Fc-specific Covalent Modification Of Antibody By Photoactivatable IgG-binding Peptide And Its Application In Immunoassay

Labeled immunoassays are the most active and fastest growing technology in current immunodiagnostic field,and heterogeneous immunoassay is a type of labeled immunoassay that requires the separation of bound and free signal-substrate,and is also the most widely used analysis method in immunoassays,such as enzyme-linked immunosorbent assay.For heterogeneous immunoassay,the immobilized antibodies are the indispensable reagents.Covalent immobilization relies on the chemical reaction of amino acid residues(amine/carboxyl groups)within antibody,and antibodies can be stably immobilized onto substrates via chemical cross-linkers,and stability of covalently immobilized antibodies is much higher than that of physically adsorbed antibodies.Covalent conjugation strategy is efficient but suffers from non-specificity because all functional groups of antibody can possibly react.Due to uncontrolled and random immobilization,these immobilization methods result in a low density of antigen binding sites,and increasing the amount of antibodies adsorbed on the support cannot effectively increase the antigen binding capacity.The development of antibody conjugation strategy with site-specific pattern is of great significance to further improve the sensitivity and specificity of immunoassay.Herein,we describe a general and effective approach for sites-pecific and covalent conjugation of native IgGs photoactivatable Fc-binding protein-assisted conjugation strategy.In this study,the Z domain,a well-known Fc-binding molecule,was incorporated in a photoactivatable probe(p-benzoyl-L-phenylalanine,Bpa)and then tagged with Cys handle.Then,genetic code expansion technology was used to obtain the photoactivatable ZBpa-Cys recombinant.Subsequently,by employing the bio-affinity with Fc region and covalent bond forming ability of the ZBpa moiety,the unique functional Cys handle was covalently conjugated to native antibody,resulting in the Fc-specific thiolated antibody.On the basis of the reaction between sulfhydryl and maleimide groups,the thiolated antibody was covalently immobilized onto the maleimide modified solid support.Thus,a general platform for directional immobilization of antibody with a covalent and homogeneous patternwas developed.This research contents were as follows.(1)Design,construction and production of photoactivatable ZBpa-Cys recombinantsZ-domian,a well-known Fc-binding molecule,is a derivative of the B-domain of Staphylococcus aureus protein A(SpA),and without cysteine(Cys)residue in its amino acid sequence.The sulfhydryl group is a useful reactive group for protein conjugation,thus,sulfhydryl group(provided by cysteine residues)is selected as the specific functional group in this project.On the basis of consulting relevant literatures on affinity binding information between Z-domain and IgG,the PyMOL software(1.0.0.0)was used for the simulation of affinity binding.The Fc-facing amino acids in α1(4-18)and α2(24-36)helixes of Z-domain,were selected as the objects inserted for Bpa replacement.The corresponding amino acid codon in the coding DNA sequence was mutated into amber codon(TAG).The target gene sequences were obtained by chemical synthesis and subcloned into the pTBX1 plasmid,yielding 12 pZTAG-Cys expression vectors with different Bpa sites.Those pZ TAG-Cys vectors were co-transformed into E.coli BL21(DE3)cells with pEVOL-pBpF plasmid,respectively,yielding 12 genetic engineering bacteria.The genetic code expansion technique was used to express the various photoactivatable ZBpa-Cys recombinants.Then the target protens were purified by immobilized metal ion affinity chromatography(IMAC),and Bpa site in the target protein sequence was detected by mass spectrometry.In addition,the cultivation conditions of genetically engineered bacteria were optimized using orthogonal design.The results showed that,those selected codons were successfully mutated to amber codon,and the pZ TAG-Cys plasmid expression vectors were successfully constructed.The host E.coli cells harboring pZTAG-Cys and pEVOL-pBpF were cultured in the presence of Bpa,those target proteins were successfully expressed with a molecular weight of 9 kDa,which is consistent with the theoretical value,respectively.Mass spectrometry results indicated that Bpa was substituted and inserted into the Z-domian peptide chain at the selected amino acid residue site.Under the shake flask culture in this experiment,the optimal expression conditions were as follows:after overnight activation,genetic engineering bacteria were transferred to fresh LB broth at 37℃ and 220 r/min at 1:50 for 3 h,then Bpa with final concentration of 3 mmol/L and 0.2%(w/v)arabinose were added to culture for 0.5 h,then IPTG with final concentration of 0.25 mmol/L was added to induce expression for 4 h.Under optimized expression conditions,approximately 20-30 mg of target proteins was harvested after purification by IMAC from 1 L LB broth.(2)Photo-conjugation of ZBpa-Cys recombinants with IgGsThe photo-conjugation of 17thZBpa-Cys and rabbit IgG was used as the research model to optimize molar ratio of ZBpa-Cys and IgG.To assess the molar ratio of ZBpa-Cys to IgG and the excitation time of 365 nm UV on the crosslinking,the photo-conjugates were examined by non-reducing and reducing SDS-PAGE analysis during UV exposure.To further verify the Fc-selective conjugation,the control experiment of photo-conjugation between 17thZBpa-Cys and rabbit F(ab’)2 was implemented.Furthermore,the coupling product was digested by papain for the further validation of Fc-selective conjugation.Each ZBpa-Cys mutant was paired with rabbit IgG,mouse IgG1,IgG2a,IgG2b and IgG3,and then the photo-conjugation efficiency was screened by reducing SDS-PAGE analysis under the optimized conditions.To discuss the correlation mechanism between Bpa functional site and photo-conjugation,mouse IgG2a was selected as the model,SPR technology was used to analyze the affinity constants of 5thZBpa-Cys,17thZBpa-Cys and 32thZBpa-Cys,which with different photo-conjugation efficiency to mouse IgG2a.The results showed that,17thZBpa-Cys and rabbit IgG were mixed at the optimal molar ratio at least 3:1 and incubated at 4℃ for bio-affinity coupling.Then,they were exposed to 365 nm UV for photo-conjugation.After 2 h irradiation,the image analysis indicated that up to 90%of IgG was crosslinked with ZBpa-Cys.The results of control experiment and papain digestion analysis indicated that ZBpa-Cys was covalently conjugated to the Fc region of IgG.After pairing selection,rabbit IgG and mouse IgG that commonly used in immunoassay,could be covalently modified by ZBpa-Cys mutants with different Bpa functional sites,that is,17thZBpa-Cys could be candidate for photo-conjugation of rabbit IgG,mouse IgG2a,IgG2b,and IgG3,and 32thZBpa-Cys could be candidate for mouse IgG1.Thus,a Cys handle was successfully conjugated to the Fc region of native antibody by employing Fc-binding protein-assisted photo-conjugation,and the Fc-specific modified IgG was namely IgG-CysFc.SPR results suggested that there was no association between the affinity of photoactivatable ZBpa-Cys to IgG,and the possible mechanisms affecting the photo-conjugation efficiency might be the space distance between the Bpa functional site in the ZBpa-Cys and amino acid residues in IgG,which provides-H for forming covalent bond with Bpa.(3)Immobilization of thiolated IgGs onto maleimide-modified solid support and its applications in immunoassayThe thiolated IgGs(IgG-CysFc)were directionally conjugated and immobilized on the surfaces of maleimide functionalized PS microplate and MNPs by employing the specific reaction between sulfhydryl and maleimide group,trespectively.And the detection of carcinoembryonic antigen(CEA)was used as a research model to investigate the detection efficiency of the directionally immobilized antibody.In the microplate ELISA,IgG-CysFc(Rabbit anti-CEA IgG)was specifically immobilized on the maleimide modified surface of PS microplate,and then was used for the determination of CEA.To obtain the maleimide-modified MNPs,the commercial NH2-MNPs were modified by Sulfo-EMCS.For immobilization of the thiolated IgG onto Mal-MNPs,Mal-MNPs was mixed with various concentration of the thiolated IgG in conjugation buffer and incubated at 4℃ for 2 h with gentle rotation for crosslinking reaction.The amount of conjugated protein onto MNPs was monitored by color reaction of BCA reagent.The IgG-CysFc coated Mal-MNPs were namely IgG-CysFc@MNPs and submitted to SDS-PAGE analysis.For control experiment,rabbit anti-CEA IgG was conjugated onto NH2-MNPs with glutaraldehyde two-step conjugation method and resulted in IgG@MNPs.Thus,IgG-CysFc@MNPs and IgG@MNPs were used to establish a sandwich MNPs-based ELISA for the determination of CEA.The detection efficiency of IgG-CysFc@MNPs-based ELISA for detection of CEA was evaluated by examining limit of detection,specificity,stability and anti-serum IgG interference.The results showed that,the BCA analysis results indicated that the maximum coupling amount of IgG-CysFc on the surface of Mal-MNPs was approximately 50 μg protein per mg MNPs for IgG-CysFc@MNPs,and the maximum coupling amount of IgG on the surface of NH2-MNPs was about 60 μg IgG per mg MNPs for IgG@MNPs.The SDS-PAGE results indicated that the conjugated IgG-CysFc onto Mal-MNPs was mediated by the specific reaction between sulfhydryl and maleimide group.The results of microplate ELISA presented the detection limit was 0.7 ng/mL of the site-specific and covalent immobilized antibodies,which was approximately 3-fold more sensitive than passive adsorption of coated antibody(2.0 ng/mL).Another,the IgG-CysFc@MNPs-based ELISA presented the detection limit was 0.02 ng/mL,which was approximately 20-fold more sensitive than that of IgG@MNPs(0.4 ng/mL).The intra day and inter day spiking recoveries of IgG-CysFc@MNPs for CEA determination were from 103.25%to 107.65%and from 97.33%to 101.81%,respectively.And the relative standard deviations were from 1.93%to 6.79%and from 5.54%to 10.6%,respectively.The results suggested that the proposed IgG-CysFc@MNPs-based ELISA was reliable in terms of selectivity and precision for the detection of CEA without interference of the intrinsic IgG in serum.Five clinical serum samples were determined by the proposed IgG-CysFc@MNPs-based ELISA,and the results showed absolute relative deviation between the measured results and clinical test values was within 10%,and the two analytical methods methods have good correlation(R2=0.9901),which suggested that the proposed IgG-CysFc@MNPs has a wide range of potential application in immunological diagnosis.In summary,in this project,12 photoactivatable ZBpa-Cys recombinants with various Bpa functional sites were constructed by genetic code expansion technology.Then,take the photoactivatable ZBpa-Cys recombinant as a bridge,the specific and functional Cys handle was successfully conjugated to the Fc region of rabbit IgG and mouse IgG by employing Fc-binding protein-assisted photo-conjugation strategy.The proposed strategy fully utilized the advantages of the site specificity of Z-domain binding to IgG and the stability of chemical covalent conjugation.Thus,the specific and selective functional group for further antibody conjugation was provided,and a general platform was developed for covalent,oriented immobilization of antibodies onto maleimide group-modified supports.The feasibility and effectiveness of the proposed antibody immobilization platform for enhancing the sensitivity of detection were discussed via the detection of CEA.In conclusion,the proposed approach could mediate different functional groups for Fc-specific covalent modification of antibody and combined with solid phase carrier surface modification,it has broad application prospects in antibody chips and immunosensors.Furthermore,this strategy also has potential applications in the field of targeted drug delivery,such as antibody-drug conjugates research.