Label-Free Detection and Characterization of Heparin-Induced Thrombocytopenia (HIT)-like Antibodies
Authors: Uwe Pliquett
Holger Rothe
Nida Zaman Khan
Li-Yu Chen
Annerose Lindenbauer
Thi-Huong Nguyen
Journal: ACS Omega (2021)
Discipline: CHEMISTRY
Keywords: Heparin-induced thrombocytopenia; Antibodies; Biosensor-based electrical detection; Monoclonal Abs; Platelet factor 4/Heparin antigens
Affiliations: Institute for Bioprocessing and Analytical Measurement Techniques (iba), 37308 Heiligenstadt, Germany, Institute for Chemistry and Biotechnology, Faculty of Mathematics and Natural Sciences, Technische Universität Ilmenau, 98694 Ilmenau, Germany, Institute of Microbiology, Friedrich Schiller University, 07745 Jena, Germany

Abstract:  Heparin-induced thrombocytopenia (HIT) antibodies (Abs) can mediate and activate blood cells, forming blood clots. To detect HIT Abs, immunological assays with high sensitivity (≥95%) and fast response are widely used, but only about 50% of these tests are accurate as non-HIT Abs also bind to the same antigens. We aim to develop biosensor-based electrical detection to better differentiate HIT-like from non-HIT-like Abs. As a proof of principle, we tested with two types of commercially available monoclonal Abs including KKO (inducing HIT) and RTO (noninducing HIT). Platelet factor 4/Heparin antigens were immobilized on gold electrodes, and binding of antibodies on the chips was detected based on the change in the charge transfer resistance (R ct). Binding of KKO on sensors yielded a significantly lower charge transfer resistance than that of RTO. Bound antibodies and their binding characteristics on the sensors were confirmed and characterized by complementary techniques. Analysis of thermal kinetics showed that RTO bonds are more stable than those of KKO, whereas KKO exhibited a higher negative ζ potential than RTO. These different characteristics made it possible to electrically differentiate these two types of antibodies. Our study opens a new avenue for the development of sensors for better detection of pathogenic Abs in HIT patients. 

Retracted: No

PubPeer comments:
 Overall scientific terminology: -tSAM layer: SAM stands for  self-assembled monolayeru201c, therefore, adding  layeru201c is redundant. -tEIS stands for u201eelectrochemical impedance spectroscopyu201c. Please do not add  measurementu201c,  experimentu201c,  testu201c, or  detectionu201c after  EISu201c, as you did. -t reduce resistance transferu201c: Can you please explain how a resistance is transferred in EIS? -t stickiness of the PF4 H antigen coated on the sensoru201c: Do I need to comment on this? Data presentation: -tFigure 4 A: In the presented plot, the measurement curves were shifted to start at the coordinate origin as if the solution resistance was zero. Please do not alter measurement curves like this! -tFigure 1 E: The authors generally seem to not understand where Rs and Rct are expressed in Nyquist Plot, as Figure 1 E u2013 while here showing a Rs 0 u2013 the sum of Rs and Rct cannot be found in the place they indicated. -tFigure 5 A: Fitting of the AFM data is adventurous, considering that especially for KKO a significant amount of data points was excluded. Protocols and performance: -tWhy did you perform EIS with 13 electrodes for RTO investigation and only 11 for KKO? -tZeta potential should never be measured in pure water, as you need the continuous phase to be conductive to measure mobility. Poor conductivity furthermore leads to electrode polarization, which destabilizes the system. -tIncubation times for the construction of the sensor are not given, except for cysteamine. In order to obtain a SAM of high quality, usually concentrations around 1 mM are used and incubated for several hours to allow the formation of highly organized and densely packed monolayers. A concentration of 30 mM cysteamine is therefore way too high, while 15 min incubation is way too short, which is probably one reason for the observed large deviations of the results from EIS. -tSpeaking of deviations, if you see that piranha treatment etches the surface and leads to significant reduction in Rct with each reuse of the electrode, why do you proceed like this, provoking large deviations of the obtained results? -tAs regeneration protocol, washing with glycine was used in order to remove bound antibodies from PF4 H. Simply the fact that the authors decided to present u0394Rct as Rct(antibody) - Rct(glycine) indicates that the Rct does not drop to its original value as it should for successful regeneration. Therefore, after incubation in 2.5, 5, and 7.5 u00b5g ml antibody, the surface seemed saturated, so that *less binding* was detected for a higher concentration of 10 u00b5g ml. With a successful regeneration protocol, *higher or similar values* for 10 u00b5g ml should be achieved. The fact that bound antibodies could not be removed completely makes obtained data for concentrations 2.5 u00b5g ml, which were measured after glycine treatment, unreliable. Major issues with binding specificity: -tUnspecific binding of biological matter is a ubiquitous problem when working with gold surfaces. Already in the results from the first CLSM, no significant difference between unspecific anti-PF4 FITC-labeled antibody binding to the SAM and its specific binding to PF4 H can be seen (Figures 3 A- C). This already proves issues with binding specificity that are present throughout the article. -tFor EIS, a control of 1:200 diluted blood sera was deducted, of which was claimed that  no binding of control antibodies to the PF4 H antigen-coated sensoru201c took place and u201cplasma proteins were thoroughly washed awayu201d. However, the obtained u0394Rct of the control was in the exact same range as the u0394Rct of RTO and KKO binding. The authors therefore either lack the necessary knowledge for the performance and evaluation of their experiments, or the honesty in discussing the obtained results. If unspecific binding of random proteins from the control led to the same u0394Rct as RTO and KKO binding, how can the authors state that any of the RTO KKO binding was specific and due to interaction with PF4 H? Overall goal of the article: -tThe aim of this article was to differentiate between KKO and RTO by electrochemical impedance spectroscopy. The large deviations for antibody binding at various concentrations (Figure 4 C) demonstrate that, however, *no differentiation can be made*. A properly designed sensor would allow estimating the concentration of the measured antibody as well as, in this case, if RTO *or* KKO are present, which is not the case for the presented data. Furthermore, *RTO or KKO cannot even convincingly be detected*, as the control yields u0394Rct in the same range. -tThe demonstrated arts and crafts with the introduced Q-value does not solve this problem, as only the results from the incubation with 7.5 u00b5g ml showed a significant difference between RTO and KKO for the Q-value. For the analysis of patient samples, however, the antibody concentration is unknown, so that the linear measurement curve u2013 which, by the way, is missing in this study u2013 is needed to calculate the antibody concentration. The necessity for introducing the Q-value was justified by the variation of the electrode size, which equaled 2.0% in width and 0.8% in length. I cannot comprehend how the authors, the editor, and the reviewers accepted this variation as the cause for the massively high deviations in u0394Rct, which even includes values below zero. -tThe applicability of the presented sensor for real patient sera is seemingly impossible. Since the included control was 1:200 diluted blood sera, it can easily be assumed that the u0394Rct of real patient sera is orders of magnitude higher, so that unspecific protein binding dominates the measured Rct and no information regarding the presence of HIT or non-HIT antibodies can be obtained. The authors have stated that u201ctests showed the same trend of changes in Rct values when RTO or KKO were detected in the purified system or were mixed in sera of healthy donors (data not shown).u201d For this claim, the authors should include their results. Concluding remark: From CLSM, a higher binding density of RTO can be concluded compared to KKO, which is in disagreement with the higher mass change of KKO in QCM (did somebody consider the molecular weights?). Due to higher mean u0394Rct for RTO binding in EIS, it was concluded that RTO would bind in higher density u2013 although the more negative zeta potential of the KKO PF4 H complex should lead to higher electrostatic repulsion of the also negatively charged redox mediator ferri ferrocyanide, which should cause a higher u0394Rct of KKO compared to RTO. , 
 1. The authors show the binding of KKO and RTO in the presence of PF4H complex in the QCM experiments, but it is not clear whether they bind via the complex or directly. The absence of control experiments makes judgement difficult. 2. The authors claim to see the higher binding of KKO antibody as compared to RTO antibody. However, on a careful inspection, the average +- SD mentioned in the manuscript looks a bit misleading. Given the higher distribution range of values of binding force and a quite visible small second peak, the author s derivations are questionable. , 
 Authors have used white light interferometry to measure roughness of the gold surface. The washing cycles shows clear etching of the surface thereby increasing chance of unspecific binding. How do the authors forsee the application of their protocol in developing a sensor with good specificity? , 
 Could the authors elaborate on the control tests pictured in Figure 4 C, where RTO and KKO interacted with the SAM? The presented data implements that antibody concentrations ranging 2.5 to 10 u00b5g ml show low changes in Rct when incubated on the cysteamin glutaraldehyde SAM in the absence of their antigen PF4 H. What does the data pictured at 0 u00b5g ml antibody refer to? How can the incubation with an antibody concentration of 0 u00b5g ml lead to a higher Rct increase than the majority of antibody concentrations 0 u00b5g ml binding on the PF4 H decorated SAM? Did you confuse the labels? If you actually incubated with antibody at 0 u00b5g ml, did you use the same sensor to test the other antibody concentrations after glycine treatment? Since glycine can only release antibodies from their antigens but not from unspecific binding sites on the sensor, incubating a sensor that is already saturated with unspecifically adsorbed RTO or KKO in higher concentrations would of course not lead to an increase of Rct. The CLSM images in Figure 3 A and B compliment the unreliable presentation of antibody binding to undecorated SAM: How is it possible that unspecific binding of RTO and KKO antibodies to the SAM without PF4 H induce such a low Rct change compared to their specific binding to PF4 H decorated SAM, while the fluorescent-labelled antibody showed no significant difference between specific binding to PF4 H and unspecific adsorption to the undecorated SAM? Major issues with binding specificity u2013 and truthful discussion of the obtained results - can also be seen from the control experiment with human sera: How can the authors state that the control antibodies present in sera do not bind to the PF4 H decorated SAM, when the corresponding Rct increase is higher or equally high as the increase due to KKO binding? Careful evaluation of the unprocessed data should be carried out by the editorial team to shed light to the contradicting data presented in this publication. , 
 In the QCM studies, the authors demonstrate the binding of KKO and RTO in the presence of PF4H complex, however it is unclear whether they bind via the complex or are fouling directly. Lack of a control group in experiments complicates evaluation. The authors conclusions seem dubious! ,