies against the infection can be determined for each patient, especially to determine and predict the severity of COVID 19. The dual gradient method can be applied to measure two targets simultaneously.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 275,581, filed November 4, 2021, the entire contents of which are incorporated by reference herein to the extent permitted. [Background technology]

[0002] background FIELD OF THEINVENTION The present invention relates to a method for assessing the concentration and affinity / avidity parameters of label-free antibodies against infectious diseases using gradients of ligand density detected by surface plasmon resonance imaging. The quantity of antibodies generated against infectious diseases at high ligand density and the quality at low but immobilized ligand density can be determined for each patient, in particular to determine and predict the severity of COVID-19.

[0003] 2. Description of Related Art Nearly two years have passed since the start of the COVID-19 epidemic, but it is still unclear how humoral responses affect disease progression. We investigated the strength of humoral antibody responses to specific SARS-CoV2 proteins, their relationship with COVID severity and clinical information. For this purpose, we developed a new method to analyze the concentration and avidity parameters of label-free antibodies in a plug-and-play manner, taking advantage of the gradient of ligand density detected by surface plasmon resonance imaging. We used surface plasmon resonance imaging (SPRi) to measure specific antibodies of isotypes IgM, IgG and IgA, as well as the binding strength to the SARS-CoV2 antigens RBD (receptor binding domain) / NCP (nucleocapsid protein), NCP, S (spike)1 and S1S2 in the serum of 76 COVID-19 patients. A positive correlation was observed between severity and IgG antibody titers to all SARS-CoV2 proteins, as well as IgM and IgA antibody titers to RBD / NCP. Interestingly, in contrast to antibody titers, avidity decreased with increasing disease severity. In the severe patient group, a positive correlation with pulmonary embolism, d-dimer values, and antibody titers was observed. We hypothesized that antibody production and / or maturation may be ineffective in severe patients. This approach may be useful for COVID-19 severity prediction by measuring the quantity and quality of antibody responses using label-free biosensing technology. The present invention discloses a method to measure concentration and affinity parameters in a single test in plug-and-play mode in about 10 minutes. During the course of the disease, IgG levels and binding strength increased, while overall IgM and IgA levels decreased. All recovered patients show high avidity of IgG type for RBD / NCP proteins. The SPRi assay of anti-RBD / NCP immunoglobulins provides new insights into the immune status of patients who have recovered from COVID-19 and can be applied to evaluate immune responses of healthy individuals in vaccination programs. The results showed that at least two SARS-CoV-2 proteins (RBD and NCP) should be applied to measure polyclonal immune profiles.The strength of binding by off-rate values ​​correlated with the prediction of COVID-19 severity. The present invention applies the dissociation of bound antibodies simultaneously for all patients, not only for spike proteins (e.g., receptor binding domain (RBD / NCP), S1, S2, S1S2 and sub-epitopes), but also for highly immunogenic nucleocapsid proteins (NCPs) and epitopes derived from NCPs. In general, by applying strongly immunogenic epitopes of viruses, bacteria or other foreign invaders in this way, the quantity and quality of antibodies made against the infection can be determined for each patient, in particular to determine and predict the severity of COVID-19. This parameter, the ratio of the strength of binding and off-rates for at least two immunogenic proteins, was proven for the first time as an important indicator of the severity of COVID-19. However, although the off-rate may be sufficient to determine the binding strength, it is better to obtain a reliable indicator of the equilibrium dissociation constant by measuring the on-rate in addition to the off-rate. A new double-gradient method was applied to quantitatively and accurately measure the on-rate and off-rate. This method is described herein.

[0004] The coronavirus 2019 (COVID-19) pandemic has disrupted global societies, placed severe strain on health systems, and resulted in relatively high mortality and morbidity rates, with continuing high demands on patient care. Although a year and a half of intensive international scientific efforts have provided much information, much remains unknown about the underlying pathogenesis, the patient-specific factors that determine disease severity, and the protective or destructive role of the humoral immune system.

[0005] Indeed, there is evidence that the humoral response can have a detrimental effect on disease severity. Thus, there is a delicate balance between an overactive immune system, leading to organ / tissue damage and potential death, and a protective effect.

[0006] However, the factors that determine the balance between disease attenuation and disease amplification remain poorly understood. For example, most papers focus on single viral proteins, specifically spike (S) or nucleocapsid (NCP). Therefore, there is limited information on the kinetics of antibody responses to specific viral targets (e.g., receptor binding domain (RBD), S1, S2 or NCP) and the ratio of responses to these viral proteins. Furthermore, several studies are inconsistent with each other regarding the longitudinal trends of antibody production and titers in mild versus severe disease. Such limitations and conflicting results are partly due to the lack of consistency of comparison groups, study design, heterogeneity of the assays used, and indirect antibody measurements. Antibody measurements are generally performed using ELISA or related immunoassays. In addition to the relatively long measurement times, testing for IgM, IgG and IgA isotopes requires them to be measured separately by either technique. Moreover, standard immunoassays only provide indirect information on antibody kinetics and affinity.

[0007] An attractive alternative is surface plasmon resonance imaging (SPRi). In previous work, we have demonstrated a high-throughput SPRi assay that quantitatively measures IgM, IgG and IgA antibodies and their apparent polyclonal affinities in the serum of COVID-19 patients in a single test within 30 minutes. This method is ideal for measuring the concentration of antibodies in patients as well as measuring the binding strength of antibodies using off-rate detection.

[0008] The immune response to the coronavirus generally provides immunity via neutralizing antibodies when exposed to the virus a second time. However, the ability to produce neutralizing antibodies alone is insufficient to predict the severity of COVID-19. To measure the immune response to these proteins, at least two highly immunogenic viral proteins should be used. Serological antibody testing is essential to know whether one has been infected with SARS-CoV-2. The quality of the immune response is determined not only by the amount of antibodies but also by the overall binding strength of the neutralizing antibody pool that binds to the immunogenic proteins of the coronavirus. Recently, the most potent and affinity-neutralizing antibodies have been shown to target the receptor-binding domain (RBD). The RBD of this SARS-CoV-2 domain binds to the angiotensin-converting enzyme 2 (ACE2) receptor expressed on target cells. The affinity of the RBDs of SARS-CoV and SARS-CoV-2 for the ACE2 receptor was approximately 10 nM K D value as determined by surface plasmon resonance. Therefore, the quality of antibodies against SARS-CoV-2 must be more than six times better than those against SARS-CoV to prevent the virus from binding to the receptor. Generally, antibodies in this range effectively block the virus monovalently. It is necessary to additionally generate, if possible, high-affinity (<nM) polyclonal supporting antibodies that inhibit the binding of the RBD of SARS-CoV-2 to the ACE2 receptor and can remove the virus via domains such as the nucleocapsid domain. If the affinity of these antibodies is low, there is a dominant Fc response and the immune system is overactivated.

[0009] An attractive alternative for antibody detection is photonic devices based on evanescent fields, for example photonic sensors such as surface plasmon resonance imaging (SPRi). SPRi is a label-free sensing technology that is highly sensitive and can quantitatively and qualitatively measure interactions between biomolecules, such as the interaction of antibodies with their respective antigens. More importantly, the strength of binding, as measured by the off-rate, can be measured in a single assay, giving an indication of the quality of the total polyclonal antibody response. The discrimination and ratio between at least two highly immunogenic proteins from SARS-CoV-2 predicts the severity of COVID-19 as described herein. Summary of the Invention

[0010] Summary of the Invention We describe a high-throughput surface plasmon resonance imaging (SPRi) assay to quantitatively measure IgG, IgM and IgA antibodies and their apparent polyclonal affinities that bind to the RBD spike protein and nucleotide capsid protein (NCP) in the serum of COVID-19 patients. At low concentrations of specific antibodies, specific interactions interfere with nonspecific interactions, resulting in false positive results. However, we have found that the avidity of specific biomolecular interactions is often stronger than nonspecific interactions. The core of the present invention is to predict disease severity by measuring the ratio of the levels and affinities of at least two immunogenic proteins. In addition to this core of the present invention, simultaneous but partial elution of antibodies bound to specific antigens can easily reveal the difference in off-rates between antibodies bound to immunogenic proteins and nonspecifically bound antibodies. This not only reveals the strength of binding of three isotype antibodies (M, G, A) to at least two immunogenic proteins of the virus, but also reduces the number of false positives.

[0011] The core of the present invention concerns a plug-and-play approach to concentration (potency) and avidity parameters using a continuous gradient of ligand density applied to the biosensor surface. Previous studies have investigated the humoral antibody response and binding strength to specific SARS-CoV2 proteins. Surface plasmon resonance imaging (SPRi) was used to measure the binding strength of IgG, IgM and IgA to the receptor binding domain and nucleocapsid (RBD / NCP) of SARS-CoV-2 in the serum of 119 COVID-19 patients. Interestingly, in contrast to the antibody titer, the avidity (increasing off-rate) decreased with increasing severity. The present invention provides a method to measure concentration and affinity / ability parameters in a single test in a plug-and-play approach, e.g. in 10 minutes. According to well-known observations, ligand density is a key parameter that has a large impact on the off-rate value. A factor 10 mismatch is easily obtained. It is necessary to measure not only the off-rate but also the on-rate at the immobilized ligand density, where the effective concentration must also be known. The core of the invention is the application of a continuous gradient to the sensor surface to determine the antibody titer and avidity, resulting in a constant but low R max Measure the avidity parameter with a high R value max The goal of the method is to measure the concentration of antibodies at a concentration of 0.01 mg / kg / dL. In this way, the quantity (in the case of high ligand density) and quality (in the case of low but immobilized ligand density) of produced antibodies against the infection can be determined for each patient, and in particular the severity of COVID-19 can be judged and predicted. The dual gradient method can be applied to measure two targets simultaneously.

[0012] In one aspect, the present invention provides a method for predicting the severity of an infectious disease by a combination of specific antibody concentration and binding strength of total antibody isotypes from a bodily fluid sample taken from a patient, the method comprising the steps of: exposing a bodily fluid sample from a patient with an infectious disease to an immunogenic antigen immobilized in a gradient on a label-free and real-time imaging biosensor; determining the concentration at high ligand density using the initial slope of the binding curve under mass transport limited conditions; and determining the binding strength at low but immobilized R on the ligand density gradient. max Determining an avidity parameter under conditions, wherein a ratio of the levels and affinities of at least two immunogenic proteins predicts the severity of infection in a patient.

[0013] In some embodiments, the label-free, real-time biosensor is based on any evanescent field optical phenomenon. In some embodiments, the evanescent field-based biosensor is an optical device based on surface plasmon resonance (SPR) imaging.

[0014] In some methods according to the invention, controlled injection of ligand utilizes at least one double flow of the sample through a flow path in contact with the sensor surface to create a gradient of ligand density due to differences in contact time.

[0015] While this method may be a convenient way to create the required ligand gradient, for example to perform this online in a device, there are other ways that the gradient may be created. For example, to create sensors for point-of-care applications, the ligand gradient may be created using other fabrication methods. For example, the gradient may be created offline in the fabrication environment by using a depletion zone or by immobilizing the ligand using a photoactivated coating. Then, by exposure to high intensity light, a high ligand density occurs, and gradually lower intensity light results in a very low ligand density. Thus, in some embodiments, a sensor coating is applied to create one or more gradients, using a gradual depletion zone, or by applying a photoactivated coating and gradually exposing it to light, or by any other means, to create either a dynamic or static gradient in the ligand density on the sensor.

[0016] In one example, the fluidics are designed to allow samples to simultaneously fit into at least two or more channels in a gradient to simultaneously measure concentration and binding and dissociation rates for at least duplicate measurements of the same sample.

[0017] The method of the present invention can be used when the infectious disease is COVID-19. In some examples, the immobilized immunogenic antigen is at least the receptor binding domain and nucleocapsid (NCP) of SARS-CoV-2. In such examples, the nucleocapsid antibody binding constant and the RBD binding constant can predict the severity of the infectious disease in a patient.

[0018] In some methods according to the invention, the sensor is rotated 180 degrees to apply half of the flow cell with timed exposure of ligand from inlet to outlet to create a gradient of ligand density for a first immunogenic protein, and while the first gradient is in the down section, the top section is timed to expose from inlet to outlet of the flow cell to allow subsequent immobilization of a second protein, thereby configuring the sensor with a multiplex flow cell for simultaneously measuring the strength of binding to at least two immunogenic proteins and the isotype of the bound antibodies.

[0019] In one embodiment, the immobilized R max The measurement of biomolecular interactions on the gradient to obtain values ​​is performed by measuring the immobilized R for all ligand gradients while simultaneously measuring the concentration at other positions in the gradient. max These include the analysis of on-rates and off-rates using sensorgrams with values. In such cases, biomolecular interactions on the gradient can be exposed to a co-injection of an anti-isotype antibody.

[0020] Several methods according to the invention can be utilized to establish the strength of binding of antibody isotypes using SPR imaging to reduce the number of false positives characterized by a gradient print of ligand exposed to patient samples.

[0021] In another aspect, the invention provides an apparatus that includes an SPR imager and a line printer for forming a gradient in a track, where a portion of the printed line or track is exposed to a patient sample.

[0022] In some embodiments of the apparatus of the present invention, a single channel is applied over the printed track and the sensor prism is repositioned to cover a portion of the track.

[0023] In another embodiment of the device of the invention, a single channel is applied over the printed track and the flow cell is repositioned to cover a portion of the track.

[0024] In a further embodiment, by swapping the position of the sensor prism or flow cell, a criss-cross track is applied to create a ligand density gradient.

[0025] The present invention is described in further detail below by reference to certain preferred embodiments, which are provided by way of example only. [Brief description of the drawings]

[0026] [Figure 1] Justification for gradient plug-and-play parameter analysis. Measurement times are approximately 1 h with current standard procedures (Panel A) versus approximately 5 min with the present invention (Panel B). [Diagram 2] Representative images of using contact time differentials to create a gradient on the sensor prism. (Panel A) Ligand is immobilized on the right side more than the left. The ligand flows in one direction and back again, allowing for contact time differentials. (Panel B) contains representative images A, B, and C showing the contact time gradient within the flow cell. [Diagram 3] Three regimes of gradients for measuring biomolecular interactions are presented. [Figure 4] Representative images of two gradients generated in opposite directions. [Diagram 5] Image of the sensor prism holder for printing the double gradient. (A) The sensor holder is shown with the positioning cross (B) and bayonet closure (C). [Figure 6]Principle of anti-SARS-CoV-2 immunoglobulin isotype SPRi assay. SPRi reflectance images (C) were obtained during spotting of serum (A) onto the RBD and NCP binding surface of the MX96 SPRi instrument (B). In (D) sensorgrams are shown for injection of three antisera to measure IgM, IgG, and IgA antibody responses. In panel (E) an overlay of injection of anti-IgM, IgA, and IgG antibodies of one spotted serum is shown to calculate the Rmax values ​​of IgM, IgG, and IgA binding. [Figure 7] Infographic outlining the test design to determine the presence or absence of SARS-CoV-2 antibody responses in patient samples using SPRi. A) IgM, IgG and IgA antibody responses to RBD, NCP, S1S2 and S2 proteins are measured sequentially using SPRi. Patient plasma is first incubated on a specific protein-binding sensor, then anti-IgM, anti-IgG and anti-IgA are injected sequentially and the associated signals are measured in real time. B) The affinity of a patient polyclonal antibody pool to RBD, NCP, S1S2 and S2 proteins was measured. Patient plasma is injected onto the protein-binding sensor and the interaction is measured in real time. The koff constant determines the strength of binding and is determined during the dissociation phase. [Figure 8] The dissociation constant of anti-RBD antibodies is shown as a function of days since onset. The central trend line is the overall line for all samples measured. The other lines are the duplicates of the four longitudinal samples. This principle is the classical method to measure the total affinity. [Figure 9] In moderate and severe patients, anti-NCP antibodies have lower avidity (higher dissociation rate) than anti-RBD antibodies. Data taken from Hendriks et al. Association and dissociation rates are measured label-free and in real time using any kind of photonic device. [Figure 10]Multiplexed measurements of antibody responses against four antigens of SARS-CoV-2. A) Total immune response (IgM, IgG, IgA) SPRi measurements of COVID-19 positive sera against RBD / NCP antigens. B) IgG immune response SPRi measurements of NCP, S1S2 and S2 antigens. Box plots represent median, p25 and p75 values, black dots represent mean SPRi RU. Comparability of each group was analyzed by Mann-Whitney U-test. The Bonferroni-Holm method was used to correct for multiple comparisons between groups (*p < 0.05 and **p < 0.01 vs. mild group, #p < 0.05 vs. moderate group). [Figure 11] Measurement of binding strength of four SARS-CoV-2 antigens. Off-rates of antibodies binding to the four antigens were measured for each severity group. Box plots represent median, p25 and p75 values, and black dots represent mean SPRi RU. Comparability of each group was analyzed by Mann-Whitney U-test. Multiple comparisons between groups were corrected using the Bonferroni-Holm method (*p<0.05 and **p<0.01 vs. mild group, #p<0.05 and #p<0.01 vs. moderate group by Mann-Whitney U test). [Figure 12] Correlation between binding strength and gender. Boxplots on the left for severe patients and on the right for moderate patients. As can be seen from the figure, there is a significant difference between females and males in the severe RBD patient group (p=0.043). The others were not significant. Boxplots represent the median, p25 and p75 values, and black dots represent the mean SPRi RU (*p < 0.05: Mann-Whitney U test). [Figure 13] COVID-19 survivors or COVID-19 deceased patients show different immune quality profiles as shown in this sketch. It is still unclear why the human body mounts such a diverse polyclonal immune response to SARS-CoV-2. Therefore, immune fingerprints such as antibody levels and binding strength should be tested as key parameters to predict the severity of COVID-19. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Detailed Description of the Invention This invention was supported in part by the ATTRACT project under grant 777222 from the European Council (EC) and by the European Regional Development Funded grant 10.13039 / 5011000008530.

[0028] The study was carried out in accordance with the guidelines for patient data sharing in surveillance scientific studies in emergency situations published by the Code of Conduct Committee of the Dutch Medical Scientific Association ( https: / / www.federa.org / federa-english ).

[0029] As described herein, we describe a high-throughput surface plasmon resonance imaging (SPRi) assay to quantitatively measure IgG, IgM and IgA antibodies and their apparent polyclonal affinities that bind to the RBD spike protein and nucleocapsid protein (NCP) in the serum of COVID-19 patients. At low concentrations of specific antibodies, specific interactions interfere with non-specific interactions, resulting in false positive results. However, it has been found that the avidity of specific biomolecular interactions is often stronger than non-specific interactions. The core of the invention is to predict disease severity by measuring the ratio of the levels and affinities of at least two immunogenic proteins. In addition to this core of the invention, simultaneous but partial elution of antibodies bound to specific antigens can easily reveal the difference in off-rates between antibodies bound to immunogenic proteins and non-specifically bound antibodies. This not only reveals the strength of binding of three isotype antibodies (M, G, A) to at least two immunogenic proteins of the virus, but also reduces the number of false positives.

[0030] The need for gradient plug-and-play mode parameter analysis is illustrated in Figure 1. Figure 1 Panel A shows the current standard procedure, with a measurement time of about 1 hour including regeneration. Overlay plots of eight analytes injected in serial dilutions have the same ligand density in all curves, but the initial slopes are different. As a result, the curvature ligand density becomes too high, and the wrong kinetic constants are measured. The present invention provides a measurement time of about 5 minutes without regeneration (Figure 1 Panel B). From a single analyte injection, the initial slopes of all curves are the same, while the ligand densities are different. Thus, the ligand density is not high. The evaluation requires R max = 50RU. If we analyze the kinetic parameters in this way, one measurement is sufficient.

[0031] Thus, the present invention provides methods and devices for evaluating the concentration and avidity detection of antibodies to infectious diseases measured with an evanescent field-based biosensor using a continuous gradient of ligand density. EXAMPLES

[0032] Surface Plasmon Resonance Imaging and Spotting Apparatus The IBIS MX96 instrument (IBIS Technologies, Enschede, The Netherlands) provides continuous sample injection in a valveless manner. The "back-and-forth" flow-based flow path allows unlimited interaction times with 100 microliter samples. Furthermore, the MX96 applies so-called reciprocal pumping during the dissociation step, keeping the concentration of dissociating analyte at zero, thereby reducing rebinding effects. The continuous flow microspotter (CFM, Wasatch Microfluidics, Salt Lake City, Utah, USA) allows reliable printing of ligand molecules under flow conditions. SensEye (登録商標)Sensors (gel type E2S, Ssens, Enschede, The Netherlands) were printed with an array of ligand samples using pre-activated surface chemistry or with streptavidin-coated sensors. This device allows multiplexed interaction kinetics analysis of up to 96 spots. The method of the present invention for RBD and NCP is not applicable to the MX96 device because the sensor applies a hemisphere and cannot be optically replaced to create a double gradient on the same sensor using a single flow cell. It also works with MX96 if a double flow cell is applied in the MX96 system or if a movable flow cell is applied. However, in a preferred embodiment of the present invention, there is an additional advantage because the sensor prism is replaced instead of the flow cell and the position of the flow cell remains the same.

[0033] Thus, specific aspects of some embodiments of the present invention are as follows: As shown in FIG. 2, panel A, a contact time difference, where the ligand is immobilized on the right rather than the left, can be used to create a gradient on the sensor prism. The ligand flows from one direction and back again, allowing for a contact time difference. FIG. 2, panel B, shows a contact time gradient in a flow cell. In panel B(A), an SPR image is shown when the ligand is injected through an inlet on the right side of the flow cell. The sensor surface is exposed to the ligand solution entering the flow cell. On the right side of (A), the resonance conditions are changed during the injection. The injection of the ligand solution at a later stage is depicted in panel B(B). The ligand solution flows slowly from right to left. Panel B(C) is similar to (B), but almost at the edge of the flow cell. In this way, a gradient of ligand density is created by the contact time difference from the inlet to the outlet. The ligand density is maximum at the inlet and zero at the outlet.

[0034] There are three regimes for measuring biomolecular interactions in gradients (see Figure 3). Regime (1) has a low R maxThe regimes are kinetically controlled regimes with affinity parameters fixed at . Regime (2) is an intermediate regime, and regime (3) is a mass transport limited (MTL) regime at high ligand density. Here, in the middle of the gradient, abundant measurements are generated, and the software uses the R of the squares “k” to measure the kinetic parameters. max = 50 to find suitable conditions. The software finds position “k” on the gradient by analyzing multiple interactions on the gradient. In square “r” there is no ligand density, which is used as a reference. Square “c” is where the concentration is measured under MTL conditions using the initial gradient after injecting the analyte into the flow cell. The analyte is exposed to all regions at once.

[0035] Figure 4 shows a representative diagram for generating two gradients in opposite directions and a blend of the two middle gradients. (A) The first gradient is created at 1 / 3 of the width of the flow cell, off center of the sensor. (B) The sensor is rotated 180 degrees and the sensor is moved down 2 / 3 of the flow cell width and exposed to ligand 1 for different contact times. Ligand 2 is exposed for different constant times. (C) Finally, the sensor holder is moved to the middle position and the flow cell is placed in three sections.

[0036] A further embodiment is shown in Figure 5. The holder for the sensor prism for printing the double gradient is shown in a top perspective view (A) together with a positioning cross (B) for changing the position of the sensor in the light beam. A bayonet closure (C) is used to mount the sensor on the instrument.

[0037] Multiplex assays against SARS-CoV-2 antigens to determine polyclonal affinity The interaction affinity of antibodies with SARS-CoV-2 antigens was determined as described above (Figure 10). Briefly, patient serum was injected under flow onto the multiplex-coated SARS-CoV-2 antigen sensor with a 3 min binding and 1 min dissociation time. To determine the affinity constant of the polyclonal antibody pool, a 1:1 Langmuir interaction model is applied. As the concentration of polyclonal antibodies in serum is unknown and the binding curve does not show monophasic behavior, the equilibrium dissociation constant (apparent K D The exact value of the equilibrium dissociation constant (K D ) and off-rate (k d In our assay, after 30 seconds of the dissociation step, the dissociation constant (k d , s -1 ) can be determined because by dividing the slope by the response, the ligand density (RU) can be accurately measured. Measuring only the off-rate as an indicator of binding strength is probably an oversimplification, since ligand density has a large effect. Measuring the on-rate and off-rate and comparing the concentration at sites of high ligand density with that at low but fixed R max Value (e.g., R max It is better to use a gradient to measure both the kinetic values ​​at 50 RU (50 RU) and this is the core of the invention described herein.

[0038] Sensor Preparation For SPRi measurements, a multiplex SPR imaging instrument (IBIS MX96, IBIS Technologies, Enschede, the Netherlands) and a Carterra LSA platform (Salt Lake City UT, US) were used with a sensor prism (HC30M, Xantec Bioanalytics Duesseldorf, Germany), but any evanescent field-based biosensor technology can be applied to practice the present invention. Similar results were obtained with both instruments on this sensor surface. The sensor was prepared by first stabilizing and removing the protective layer in water, followed by treatment with a 1:1 aqueous solution of 100 mM N-hydroxysuccinimide (NHS) and 400 mM N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) for 10 min. After rinsing with water for 20 s, the sensors were exposed to spiked RBD / NCP His-tag (SINO biological Frankfurt, Germany) or His-tag N-protein (NCP) (SINO biological Frankfurt, Germany) in immobilization buffer (50 mM sodium acetate pH 4.8) for 20 min. Coupling with EDC-NHS resulted in reproducible sensor surfaces. After rinsing the sensors with water for 20 s, the surfaces were passivated with 1 M ethanolamine (pH 8.5) for 10 min. The sensors were then equilibrated with a running buffer consisting of PBS (137 mM NaCl, 10 mM phosphate, 2.7 mM KCl, pH 7.4) supplemented with 1% bovine serum albumin (BSA), 0.5% casein, and 0.1% Tween 20.

[0039] Spotting serum The dilution of serum samples was constant with an optimized dilution ratio of 1:100. 2 μl of each serum was diluted with 198 μl of running buffer and pipetted into a 96-well plate. For the IBIS MX96 measurements, a continuous-flow microfluidic (CFM) system (Carterra Salt Lake City, UT, US) was used to capture 96 sera onto the RBD / NCP-Spike-coupled sensors. The top of the flow cell can be RBD and the bottom NCP, or vice versa. The first 48 samples were spotted in duplicates for 3 min on the top (RBD) section and then 3 min on the bottom (NCP) section. The Carterra LSA can print 384 spots as 4 nested positions of 96 each. Here, the sensor can be immobilized with RBD on the top of the sensing area and NCP on the bottom. The top and bottom rows are supplied with the same serum. Although the operation and sample injection of both instruments are similar, the LSA allows the spotting process and dissociation rate to be tracked in real time. However, for both IBIS MX96 and Carterra LSA, only the dissociation rate can be determined, but the complete affinity data including on-rate and equilibrium dissociation constant cannot be determined. If the ligand density is not optimally adjusted, deviations in the off-rate will occur. This can be avoided by applying a gradient of ligand density, as described herein.

[0040] Measurements with IBIS MX96 The SPRI MX96 SUIT (Set Up Ibis Tool), DAX (data acquisition software) and SPRINTX (analysis software) software packages were used. After washing the sensor chip with two separate injections of patient serum from the top (RBD) and bottom (NCP), the chip was first incubated with 50-fold diluted goat anti-human IgM (aIgM approx. 4 mg / ml, 20-S5170 GND1-D0 Fitzgerald) in running buffer (200 μl per injection), followed by 100-fold diluted goat anti-human IgG (aIgG-Fc approx. 8 mg / ml, 20-S1211G001-S4 Fitzgerald) in SPRi running buffer. The third injection was performed with 100-fold diluted goat anti-human IgA (aIgA approx. 7 mg / ml, 20-S1111G000-S4 Fitzgerald). Data were transformed by local referencing, baseline zeroed, and injection points aligned for the three injections, then fitted in R using a special biphasic fit algorithm (InterFluidics, Haaksbergen, The Netherlands). max The values ​​were determined. This software tool, programmed with Microsoft 'R' Studio, allows calculation of the data from both SPR imagers. If the curves did not show exponential behavior (such as negative samples), a linear fit was applied and the average value of the linear fit was determined.

[0041] LSA off-rate measurements A total of 48 serum samples were spotted in duplicate in one run on the sensor prism surface with HC30M RBD and NCP at four dilutions (1:50, 1:100, 1:200, 1:400). During spotting, the binding signal was followed for 15 min and each serum sample was measured 8 times at four dilutions. The signal recorded in RU reflects the total bound anti-RBD / NCP antibodies. After spotting, RBD-NCP (15 μg / ml) in dilution buffer was injected for 5 min, which sufficiently dissociated the anti-RBD and anti-NCP antibodies. The global dissociation constant or global off-rate constant can be calculated for all 384 spots. In the final step, solutions of anti-IgM, anti-IgG and anti-IgA antibodies are injected sequentially. The ratio of bound immunoglobulins with respect to RBD and NCP was calculated from the binding signal of anti-isotype antibodies by the RU. max It can be calculated by determining the value of R max The value is directly related to the density of captured ligand and is directly related to the concentration of anti-RBD and anti-NCP antibodies in the serum.

[0042] There is an urgent need for effective methods to predict the course of COVID-19 disease. More recently, non-neutralizing antibodies against a 21-residue epitope in the nucleocapsid (called Ep9) have been found to be associated with severe disease, including admission to the intensive care unit (ICU), need for mechanical ventilation, or even death. Importantly, anti-Ep9 antibodies can be detected within 6 days of onset, and in some cases within 1 day, using an enzyme-linked immunosorbent assay (ELISA) with a sensitivity comparable to that of SPRi. Furthermore, anti-Ep9 antibodies correlate with various complications and features of immune enhancement and sepsis. Thus, high titers of low-affinity antibodies in COVID-19 patients are associated with disease severity.

[0043] result Simultaneous measurement of anti-SARS-CoV-2 IgM, IgG and IgA antibodies in 48 samples by SPRi for RBD and NCP Figure 6 shows the principle of the SPRi assay to determine isotype fractions. In panel A, 96 sera are spotted onto the RBD / NCP-conjugated sensor. In panel B, the sensor is placed on an SPR imager and real-time measurements are performed upon three consecutive injections of anti-IgM, anti-IgG, and anti-IgA. Panel C shows the SPR reflectance image after anti-isotype antibody injection of 96 sera. Panel D shows a typical sensorgram of the three sera. The first curve shows the highest IgM (R max Panel A shows a serum with low IgM (1940 RU), very high IgG (5012 RU), and weak IgA (243 RU). The third curve shows a serum with moderate IgM (845 RU), moderate IgG (1215 RU), and weak IgA (464 RU). The second curve shows a serum with weak IgM (203 RU), high IgG (3950 RU), and high IgA (3796 RU). Panel B shows the R of anti-IgM, anti-IgG, and anti-IgA. max Patient overlay to calculate values ​​proportional to bound IgM, IgG, IgA anti-RBD / NCP. Baseline is zeroed and injections are aligned. This allows for R of the three isotypes. max To calculate , a biphasic binding model can be applied.

[0044] Characterizing the immune response in COVID-19 patients using SPRi To investigate the immune response of COVID-19 patients, SPRi technology was used as described above. Briefly, the binding of IgM, IgG and IgA to specific SARS-CoV-2 proteins (RBD / NCP, NCP, S1S2 and S2) was measured in real time (Figure 7A), and the affinity of a polyclonal antibody pool to each protein was measured to assess the strength of binding (Figure 7B). Furthermore, a peptide library was used to investigate the binding of patient antibodies to specific epitopes on the RBD / NCP proteins (Figure 7C).

[0045] Sensorgrams were measured simultaneously for all samples and are shown as overlay plots in Figure 7. Repeated measurements using the same serum yielded RU levels with less than 5% variation.

[0046] Anti-SARS-CoV-2 spike RBD / NCP IgG, IgA and IgM binding strength measurements The density of the ligand may cause rebinding when the analyte dissociates. Rebinding results in overestimation of the off-rate values ​​or stronger binding. To reduce the rebinding effect of dissociated molecules, free RBD / NCP was added to the running buffer at a concentration of 15 μg / ml. At 5 min, a mixture of degrees of dissociation was observed for the various samples and longitudinal samples (see Supplementary Figure 7), and the dissociation or off-rate constants could be calculated and plotted as a function of symptom onset days (see Figure 8). During disease onset, a smaller off-rate was observed indicating improved antibody avidity or quality. That is, patients produce a repertoire of better quality anti-RBD / NCP polyclonal antibodies over time. This trend in off-rates is observed for all longitudinal samples (better binding strength). A paradox exists that only the weakest binding antibodies dissociate first from the sensor surface. Weakly binding antibodies block RBD but have a reduced neutralizing effect. When applying a gradient of ligand density in this type of measurement, the addition of free RBD / NCP is not necessary because the software always runs R max = 50 to find suitable conditions for measuring the kinetic parameters on-rate and off-rate. The on-rate requires a concentration parameter, which is measured under mass transport limited conditions at high ligand density on the same gradient.

[0047] SARS-CoV-2 binds more tightly to the ACE2 receptor than SARS-CoV. This means that antibodies need high affinity to compete and neutralize the virus. Our method of profiling immunity in terms of isotype concentration and strength of binding can reveal this effect in a high-throughput manner. Low concentrations of high avidity anti-RBD antibodies may be more effective at neutralizing SARS-CoV-2 than high concentrations of low affinity anti-RBD antibodies. Anti-NCP antibodies do not neutralize the virus and can be used as an indicator of infection in vaccinated individuals who express only anti-RBD antibodies.

[0048] From previous studies, it is clear that the binding of antibodies from different patients varies significantly, a parameter that, to our knowledge, has never been applied in a clinical test for specific antibody monitoring and COVID-19 severity prediction.

[0049] This result is the basis for a new type of COVID-19 prognostic biomarker that allows early identification and triage of high-risk patients, in addition to the complete NCP protein and S-RBD, using label-free sensing techniques such as SPR. Such information, including avidity, leads to effective therapeutic intervention. Patients with mild symptoms show low titers, but anti-RBD and anti-NCP can be easily detected. In our initial study, we were not able to measure vaccinated individuals, but many papers have shown that anti-NCP antibodies are absent in the serum of vaccinated individuals without SARS-CoV-2 infection. As for the ratio of anti-RBD and anti-NCP antibodies, a correlation with severity has been shown in our latest paper, but it is clear that vaccinated and non-vaccinated individuals (seropositive and seronegative) can be clearly distinguished. Furthermore, anti-RBD values ​​+ anti-NCP values ​​correlate well with the severity of seropositive patients, and anti-RBD values ​​of vaccinated individuals (negative for NCP) indicate the immune status of COVID-19. Correlating epidemiological studies of vaccinated and seropositive patients are not required, but rather validation of patient data by conventional serological tests (eg, ELISA).

[0050] Total immune responses to SARS-CoV-2 antigens by patient subgroups Multiplex SPRi measurements of four SARS-CoV-2 antigens were used to measure the total immune response (IgM, IgG, IgA) per patient. Analytical sensitivity was sufficient for IgA, IgM and IgG responses to RBD, and IgG responses to NCP, S1S2 and S2 proteins, but not for IgA and IgM responses to these antigens. Figure 9 shows immune responses classified by severity: mild, moderate or severe / critical. Patients with moderate or severe disease had significantly higher IgM, IgG and IgA responses to RBD and significantly higher IgG responses to NCP, S1S2 and S2 than patients with mild disease. Furthermore, patients with severe disease had significantly higher RBD-IgM, RBD-IgG and S1S2-IgG than patients with moderate disease. Thus, increasing disease severity was positively correlated with increasing IgG antibody titers against each antigen, as well as increasing IgM and IgA antibody titers.

[0051] Antibody binding strength Off rate (k d ) was determined to rank the avidity of the polyclonal antibody pool reacting with each antigen. d The higher the equilibrium dissociation constant (K D ) and therefore lower affinity. Figure 8 shows that with increasing severity, k d The results show that there is an increase in the affinity of antibodies to S1S2 and S2, which is significant in severe vs. mild disease for RBD and NCP, and in severe vs. moderate disease for S1S2 and S2, suggesting a decrease in maturation to higher affinity antibodies. The exception to this is that measured antibody affinity to S2 is lower in mild disease compared to moderate and severe disease, and antibody affinity to S1S2 is also not as high (see Figure 11).

[0052] Immune response, sex and avidity Already at the beginning of the COVID-19 outbreak, it was observed that men were at higher risk of worsening symptoms and death, regardless of age. When comparing the immune responses of men (n = 42) and women (n = 28), no differences were found in the amount of antibodies (Fig. S12), antibody specificity, and antibody avidity (data not shown). Only in the moderate group, the anti-RBD / NCP antibody pool in men showed significantly lower avidity than in women (Fig. S22).

[0053] In a later study, we compared data from severely and moderately diseased patients and observed that binding strength to all antigens decreased with increasing severity of disease.

[0054] Thus, antibodies against the RBD, spike and NCP are produced in greater quantities than in patients with moderate disease, but with much weaker binding avidity. Since the affinity of the RBD domain for the ACE2 receptor is significantly higher (~10 nM), this reduced affinity may have significant consequences for effective neutralization. Furthermore, there is a strong correlation between the severity of COVID-19 using antibody levels and binding strength, as well as high expression of D-dimer, C-reactive protein (CRP) and interleukin-6. Incorporating these biomarkers into a multiplex SPRi test can provide further insight into COVID-19.

[0055] An invention to determine the severity of COVID-19 The assay of the present invention is very simple to perform. A COVID19 serum sample at 1:100 dilution can be exposed to at least two proteins, already from at least two immunogenic proteins corresponding to the gradient flow path of any multichannel biosensing imaging device. However, the more specific the immunogenic epitopes, the better the immune profile for severe COVID-19 can be evaluated. The binding of specific antibodies in the serum can be tracked for 3 minutes in real time and the dissociation for 2 minutes in real time for any of these specific epitopes. The ratio of the initial slopes as a function of concentration can be calculated and the slope value of the dissociation phase of the antibodies for at least two immunogenic proteins can be calculated.

[0056] It is also noted that this approach can be easily applied to monitor immune responses in other types of infectious diseases. Any protein targeted by the immune response can be immobilized in a gradient on the sensor surface, allowing quantitative and reproducible immune characterization and application of at least two immunogenic proteins. Although more is better for detailed profiling, we found that the level of RBD to NCP and the strength of binding are already sufficient to predict the severity of COVID-19. SPRi monitors signals in real time, making it suitable for rapid deployment and optimization. Our latest findings show that not only the concentration level but also the avidity ratio of specific antibodies to immunogenic proteins is important for COVID-19 severity prediction.

[0057] This workflow allows accurate ranking and quantification of the effect of antibody affinity / avidity with the aim of improving clinical outcomes. In addition to tracking binding strength and concentration of anti-RBD / NCP antibodies in COVID-19 patients, the assay is also ideally suited for monitoring healthy individuals who have received a SARS-CoV-2 vaccine; only anti-RBD antibodies are measured, and anti-NCP antibodies are not present. The SPRi assay described herein can provide important insights in determining whether the final quality of the IgG response after vaccination is sufficient to generate neutralizing antibodies with sufficient affinity to clear the virus.

[0058] Furthermore, to have the highest success rate in developing therapeutic neutralizing mAbs, individuals and donors for passive immunization programs should be screened for the highest immune response against the immunogenic proteins of SARS-CoV-2.

[0059] The method described herein for measuring the avidity constants of at least two immunogenic proteins is reliable, concentration-independent, high-throughput and accurate for immune profiling of patients; predicting the severity of COVID-19; and dramatically reducing the number of false positives. The method reveals maturation trends and allows for the assessment of the overall quality of antibodies (see Figure 13).

[0060] Advantages of near-field optical biosensor technology in COVID-19 severity monitoring Other evanescent field-based optical biosensor techniques such as surface plasmon resonance imaging, or label-free real-time optical biosensing imaging techniques (e.g. grating coupler, holographic imaging, attenuated total reflection imaging) can be applied to track specific antibody interactions in real time within 3 minutes. This allows the most rapid fully quantitative multiplex assay compared to other labeling techniques (e.g. Elisa, EIA, ECLIA, lateral flow assays). We found that anti-nucleocapsid antibodies are strongly correlated with anti-RBD antibodies, and the strength of binding is strongly correlated with COVID-19 severity. Only label-free sensing techniques can measure this parameter in a 5-minute time window (3 minutes association and 2 minutes dissociation). SPR imaging in multiplex mode allows complete profiling of patient samples.

[0061] Consider Patients with COVID-19 showed great heterogeneity in disease severity as a result of SARS-CoV-2 infection. Although the literature suggests that humoral immune responses are involved in disease severity, their relationship has not been fully elucidated. We developed a number of SPRi-based assays to more broadly characterize patients' humoral responses and improve our understanding of their contribution to disease progression. We previously showed that SPRi can be used to detect the composition and affinity of a patient's antibody response to SARS-CoV-2.

[0062] In this study, serum samples from 76 SARS-CoV-2 patients were analyzed with SPRi using the viral antigens NCP, S1S2, S1 and RBD. For all antigens, patients with moderate or severe disease had significantly higher antibody amounts than those with mild disease. Furthermore, the data show significant differences between severe, moderate and mild disease categories for IgM, IgG and IgA isotypes. This result confirms similar findings reported in other serological studies of SARS-CoV-2 patient cohorts. In addition to measuring immune responses, SPRi can also measure the strength of antibody binding. Recently, we have demonstrated that the off-rate (kd) correlates well with the affinity equilibrium constant (KD) and can be used to rank antibody responses in terms of binding strength. Surprisingly, severe patients had significantly lower binding antibody strength against RBD compared to mild patients, and only moderate and severe patients had lower antibody strength against NCP. This contrasts with the data on binding strength for S1S2 and S2, which show that binding strength for moderate and severe is higher than for mild. However, an important point to consider here is that mild patients have a much smaller antibody response, which reduces the accuracy of the affinity measurement. If we compare only the data for severe and moderate patients, we can see that the affinity for all antigens decreases with increasing severity. Thus, severe patients produce more antibodies against RBD, S1S2, S2 and NCP than moderate patients, but with much weaker binding. Since the affinity of the RBD domain for the ACE2 receptor is very high, about 10 nM, this decrease in affinity is likely to have a significant impact on neutralization.

[0063] Most of the neutralizing antibodies isolated from SARS-CoV-2 convalescent donors target the RBD, and a subset of these antibodies block viral entry by binding to the ACE2-binding site of the RBD. Antibodies against the RBD with low affinity may not be able to compete for the interaction of RBD and ACE-2, resulting in severe disease. Beyond neutralization, antibodies can trigger various Fc-mediated immune functions, such as antibody-dependent complement deposition, cellular phagocytosis, and cell-mediated cytotoxicity. Although these are considered beneficial functions, they can also induce inflammation during viral infection. It has been reported that severe COVID-19 patients had higher antibody levels and more antibody-dependent complement deposition, but less antibody-dependent cellular phagocytosis and cytotoxicity. This indicates that not only the quantity of specific antibodies but also their qualitative characteristics play an important role. Furthermore, binding to IgA was found only in severe patients. This supports the finding that prolonged IgA responses are associated with unfavorable clinical outcomes.

[0064] From the beginning of the pandemic, it was observed that men were at higher risk of severe disease, worse prognosis, and death. However, in our cohort, the antibody responses of men and women were comparable. No gender differences were observed in the amounts of IgM, IgG, or IgG (data not shown). Similarly, there was no difference in the avidity of antibodies between men and women with severe disease. Only in the moderate group, the avidity of anti-RBD / NCP antibodies was lower in men than in women. This indicates that the primary antibody response between men and women is not the only cause of the difference in severity. Downstream gender differences in the reactivity of the innate immune system, for example the complement system, may play an important role.

[0065] The SPRi assay allowed us to characterize the details of the humoral response, including isotype, affinity, and epitope, on a single platform. We applied this to characterize COVID-19 patients, but it may be applicable to any infectious disease. A strong correlation was shown between antibody concentration and disease severity. However, these antibodies have low affinity and may be dysfunctional in maturation at neutralizing epitopes.

[0066] As the severity of specific antibodies increases d Due to the increased affinity and incomplete isotype switching, a subset of patients may have poor antibody production and maturation, resulting in the formation of a low affinity polyclonal antibody pool. This results in poor neutralization of SARS-CoV-2, higher viral loads, and increased inflammation. Furthermore, in an attempt to effectively neutralize high viral loads, antibody production increases and serum levels rise. In neutralization assays, higher antibody titers can compensate for low affinity, which may be due to antibody side effects. This is because these antibodies may find more targets that can trigger Fc-mediated immune responses, such as activation of the complement cascade, enhanced coagulation, and activation of innate immune cells. These combined factors may lead to a hyperinflammatory state that may contribute to the severity of COVID-19 patients.

[0067] SPR imaging system for determining kinetic parameters using ligand density gradients By applying the ligand solution under controlled conditions, a steep gradient of ligand density can be generated on the sensor surface. The affinity constant (k d , k a and K. D This is a major advantage in measuring affinity parameters, since the value of k ) is affected by the ligand density [6]. By gradienting the ligand density, the SPR imager can measure the ligand binding of the analyte in a spatially resolved manner over a gradient of ligand densities using a controlled injection flow method. Kinetic titration studies without a regeneration step can be applied to various binding antibodies with gradient ligand densities binding to a single antigen. Globally fitted rate constants (k d and k a ) and the dissociation equilibrium constant (K D ) can be measured, for example, R max = 100RU response level (K D R100 ) or Rmax = 50RU response level (K D R50 ) with the ligand density fixed (R max The parameters can be determined by fixing the values.

[0068] These molecular binding constants obtained from current immobilized ligand-based assays are affected by the immobilization state of the ligand. Thus, interferences due to the immobilization of the ligand cause the apparent constants thus determined to deviate from the true "solution" constants. These interferences include rebinding effects, mass transport limitations, non-specific binding, and deviations from the 1:1 model binding. The higher the ligand density, the more pronounced these interferences become, which are typically applied just above the detection limit of the biosensor device. Interferences arise when multiple analyte molecules compete to interact with one immobilized ligand molecule.

[0069] Therefore, the calculation of the "true" affinity equilibrium constant is more reliable at lower densities, preferably at a "density" of only one immobilized ligand molecule acting as a free ligand. The contribution of interferences is then zero and does not affect the kinetic and affinity equilibrium constants. The lower the ligand density, the noisier and less reliable the sensorgram becomes. Moreover, the quality of the fit to a noisy curve cannot be properly judged. It should be noted that immobilization artifacts and heterogeneity of surface binding sites must be prevented, for example, by application of high affinity anti-ligand antibodies or directional capture of the ligand by exploiting tag-anti-tag interactions.

[0070] The so-called K for determining affinity constants D R0A method has been published [7] in which the contribution of interferences is minimized or theoretically zeroed, so that the constants are better estimates of the true constants of biomolecular interactions in solution. The method is based on extrapolating the number of immobilized ligand and analyte molecules to zero, mimicking an interaction involving only one ligand and one analyte molecule, theoretically allowing a true 1:1 binding model without interferences.

[0071] Recognized practical effects are ligand immobilization artifacts and heterogeneity of surface binding sites. Methods involve capturing the ligand and allowing it to subsequently interact with the target. When harsh regeneration steps are involved, R max The value may decrease after injection of subsequent analyte concentrations, again affecting the kinetic affinity constant. Preferably, a surface regeneration step should be avoided, which is achieved using kinetic titration.

[0072] Kinetic constants were calculated from spots with varying ligand densities. Today, many users of SPR platforms adjust the ligand density so that even very low interactions with the analyte are measurable. The sensitivity of the instrument determines how low the ligand concentration can be. Users decide what they consider to be low, but since there are no rules for interpreting the quality of the fitting of the binding curve, the values ​​they make tend to diverge from each other.

[0073] According to the invention, a steep gradient of ligand density is created and the device measures the binding of the analyte on the ligand gradient. All densities are available, from very high to very low densities. Thus, if the gradient in the flow cell is divided into, for example, 1000 target regions, or even better, adjustable or kinetic target regions, the device automatically measures, for example, R max= 100 RU or 50 RU or any value for a similar set of biomolecular interactions. Instead of performing proven methods such as those published in [6] individually on a limited number of spots with low ligand density, they can now be performed on a gradient of ligand density. No more interpretation of the fitting quality by the user is required, for example by applying a 1:1 Langmuir binding algorithm. The software generates biomolecular affinity parameters measured always in the same way, with the same ligand density somewhere on the gradient. No more interpretation of the curves by the user, laboratory technician or instrument operator. The kinetic gradient method significantly improves data analysis, since the parameters are always generated in the same way.

[0074] Many more applications are possible if we can create a controlled gradient of ligand density on the sensor surface. For example, particles such as cells, viruses, organelles, vesicles, etc. contain a certain number of cell surface receptors (for CDs) that bind to anti-CD antibodies. These antibodies can bind to these particles and tests such as inhibition tests can be performed on the gradient. The higher the affinity of binding (avidity in case of multivalent interactions), the lower the ligand density that these particles will be present at. If the ligand density is zero, there will be no binding.

[0075] The T / S measurement strategies presented in the Surface Plasmon Resonance Handbook, 2nd Edition, Chapter 12.8.1, page 447, can now be applied to gradients. This can be an important strategy for avidity ranking of interactions using increasing flow protocols, as explained in Chapter 12.8.4, page 463. These detection strategies can be better applied to sensor surfaces with gradients in ligand density.

[0076] When cells are injected into the flow cell, they bind to the sensor surface. Companies developing antibodies for various cell applications need to characterize the affinity of monoclonal antibodies for live cell receptors. Direct detection of antibody binding to precipitated cell lines has not been possible due to a very unstable baseline caused by cellular activity. However, we have found that the release of cells from the sensor surface depends on several factors. For example, flow rate, number of receptors on the cell, affinity of the cell receptor for the immobilized ligand, and ligand density are important parameters. When a ligand gradient is applied in combination with increasing flow rate (shear rate), affinity ranking can be measured for multiple receptor-Ab combinations. The shear applied to the cells depends on the local velocity profile of the buffer flow over the immobilized cells. In some areas on the ligand gradient, the cells still bind, but by increasing the speed of the buffer that detaches the cells from the surface, the cells no longer bind. The faster the speed, the higher the ligand density required to keep the cells on the surface. With SPR imaging, this process can be followed in real time. By applying a uniform force to the cells, the ligand density series of anti-membrane antigens adjusts the position where the cells at a certain speed dissociate from the gradient. In this way, when different antibodies are immobilized in the ligand gradient at the same time, the affinities of the receptors on the cells can be compared and ranked against each other. This SPRi application could have a great impact.

[0077] Preferably the sensor surface consists of multiple active sites (preferably a continuous gradient) and the change in surface plasmon resonance angle due to light incidence on the sensor surface is monitored, preferably with a camera, providing reliable and multifunctional SPR imaging measurements.

[0078] SPR measurements can be performed in one flow cell or in several flow cells (e.g. 2-6 or more). When several flow cells are used, each flow cell is served by its own pumping means to form a gradient of ligand density on the sensor surface. However, the flow cell for injecting the analyte is preferably provided by a common pumping means so that all spots are exposed to the same conditions (flow rate, transport and passage of sample, buffer, thus allowing reliable automated measurements in the ligand gradient). The so-called "one over all" approach.

[0079] The mentioned and other features of the SPR measurement system and SPR measurement method according to the present invention will be further explained by various embodiments, which are given for information purposes only and are not intended to limit the scope of the invention in any way, with reference to the accompanying drawings, in which:

[0080] This method is important for example for point-of-care measurements of COVID-19 with a plug-and-play manner where the avidity parameters can be determined within 10 minutes without the problem of ligand density effects. The concentration of the analyte is found at high ligand density where mass transport occurs under controlled conditions. Both the concentration (at high ligand density) and the avidity (R max The method can automatically analyze both the concentration and avidity (at low ligand densities, e.g., =50 RU). This method could be groundbreaking for point-of-care detection, where both concentration and avidity are important parameters, e.g., for infectious diseases, especially COVID-19.

[0081] Although the present invention has been described with reference to specific embodiments, those skilled in the art will recognize that many variations are possible. For examples in specific test conditions described herein, it should be understood that the disclosure according to the present invention only shows some preferred embodiments and objects and advantages of the present invention without departing from the broader scope and spirit of the present invention. It should be understood that these discoveries according to the present invention are merely illustrative among many additional potential applications that may be envisioned by those skilled in the art, and therefore do not limit the present invention in any way. Therefore, other objects and advantages of the present invention may be apparent to those skilled in the art upon reading the detailed description together with the claims.

[0082] literature 1. Jin Y. et al. Diagnostic value and dynamic variance of antibody serum in coronavirus disease 2019. International Journal of Infectious Diseases doi.org / 10.1016 / j.ijid.2020.03.065 (2020) 2. Brouwer PJM et al., Potent neutralizing antibodies from CoViD-19 patients define multiple targets of vulnerability. Science 10.1126 / science.abc5902 (2020). 3. Schasfoort RBM, van Weperen J, van Amsterdam M, Parisot J, Hendriks J, Koerselman M, Karperien M, et al. Presence and strength of binding of IgM, IgG and IgA antibodies against SARS-CoV-2 during CoViD-19 infection. Biosensors and Bioelectronics 2021 2021 / 07 / 01 / ;183:113165 as doi: https: / / doi.org / 10.1016 / j.bios.2021.113165. 4. Schasfoort RBM, van Weperen J, van Amsterdam M, Parisot J, Hendriks J, Koerselman M, Karperien M, et al. High throughput surface plasmon resonance imaging method for clinical detection of presence and strength of binding of IgM, IgG and IgA antibodies against SARS-CoV-2 during CoViD-19 infection. MethodsX 2021 2021 / 01 / 01 / ;8:101432 as doi: https: / / doi.org / 10.1016 / j.mex.2021.101432.Hendriks, Jan, et al. “High titers of low affinity antibodies in Covid-19 patients are associated with disease severity.” Frontiers in immunology (2022): 1620. 5. Schasfoort R.B.M., Editor of the Handbook of Surface Plasmon Resonance, 2nd edition, Royal Society of Chemistry London, UK (2017) 6. Schasfoort R.B.M. et al. Interpolation method for accurate affinity ranking of arrayed ligand-analyte interactions Analytical Biochemistry 500, Pages 21-23 https: / / doi.org / 10.1016 / j.ab.2016.01.023 (2016) 7. Schasfoort, Richard BM, et al. “Method for estimating the single molecular affinity.” Analytical biochemistry 421.2 (2012): 794-796.

Claims

1. 1. A method for predicting the severity of immune-related diseases by a combination of specific antibody concentrations and total antibody isotype binding intensities from a patient's body fluid sample, comprising: a. exposing a body fluid sample from a patient suffering from an immune-related disease to an immunogenic antigen immobilized in a gradient on a biosensor capable of label-free and real-time imaging; b. Using the initial slope of the binding curve under mass transport limited conditions to determine the concentration at which the ligand density is high; and c. On a ligand density gradient, low but immobilized R ｍａｘ determining an avidity parameter under the conditions; wherein the ratio of the levels and affinities of at least two immunogenic proteins predicts the severity of an immune-related disease in a patient.

2. The method of claim 1, comprising a label-free and real-time biosensor based on any evanescent field optical phenomenon.

3. The method of claim 2, wherein the evanescent field-based biosensor is an optical device based on surface plasmon resonance (SPR) imaging.

4. 2. The method of claim 1, wherein the controlled injection of the ligand uses at least one back-and-forth flow of the sample through a flow path in contact with the sensor surface to create a gradient of ligand density due to differences in contact time.

5. The method of claim 1, wherein the fluidic system is designed to allow a sample to simultaneously flow through at least two or more channels in a gradient for simultaneously measuring concentration, binding rate, and dissociation rate for at least duplicate measurements of the same sample.

6. The method described in claim 1, wherein the immune-related disease is an infectious disease.

7. 2. The method of claim 1, wherein the immobilized immunogenic antigen is at least the receptor-binding domain and nucleocapsid (NCP) of SARS-CoV-2.

8. 8. The method of claim 7, wherein the nucleocapsid antibody binding constant and the RBD binding constant predict the severity of infection in COVID-19 patients.

9. The method of claim 1, wherein the sensor is rotated 180 degrees to apply half of the flow cell with timed exposure of ligand from inlet to outlet to create a gradient of ligand density for a first immunogenic protein, and while the first gradient is in the down section, the top section is timed to expose from inlet to outlet of the flow cell to allow subsequent immobilization with a second protein, thereby the sensor is equipped with a multiplex flow cell for simultaneously measuring binding strength and isotype of bound antibodies to at least two immunogenic proteins.

10. Fixed R ｍａｘ The measurement of biomolecular interactions on the gradient to determine the value is performed with a fixed R ｍａｘ 10. The method of claim 1, comprising analysis of on and off rates using a sensorgram having values ​​at which concentrations are measured at different positions on the gradient.

11. The method of claim 10, wherein the biomolecular interactions on the gradient are simultaneously exposed to a co-injection of an anti-isotype antibody.

12. 10. The method of claim 1, wherein SPR imaging is used to establish the strength of binding of antibody isotypes and reduce the number of false positives characterized by a gradient print of ligand exposed to patient samples.

13. a. SPR imager; b. A line printer for creating gradients on the track; wherein a portion of the printed line or track is exposed to a patient sample.

14. 14. The apparatus of claim 13, wherein a single channel is applied onto the printed track and the sensor prism is repositioned to cover a portion of the track.

15. 14. The device of claim 13, wherein a single channel is applied onto the printed track and the flow cell is repositioned to cover a portion of the track.

16. 14. The device of claim 13, wherein a crisscross track is applied to form a ligand density gradient by changing the position of the sensor prism or the flow cell.

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