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Erika Yoshihara, Ahmed Nabil, Michihiro Iijima, [Mitsuhiro Ebara](https://orcid.org/0000-0002-7906-0350)

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[A Comparative Study of “Grafting to” and “Grafting from” Conjugation Methods for the Preparation of Antibody-Temperature-Responsive Polymer Conjugates](https://mdr.nims.go.jp/datasets/929e2856-e21c-48e0-8198-01f4ea273d9b)

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A Comparative Study of “Grafting to” and “Grafting from” Conjugation Methods for the Preparation of Antibody-Temperature-Responsive Polymer ConjugatesA Comparative Study of “Grafting to” and “Grafting from”Conjugation Methods for the Preparation of Antibody-Temperature-Responsive Polymer ConjugatesErika Yoshihara, Ahmed Nabil,* Michihiro Iijima, and Mitsuhiro Ebara*Cite This: ACS Omega 2024, 9, 22043−22050 Read OnlineACCESS Metrics & More Article Recommendations *sı Supporting InformationABSTRACT: Early diagnosis of infectious diseases is still challenging particularly in a nonlaboratory environment or limitedresources areas. Thus, sensitive, inexpensive, and easily handled diagnostic approaches are required. The lateral flow immunoassay(LFIA) is commonly used in the screening of infectious diseases despite its poor sensitivity, especially with low pathogenic loads(early stages of infection). This article introduces a novel polymeric material that might help in the enrichment and concentration ofpathogens to overcome the LFIA misdiagnosis. To achieve this, we evaluated the efficiency of introducing poly(N-isopropylacrylamide) (PNIPAAm) into immunoglobulin G (IgG) as a model antibody using two different conjugation methods:grafting to (GT) and grafting from (GF). The IgG−PNIPAAm conjugates were characterized using SDS-PAGE, DLS, andtemperature-responsive phase transition behavior. SDS-PAGE analysis revealed that the GF method was more efficient inintroducing the polymer than the GT method, with calculated polymer introduction ratios of 61% and 34%, respectively. The GFmethod proved to be less susceptible to steric hindrance and more efficient in introducing high-molecular-weight polymers intoproteins. These results are consistent with previous studies comparing the GT and GF methods in similar systems. This studyrepresents an important step toward understanding how the choice of polymer incorporation method affects the properties of IgG−PNIPAAm conjugates. The synthesized polymer allowed binding and enrichment of mouse IgG that was used as a model antigenwith a clear LFIA band. On the basis of our findings, this system might help in improving the sensitivity of simple diagnostics.■ INTRODUCTIONVarious studies including ours have reported the severedrawbacks of the current commercially available SARS-CoV-2 diagnostic kits, as most of them have a high false negativerate and poor sensitivity, and even the gold standardpolymerase chain reaction (PCR) is recommended to berepeated to overcome this misdiagnosis.1−5 Many factors werereported to contribute to this problem, including fluctuation inthe viremia load of nasopharyngeal and oropharyngeal swaps(NP/OP) commonly used for SARS-CoV-2 diagnosis,6−8virions colonize the lower respiratory tract while these swapsare collected from the upper respiratory tract,9,10 and finally,the dynamics of the viral load of SARS-CoV-2 where the viralload peaked on day 10 after the onset of infection,approximately.11−13 On the other hand, PCR testing requiressophisticated equipment, high cost, time, and experts to collectmucosa samples and detect viral load and other antigenictargets. To solve these issues, point-of-care testing (POCT)has been developed, which is a simple real-time test that can beperformed by healthcare professionals themselves at thepatient’s side using a small analyzer or rapid diagnostic kits.Our team focused on the lateral flow immunoassay (LFIA) asone of the POCT approaches. LFIA is superior because it ischeap, portable, fast, and widely used in diagnostics includinginfluenza detection kits and pregnancy tests. Moreover, theresult of the LFIA test can be easily determined visually.14However, the main problem with LFIA is its limited detectionsensitivity.15 For example, lipoarabinomannan (LAM) isReceived: January 4, 2024Revised: March 1, 2024Accepted: March 12, 2024Published: May 10, 2024Articlehttp://pubs.acs.org/journal/acsodf© 2024 The Authors. Published byAmerican Chemical Society22043https://doi.org/10.1021/acsomega.4c00103ACS Omega 2024, 9, 22043−22050This article is licensed under CC-BY-NC-ND 4.0Downloaded via NATL INST FOR MATLS SCIENCE (NIMS) on December 3, 2024 at 08:17:26 (UTC).See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Erika+Yoshihara"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://pubs.acs.org/action/doSearch?field1=Contrib&text1="Ahmed+Nabil"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://pubs.acs.org/action/doSearch?field1=Contrib&text1="Michihiro+Iijima"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://pubs.acs.org/action/doSearch?field1=Contrib&text1="Mitsuhiro+Ebara"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://pubs.acs.org/action/showCitFormats?doi=10.1021/acsomega.4c00103&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.4c00103?ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.4c00103?goto=articleMetrics&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.4c00103?goto=recommendations&?ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.4c00103?goto=supporting-info&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.4c00103?fig=agr1&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.4c00103?fig=agr1&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.4c00103?fig=agr1&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.4c00103?fig=agr1&ref=pdfhttps://pubs.acs.org/toc/acsodf/9/20?ref=pdfhttps://pubs.acs.org/toc/acsodf/9/20?ref=pdfhttps://pubs.acs.org/toc/acsodf/9/20?ref=pdfhttps://pubs.acs.org/toc/acsodf/9/20?ref=pdfhttp://pubs.acs.org/journal/acsodf?ref=pdfhttps://pubs.acs.org?ref=pdfhttps://pubs.acs.org?ref=pdfhttps://doi.org/10.1021/acsomega.4c00103?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://http://pubs.acs.org/journal/acsodf?ref=pdfhttps://http://pubs.acs.org/journal/acsodf?ref=pdfhttps://acsopenscience.org/researchers/open-access/https://creativecommons.org/licenses/by-nc-nd/4.0/https://creativecommons.org/licenses/by-nc-nd/4.0/https://creativecommons.org/licenses/by-nc-nd/4.0/https://creativecommons.org/licenses/by-nc-nd/4.0/https://creativecommons.org/licenses/by-nc-nd/4.0/secreted in the urine of tuberculosis (TB) patients and is abiomarker of TB, but LAM is currently difficult to detect byLFIA due to its limited concentration in urine samples, whichis below the detection limit of LFIA (several μg/mL).Similarly, in the diagnosis of SARS-CoV-2, low viral antigenicconcentrations (<ng/mL) in (NP/OP) samples make visual-ization of positive results challenging. Therefore, it is nowevident that our primary challenge is to improve the limitedsensitivity of LFIA. This can be achieved through theenrichment of biomarkers in the specimens tested beforetheir application to LFIA. Chromatography and ultrafiltrationare common antigen concentration methods16 but are notsuitable for POCT due to their sophisticated and expensiveequipment. Therefore, we focused on poly(N-isopropylacryla-mide) (PNIPAAm), which is a temperature-responsivepolymer, as an alternative method for antigen purificationand easy enrichment easily.PNIPAAm is water-soluble at room temperature and has alower critical solution temperature (LCST), around 32 °C, soit aggregates and precipitates at temperatures above 32 °C.17Taking advantage of this property, PNIPAAm and itsderivatives can be introduced into antibodies to provideON−OFF switching functionality upon temperature stimula-tion. Antigen enrichment is expected by its trapping withspecific antibody-temperature-responsive polymer conjugates.Many previous studies, including ours, that introducedPNIPAAm and PNIPAAm-derived polymers into variousproteins, including antibodies, have already been reported.Hoffman et al. have successfully prepared conjugates with aseries of amine-containing proteins, including lysozyme,myoglobin, protein A, hemoglobin, albumin, and γ-globulinby using NIPAAm copolymer with N-hydroxysuccinimide(NHS).18,19 Okano et al. have also synthesized semitelechelicPNIPAAm with NHS groups at the ends of the polymer andsuccessfully conjugated it with antibodies.20,21 Recently, ourgroup reported the preparation of polymeric antibodyconjugates in real biological samples by adding functionalgroups to proteins by modifying PNIPAAm-derived copoly-mers using click chemistry.1,10 These temperature-responsiveprotein-polymer conjugates were prepared by a method called“grafting-to” (GT), in which a presynthesized polymer isconjugated to a functionalized protein by a coupling reaction.Although this method seems to be simple and convenient, itrequires a complicated process that includes the synthesis ofPNIPAAm-derived polymers in multiple steps, in addition tothe functionalization of the polymer end groups and thepurification that accompanies this process. In addition, theefficiency of the introduction of polymers into protein is lowdue to steric obstacles; therefore, the addition of excess freepolymers is necessary to improve its enrichment efficiency.22In addition to the GT method, there is another “grafting-from” (GF) method, which is also used for introducingpolymers into proteins. The GF method is a technique that hasbeen attracting attention in recent years because it introducespolymers directly by polymerizing monomers in solution usingfunctionalized proteins as the starting point for polymerization,thus overcoming the previously mentioned issues of the GTmethod, including steric hindrance and excess addition of freepolymers.23,24 Studies comparing the GT and GF methods ofintroducing polymers into RNA, cellulose, and graphene havegenerally concluded that the GF method is the most efficientway to introduce polymers.25−27 GF basically uses living-radical polymerization in water to introduce the polymer. Insome reported studies, a chain transfer agent (CTA) wasintroduced into bovine serum albumin (BSA) and lysozyme byreversible addition−fragmentation chain transfer polymer-ization (RAFT).28−30 This demonstrated that the proteinfunctionality of the conjugates was retained after the NIPAAmpolymerization from proteins and the introduction ofPNIPAAm by living radical polymerization. We have alsosucceeded in preparing antibody-temperature-responsive poly-mer conjugates by introducing CTA into antibodies andperforming NIPAAm polymerization with antibody as theinitiation point of polymerization.31 This conjugate that wasprepared by the GF method showed nanoparticle morphology,which was also reported to be useful in improving variousphysical properties including stability against enzymes, highantigen−antibody reaction activity, and applicability fordifferent medical applications, including drug delivery systems(DDSs). However, there are no previous comparative studiesbetween the GT and GF methods, which can give anindication whether antibody-polymer conjugates produced bythe GF method are inferior to antibody-polymer conjugatesproduced by the GT method or vice versa.Therefore, the objective of this study is to clarify theperformance of antibody polymer conjugates (APCs)produced by the GF method compared to the GT methodby introducing thermoresponsive polymers to antibodies usingeach of the GT method and the GF method, which wereestablished in our previous studies, and by comparing andevaluating the polymeric introduction capacity and thepolymeric conjugate efficiency (thermal precipitation effi-ciency, activity against antigens, etc.). In addition, this studyaimed to improve the medical applications of GF by increasingthe sensitivity and accuracy of antigen testing by antigenenrichment using the IgG−PNIPAAm conjugate. To the bestof our knowledge, this report may be the first to conduct acomparative study between GT and GF methods in addition toevaluation of their biomedical performance.■ MATERIALS AND METHODSMaterials. N-Isopropylacrylamide (NIPAAm, FujifilmFujifilm Wako Pure Chemical, 97%) was recrystallized fromhexane and dried under vacuum prior to use. 2,2′-Azobis [2-(2-imidazolin-2-yl) propane] dihydrochloride (VA-044, TokyoKasei, 98.0%) was recrystallized from methanol and driedunder vacuum before use. 2-(Dodecylthiocarbonothioylthio)-2-methylpropionic acid N-hydroxysuccinimide ester (NHS-CTA, Sigma-Aldrich, St. Louis, MO, USA), poly(N-isopropy-lacrylamide), N-hydroxysuccinimide (NHS) ester terminated(PNIPAM-NHS, , Mn 2,000, Sigma-Aldrich, St. Louis, MO,USA), poly(N-isopropylacrylamide), N-hydroxysuccinimide(NHS) ester terminated (NHS-PNIPAAm, Mn 2000, Sigma-Aldrich, St. Louis, MO, USA), N,N-dimethylformamide (DMF,99.5%, Fujifilm Wako Pure Chemical), dulbecco phosphatebuffered saline (PBS, Aldrich), goat polyclonal secondaryantibody to mouse IgG−H&L (HRP) (IgG, abcam), dimethylsulfoxide (DMSO, Fujifilm Wako Pure Chemical, 99.0%), DL-2-aminobutyric acid (Tokyo Kasei, 99.0%), fluorescamine(Tokyo Kasei), sodium ascorbate (Aldrich), tris (hydrox-ymethyl) amino methane (Tris, 99.8%, Aldrich), hydrochloricacid (1.0 mol/L, Fujifilm Wako Pure Chemical), methanol(99.8%, Fujifilm Wako Pure Chemical), 10 × tris/glycine/SDSbuffer (BIO-RAD), coomassie brilliant blue R-250 (CBB, BIO-RAD), laemmli sample buffer (BIO-RAD), 2-mercaptoethanol(Fujifilm Wako Pure Chemical, 99%), precision plus proteinACS Omega http://pubs.acs.org/journal/acsodf Articlehttps://doi.org/10.1021/acsomega.4c00103ACS Omega 2024, 9, 22043−2205022044http://pubs.acs.org/journal/acsodf?ref=pdfhttps://doi.org/10.1021/acsomega.4c00103?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-asunstained standards (BIO-RAD), goat antibody to mouse IgG(1.96 mg/mL, abcam), goat antibody to mouse IgG−H&L(2.06 mg/mL, abcam), mouse IgG (antigen, abcam), hydro-chloric acid (Stop solution, Goat antibody to mouse IgG (1.96mg/mL, abcam), 1.0 mol/L), goat anti-mouse IgG H&L(Biotin) (2 mg/mL, abcam), streptavidin (HRP) (1 mg/mL,abcam), 3′,5,5′-tetramethylbenzidine (TMB) solution (BIO-RAD), and the QuickQuant Mouse IgG Quantification Kit(funakoshi) were purchased and used as received. Poly-(oxyethylene sorbitan monolaurate) (Tween 20, Tokyo Kasei)was used after diluting to a concentration of 0.5% in PBS afterpurchase. This is a purified goat polyclonal antibody (IgG),prepared by injecting whole mouse IgG into a healthy goat.The product specifically targets mouse IgG. This antibody hasbeen shown to react with mouse IgG in ELISA (1:10000) andhas been evaluated for activity using our previous reports.10,31ELISA coating buffer (abcam) was used after diluting 10 timeswith ultrapure water after purchase.Methods. IgG−PNIPAAm Conjugate Preparation by theGF Method.30,31 Synthesis was performed according to ourpreviously established reports, as we allowed conjugationbetween NHS-CTA and IgG, and an alkaline buffer was usedto facilitate carbodiimide chemistry (Scheme 1). The bufferwas prepared by dissolving NaHCO3 (105 mg, 1.25 mmol) inultrapure water (resistivity value: 18.2 Ω cm, 20 mL).Subsequently, an aqueous NaOH solution (1 mol L−1) wasadded dropwise to the buffer until the pH of the solutionreached 8.6. Before conjugation, IgG (400 μg, 0.0026 μmol)was diluted to 1 mg/mL in NaHCO3 buffer, then NHS-CTAdissolved in DMF (0.26 μmol, 123.1 μg) (21 μL) was added,and the reaction was continued for 24 h in a 25 °C block bathshaker. After the reaction, the byproducts were removed byultrafiltration (Amicon Ultra Centrifugal Filter, MWCOScheme 1. Synthesis of Antibody-Temperature-Responsive Polymer Conjugates by the “Grafting to” and “Grafting from”MethodsACS Omega http://pubs.acs.org/journal/acsodf Articlehttps://doi.org/10.1021/acsomega.4c00103ACS Omega 2024, 9, 22043−2205022045https://pubs.acs.org/doi/10.1021/acsomega.4c00103?fig=sch1&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.4c00103?fig=sch1&ref=pdfhttp://pubs.acs.org/journal/acsodf?ref=pdfhttps://doi.org/10.1021/acsomega.4c00103?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-as10000, 0.5 mL), and the IgG conjugates were washed threetimes with PBS. The synthesized IgG-CTA (400 μg, 0.0026μmol) was mixed with 200 μL of PBS (pH 7.5) in a 1.5 mLmicrotube. Then NIPAAm (18 mg, 160 μmol) and 200 μL ofPBS containing (3.6 μmol, 1.2 mg) of the initiator VA-044were added and dissolved in the previous solution. Thereaction was then carried out in a shaking water bath at 32 °Cfor 24 h.IgG−PNIPAAm Conjugate Preparation Using the GTMethod.20 IgG (400 μg, 0.0026 μmol) was diluted to 1 mg/mL by replacing the solution with NaHCO3 buffer. PNIPAAmwith an NHS end group (NHS-PNIPAAm) (0.26 μmol) wasdissolved in DMF (21 μL) and added to the IgG solution(Scheme 1). The solution was then reacted at 4 °C for 12 h,purified by centrifugal dialysis, and the solution was replacedwith PBS. For molecular weights of 25,000 and 50,000,COOH-terminal PNIPAAm was synthesized and conjugatedto the antibody by active esterification to form amide bonds.The synthesized polymers are mentioned in the SupportingInformation.Sodium Dodecyl Sulfate-Polyacrylamide (SDS-PAGE)Measurement. The IgG polymeric conjugates were thencharacterized by SDS-PAGE compared to IgG and the polymersolution, alone as previously mentioned in our previouspublications.1,10DLS Measurement. The conjugate solutions prepared werediluted and adjusted with PBS to the IgG concentration of 0.05mg/mL and measured using a Malvern Zetasizer-Nano ZSP atλ = 633 nm, scattering angle 173°, and temperature 25 °C. Thediameter and aggregation state of the samples were evaluated.Lower Critical Solution Temperature (LCST) Measure-ment. The temperature dependence of the transmittance inthe prepared samples was measured by using a spectropho-tometer. The prepared conjugate solution was diluted andadjusted to 0.5 mg/mL IgG concentration with PBS. Thesample solution and the stirring bar were subjected toabsorbance measurement using a spectrophotometer in anitrogen atmosphere at a wavelength of 450 nm, a temperaturerange of 25−40 °C, and a temperature increase rate of 0.2 °C/min.Evaluation of the IgG Recovery Ratio. The conjugatesolutions prepared were diluted with PBS to an IgGconcentration of 0.5 mg/mL, and then 300 μL was added toa 1.5 mL microcentrifuge tube and centrifuged at 37 °C and15,000 rpm for 15 min. After 240 μL of the supernatant wasaliquoted, 240 μL of PBS was added, and the solution wasredissolved. The IgG concentration in the solution was thenmeasured by the BCA method, and the recovery ratio wascalculated.BCA Method for the IgG Concentration Assay. Theconjugate solutions prepared were diluted with PBS andadjusted to a concentration of IgG of 0.05 mg/mL. Then 25μL of this solution and 200 μL of working solution (MicroBCATM Protein Assay Kit) were added to 96 well plates andincubated at 37 °C for 30 min. The absorbance of each samplewas measured at a wavelength of 562 nm, and the BSAconcentration was calculated according to the manufacturer’sinstructions based on the IgG calibration curve (0, 0.0125,0.025, 0.05, and 0.1 mg/mL).Evaluation of the Antibody Temperature ResponsivePolymeric Conjugate Binding Constant to Antigen. Asandwich enzyme-linked immunosorbent assay (ELISA,developed in-house) was used to measure the apparentbinding affinities of the IgG-CTA and IgG−polymerconjugates. The primary antibody (1.0 mg mL−1) was firststabilized into a 96-well plate and incubated overnight.Following stabilization, the plate was washed five times withPBS containing 0.5% Tween 20. Second, the plate was blockedwith 200 μL of blocking buffer and incubated for 30 min. Theplate was then washed five times with PBS containing 0.5%Tween 20. Third, 100 μL of antigen was added to the 96-wellplate by varying the antigen concentration from 10 to 118 pgmL−1 and incubated for 1 h. Mouse IgG was used as the modelantigen. In the next step, the plate was washed five times withPBS containing 0.5% Tween 20. Thereafter, 1 mg mL−1 IgG-CTA biotin-labeled or conjugates were added and incubatedfor 30 min. After that, HRP-conjugated streptavidin was added,and the mixture was incubated for 30 min. The binding affinitywas evaluated. After five washes with PBS containing 0.5%Tween 20, TMB was added. The final assay signals wererecorded by measuring the absorbance at 450 nm after 10 minof incubation with an acid treatment to stop the enzymaticreaction.Evaluation of the Antibody Temperature-Responsivenessof Polymer Antigenic Enrichment Efficacy Using a LateralFlow Immunoassay Strip (LFIA). Different concentrations ofmouse IgG were tested by using LFIA as a model antigen.QuickQuant Mouse IgG Quantification Kit (funakoshi) wasused. Antigen concentrations were (200, 100, 50, 25, 10, and 5ng/mL) to determine the lower detection limit of this LFIA.Samples that were negative tested using the LFIA QuickQuantMouse IgG Quantification Kit (funakoshi) were used and wereretested again after polymeric enrichment (polymer preparedby the GF method). Mouse IgG purified (10 ng/mL) 900 μLin PBS was mixed with antibody-polymer conjugate andincubated for 1 h, then 3 mg of free polymer was added andcentrifuged in microtubes at 37 °C and 13 000g for 15 min.The supernatant (900 μL) and the precipitate (100 μL) werecollected, and the concentrated portion was again evaluated byLFIA.■ RESULTS AND DISCUSSIONEvaluation of the Efficiency of Introduction ofImmunoglobulin G (IgG)−poly(N-Isopropylacrylamide)(PNIPAAm). IgG−polymeric conjugate products were appliedto SDS-PAGE to confirm the molecular weight shift due to theintroduction of the polymer, as shown in Figure 1. In the caseof IgG−PNIPAAm that was prepared by the GT method, amolecular weight shift was observed with smeared migrationFigure 1. SDS-PAGE analysis for IgG−PNIPAAm conjugates: (lanes:(1) protein standard, (2) IgG, (3) to 2,500, (4) from 400 mM, and(5) protein standard).ACS Omega http://pubs.acs.org/journal/acsodf Articlehttps://doi.org/10.1021/acsomega.4c00103ACS Omega 2024, 9, 22043−2205022046https://pubs.acs.org/doi/suppl/10.1021/acsomega.4c00103/suppl_file/ao4c00103_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acsomega.4c00103/suppl_file/ao4c00103_si_001.pdfhttps://pubs.acs.org/doi/10.1021/acsomega.4c00103?fig=fig1&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.4c00103?fig=fig1&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.4c00103?fig=fig1&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.4c00103?fig=fig1&ref=pdfhttp://pubs.acs.org/journal/acsodf?ref=pdfhttps://doi.org/10.1021/acsomega.4c00103?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-aspatterns between 50 and 75 kDa, confirming the increase inmolecular weight due to the introduction of the polymer. Theratio of polymer introduction to antibody was calculated fromthe area of the unreacted 50 kDa band and the shifted band ofpolymeric conjugates using ImageJ and was found to be 34% asshown in Table 1. This value is relatively close to theintroduction efficiency of conventionally used polymers withNHS groups at the ends, suggesting that conjugation at thepolymeric ends by the GT method has low introductionefficiency. On the other hand, in IgG−PNIPAAm prepared bythe GF method, a wide band widening range appeared between50 and 250 kDa, as mentioned before in our previousstudies.1,10,30,31 Similarly, the polymer introduction ratio wascalculated to be 61%, which is higher than that of the GTmethod. Compared to the GT method, GF is less affected bysteric hindrance and more efficient in polymer introduction, aspolymers are directly introduced by polymerization frommonomers. Furthermore, in the 400 mM band, a highermolecular weight shift was shown when compared to the GTmethod, suggesting that the GF method can introduce highmolecular weight polymers to proteins. The band visibility inour SDS-PAGE results and the polymer introduction efficiencydifferences between GT and GF conjugates were consistentwith the previous report by Lin et al. They tried to comparethe GT and GF methods in small interfering ribonucleic acidconjugation to polymer in a similar PPC system.25 Anothercouple of interesting studies had compared GT and GFmethods for the introduction of polymers from cellulosesubstrates or graphene surfaces and both concluded that theGF method was superior to controlling the surface distributionof polymers than the GT method.26,27Temperature-Responsive Phase Transition Behavior.To confirm whether the introduction of PNIPAAm into theantibody provided temperature responsiveness, we measuredLCSTs and observed the temperature-responsive phasetransition. LCST was defined as the temperature at which50% transmission was observed. The LCST of IgG−PNIPAAmproduced by the GT method decreased with increasingmolecular weight: 40 °C, 30.5 °C, and 29.5 °C when thelength of the polymer chain introduced into the antibody waschanged to 2,500, 25,000, and 50,000, respectively (Figure 2).These findings were parallel with Hazer et.al., previous study asthey reported that the LCST of the PNIPAAm-PEGcopolymer showed a significant increase as the percentage ofPEG in the copolymer increased, indicating that the percentageof the hydrophilic conjugate to PNIPAAm affects LCST.32The LCST of IgG−PNIPAAm produced by the GF methodwas 31 °C, which was close to that of IgG−PNIAAm producedby the GT method, which introduced a polymer with a chainlength of tens of thousands. Field flow fractionation measure-ments from our previous case study, combined with the resultsof this experiment, suggest that the molecular weight of thepolymer in the conjugate, prepared using the GF method, is inthe tens of thousands.31Characterization of IgG−PNIPAAm Using DLS. DLSmeasurements were performed to confirm the change inparticle size as a result of polymer conjugation. The samplewith a polymer chain length of 50,000 prepared by the GTmethod showed a particle size of 29.2 ± 10.1 nm below LCST,which is larger than the other samples (Figure 3A). This resultsuggests that amide hydrogen bonds were induced because thedistance between the chain lengths of PNIPAAm was closer.33This leads to a larger particle size. We reported that the IgG-CTA and IgG−PNIPAAm precursor prepared by the GFmethod had particle sizes of 100−200 nm in our previousstudy (Figure 3B).31 The investigation approved the formationof 100−200 nm particle size by the GF method in APCs.Thermal Precipitation and Recovery Ratios of IgG−PNIPAAm Temperature-Responsive Polymeric Conju-gates. To compare the effect of different conjugation methodson the thermal precipitation efficiency, we evaluated therecovery ratio of IgG. All conjugates were thermally stimulated,followed by centrifugation to allow the sedimentation of thepolymeric conjugates. IgG concentration in the supernatantwas measured to calculate the IgG recovery ratio. In the samplewith a molecular weight of 2,500 prepared by the GT method,IgG could not be recovered because thermal precipitation didnot occur (Figure 4A). In samples with molecular weights of25,000 and 50,000, IgG recovery was about 40% and 70%,respectively, and the recovery ratio increased with increasingmolecular weight. For samples prepared using the GF method,the recovery ratios were approximately 45% at 250 mM andapproximately 70% at 400 and 550 mM, respectively (Figure4B). All samples prepared by the GF method, as well as thosewith a polymer chain length of 50,000 prepared by the GTmethod, exhibited high recovery values, which can indicate theformation of particles. The investigation implies an improve-ment of the thermal precipitation efficiency that could havebeen caused by entanglement between chain lengths when thePNIPAAm chain length introduced and the amount of freePNIPAAm in solution were increased.Evaluation of the IgG−PNIPAAm Antigen BindingConstant. The apparent binding affinities of the sample with aTable 1. Characterization of Conjugates of IgG−PNIPAAmsamplenameintroducedmolecularweight inGT method(g/mol)monomerconcentrationfor synthesisin GF method(mM)conjugationyield (%)LCST(°C)IgGrecoveryratio (%)GT-2,5002,500 − 34 40 −GT-25,00025,000 − − 30.5 37.6GT-50,00050,000 − − 29 72.8GF-250 − 250 − 31 45.3GF-400 − 400 61 31 66.5GF-550 − 550 − 30 71.3Figure 2. Transmittance of the IgG−PNIPAAm solutions againsttemperature (thermal-response; solvent: PBS (pH: 7.4), IgGconcentration: 0.5 mg/mL, heating rate: 0.2 °C/min, wavelength:450 nm).ACS Omega http://pubs.acs.org/journal/acsodf Articlehttps://doi.org/10.1021/acsomega.4c00103ACS Omega 2024, 9, 22043−2205022047https://pubs.acs.org/doi/10.1021/acsomega.4c00103?fig=fig2&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.4c00103?fig=fig2&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.4c00103?fig=fig2&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.4c00103?fig=fig2&ref=pdfhttp://pubs.acs.org/journal/acsodf?ref=pdfhttps://doi.org/10.1021/acsomega.4c00103?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-aspolymer chain length of 20,000 prepared by the GT method(GT-25,000) and prepared by the GF method at 400 mMconjugates (GF-400) were evaluated using a sandwich ELISA(developed in-house). The results of binding affinities to thefree antigen are listed in Figure 5. When the maximum bindingratio of native IgG to the antigen was 100%, the maximumbinding ratio of the GT-2,500 conjugate and the GF-400conjugates was 90% and 80%, respectively. The maximumbinding ratio of the GF-400 conjugate was similar to that ofour previous study, suggesting that the introduction ofpolymers slightly decreased the binding affinity of theantibody. However, in the GT-2,500 conjugate, the maximumbinding ratio was slightly lower than the free antibody and asgood as the GF method one, presumably due to the closestructure of the conjugate produced by the GT and GFmethods. Both the GT and GF methods, in most cases, resultin similar cyclic peptide−polymer conjugates. This has beendiscussed in studies where peptide−polymer conjugates weresystematically compared using both the GT synthesis routeand the GF polymer synthesis route.34Previously, our team succeeded in the introduction ofpolymeric antibodies by click reaction in a GT strategy withhigh polymer introduction efficiency, but the maximumantigenic binding ratio decreased markedly to approximately30% after polymeric conjugation.10 These findings suggest thatantibody modification at the polymer end is less affected by theantigen−antibody reaction than polymer modification at thepolymer side chain.Evaluation of Antigen Enrichment Using a LateralFlow Immunoassay Strip. Antibody-temperature-responsivepolymer conjugates prepared by the GF method were used toenrich antigens to evaluate their antigen enrichment capacityand their potential in improving the diagnostic sensitivity ofLFIA. Mouse IgG was used as the model antigen. Theminimum detection limit of mouse IgG using LFIA was 25 ng/mL, as shown in Figure 6A while lower concentrations,including 10 and 5 ng/mL, showed false negative results. Afterpolymer introduction, antigen−antibody reaction was allowed,and antigens were enriched in the misdiagnosed sample of 10Figure 3. Confirmation of the particle size of IgG−PNIPAAm conjugates using DLS at 25 °C and IgG concentration of 0.05 mg/mL. (A) Samplesproduced by the GT method. (B) samples by the produced GF method.Figure 4. Effects of different conjugation methods on the IgG recovery ratio (mean ± SD, n = 3). (A) Samples produced using the GT method. (B)Samples produced by the GF method.Figure 5. Effect of antigen concentration on antigen−antibodyaffinities measured by ELISA (mean ± SD, n = 3).Figure 6. Lateral flow immunoassay (LFIA) evaluation for mouse IgGdetection using our developed antibody-temperature-responsivepolymer enrichment strategy. (A) Different concentrations ofmouse IgG were tested using a lateral flow immunoassay. (B)Comparison of misdiagnosed samples before and after polymericenrichment using the mouse antibody-temperature-responsive poly-mer.ACS Omega http://pubs.acs.org/journal/acsodf Articlehttps://doi.org/10.1021/acsomega.4c00103ACS Omega 2024, 9, 22043−2205022048https://pubs.acs.org/doi/10.1021/acsomega.4c00103?fig=fig3&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.4c00103?fig=fig3&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.4c00103?fig=fig3&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.4c00103?fig=fig3&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.4c00103?fig=fig4&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.4c00103?fig=fig4&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.4c00103?fig=fig4&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.4c00103?fig=fig4&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.4c00103?fig=fig5&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.4c00103?fig=fig5&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.4c00103?fig=fig5&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.4c00103?fig=fig5&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.4c00103?fig=fig6&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.4c00103?fig=fig6&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.4c00103?fig=fig6&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.4c00103?fig=fig6&ref=pdfhttp://pubs.acs.org/journal/acsodf?ref=pdfhttps://doi.org/10.1021/acsomega.4c00103?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-asng/mL by thermal stimulation followed by centrifugation. Thelateral flow band of the 10 ng/mL misdiagnosed sample beforeand after enrichment, which was not visible before enrichment,became clearly visible after enrichment (Figure 6B).Interestingly, polymeric enrichment allowed different sub-classes of mouse IgG to be enriched and visualized for the firsttime compared to the highest concentration of the antigen thatdid not appear without our strategy. The enrichment capacityin this experiment was up to 10 times that of the originalconcentration. The sample with an antigen concentration of 10ng/mL, which was misdiagnosed without polymeric enrich-ment, was enriched to an antigen concentration equivalent to100 ng/mL and the test line became visible after applying ourstrategy. These data showed good agreement with our previousLFIA results, as we succeeded in antigenic enrichment andavoided LFIA misdiagnosis in our previous reports.1,10Furthermore, there are no other reports of attempts toenhance the sensitivity of lateral flow immunoassays byenriching antigens using antibody-PNIPAAm conjugatesproduced by the GF method. In addition, it is known thatimmobilization of antibodies on the surface of nanoparticlesenhances the diagnostic performance of ELISA. This trend isalso observed in nanoparticle-produced antibody-PNIPAAmconjugates produced by the GF method reported in ourprevious study.31 From this, it is suggested that the GF methodis a superior technique in terms of enhancing the diagnosticperformance of lateral immunoassays after antigen enrichment.Finally, what makes the GF method superior to the GTmethod is that in the GF method, antibodies are conjugated toresponsive polymers to produce PPCs before antigen−antibody interactions, which allow its involvement in a varietyof applications.1,10 On the basis of the above, the GF method isexpected to highly contribute to LFIA diagnostic sensitivityimprovement by antigen enrichment.■ CONCLUSIONSThis research describes the evaluation of IgG−PNIPAAmconjugates produced by two different methods, GT and GF,with respect to their efficiency and temperature-responsivephase transition behavior. The efficiency of polymerintroduction to antibody was evaluated by SDS-PAGE analysis,and the ratio of polymer introduction to antibody wascalculated. The GT method showed a lower introductionefficiency than the GF method. LCSTs were measured toobserve the temperature-responsive phase transition, and theLCST of IgG−PNIPAAm produced by the GF method wasfound to be close to that of IgG−PNIPAAm produced by theGT method, which introduced a polymer with a chain lengthof tens of thousands. DLS measurements were performed toconfirm the change in particle size as a result of polymerconjugation. The particle size of the IgG−PNIPAAmconjugate produced by the GT method increased with anincreasing molecular weight of the polymer chain length.However, the IgG−PNIPAAm conjugate produced by the GFmethod had a particle size of 100−200 nm, confirming theformation of APCs. In summary, both methods offer uniqueadvantages and challenges. The GT method providessimplicity but demands careful design and purification, whereasthe GF allows precise control but requires appropriate surfacechemistry for initiation. We need to select the mostappropriate method based on specific applications and desiredsurface modifications. In general, these findings provideimportant insight into the design and development of novelbiomaterials that respond to temperature.■ ASSOCIATED CONTENT*sı Supporting InformationThe Supporting Information is available free of charge athttps://pubs.acs.org/doi/10.1021/acsomega.4c00103.1H NMR spectrum of COOH-PNIPAAm in DMSO(Figure S1) and GPC results of COOH-PNIPAAm(Figure S2 and Table S1) (PDF)■ AUTHOR INFORMATIONCorresponding AuthorsMitsuhiro Ebara − Research Center for Macromolecules andBiomaterials, National Institute for Materials Science(NIMS), Tsukuba 305-0044, Japan; Graduate School ofPure and Applied Sciences, University of Tsukuba, Tsukuba305-8577, Japan; Graduate School of Industrial Science andTechnology, Tokyo University of Science, Shinjuku, Tokyo162-0825, Japan; orcid.org/0000-0002-7906-0350;Email: ebara.mitsuhiro@nims.go.jpAhmed Nabil − Research Center for Macromolecules andBiomaterials, National Institute for Materials Science(NIMS), Tsukuba 305-0044, Japan; Biotechnology and LifeSciences Department, Faculty of Postgraduate Studies forAdvanced Sciences (PSAS), Beni-Suef University, Beni-Suef62511, Egypt; Egyptian Liver Research Institute and Hospital(ELRIAH), El Mansoura 35511, Egypt; orcid.org/0000-0002-5617-4726; Email: tolba.ahmednabil@nims.go.jpAuthorsErika Yoshihara − Research Center for Macromolecules andBiomaterials, National Institute for Materials Science(NIMS), Tsukuba 305-0044, Japan; Graduate School ofPure and Applied Sciences, University of Tsukuba, Tsukuba305-8577, JapanMichihiro Iijima − Department of Materials Chemistry andBioengineering, National Institute of Technology, OyamaCollege (NIT, Oyama College), Oyama 323-0806, JapanComplete contact information is available at:https://pubs.acs.org/10.1021/acsomega.4c00103NotesThe authors declare no competing financial interest.■ ACKNOWLEDGMENTSThe authors of this manuscript express their great gratitude toNanotechnology Innovation Station at NIMS for their support.The authors are grateful to Prof. Allan S. 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