A high-affinity gold-binding camel antibody: antibody engineering for one-pot functionalization of gold nanoparticles as biointerface molecules

Antibodies, with their high affinity and specificity, are widely utilized in the field of protein engineering, medicinal chemistry, and nanotechnology applications, and our recent studies have demonstrated the recognition and binding of antibody for the surface on inorganic material. In this study, we generated a high-affinity gold-binding antibody fragment by a combination of peptide-grafting and phage-display techniques and showed the availability of the material-binding fragment for one-pot functionalization of nanoparticles as interface molecules. After a gold-binding peptide sequence was grafted into one of the complementarity determining regions of a single variable domain of a heavy-chain camel antibody, a combinatorial library approach raised by 20 times the affinity of the peptide-grafted fragment. The high-affinity gold-binding fragment (E32) spontaneously adsorbed on gold nanoparticles, and consequently the nanoparticles formed a stable dispersion in a high-ionic-strength solution. Multivalent and bispecific antibodies constructed on the E32 platform by means of fusion technology functionalized gold nanoparticles in one pot, and these functionalized nanoparticles could be used to obtain surface plasmon resonance scattering images of cancer cells and to spontaneously link two different nanomaterials. Here, we propose the bispecific antibodies as convenient interface molecules in the nanosized world.     

双专－性抗体介导的纤溶作用

用双功能团试剂将抗尿激酶单克隆抗体Ｎ３４的ＩｇＧ和抗人活化血小板α－颗粒膜蛋白ＧＭＰ－１４０单克隆抗体ＳＺ－５１的Ｆａｂ片段通过二硫键共价偶联,偶联的抗体保留了对各自抗原的亲和性。这种对尿激酶和血栓同时具有亲和活性的双专一性抗体（Ｎ３４－ＳＺ－５１）提高低分子量尿激酶的溶栓效率３８倍,且对血浆中纤维蛋白原的含量基本上不影响。     

Antibody-directed fibrinolysis. An antibody specific for both fibrin and tissue plasminogen activator

An investigation was made to determine whether it is possible to attract tissue plasminogen activator (tPA) to the site of a thrombus by means of an antibody with affinites for both tPA and fibrin. A bispecific antibody conjugate was constructed by cross-linking two monoclonal antibodies: one specific for tPA, the other specific for fibrin. The bispecific antibody enhanced fibrinolysis by capturing tPA at the site of a fibrin deposit. In an in vitro quantitative fibrinolysis assay, the relative fibrinolytic potency of tPA bound to the bispecific antibody was 13 times greater than that of tPA and 200 times greater than that of urokinase. When fibrin was treated with the bispecific antibody before being mixed with tPA, the relative fibrinolytic potency of tPA was enhanced 14-fold. This capture also occurred when the concentration of tPA present in the assay was less than the concentration of tPA present in normal human plasma. In a human plasma clot assay, samples containing both the bispecific antibody and tPA exhibited significantly more lysis than did samples containing tPA alone. In spite of the increased clot lysis effected by the presence of bispecific antibody, there was no significant increase in fibrinogen or alpha 2-antiplasmin degradation at equal tPA concentrations. The ability of the bispecific antibody to concentrate exogenous tPA in vivo was then examined in the rabbit jugular vein model. Systemic infusion of a small amount of tPA (10,000 units) produced no significant increment in thrombolysis over the level of spontaneous lysis (14 +/- 8%). However, the simultaneous infusion of 10,000 units of tPA and 2 mg of bispecific antibody resulted in 42 +/- 14% (p less than 0.01) lysis. These results suggest that a molecule capable of binding both fibrin and tPA with high affinity could enhance thrombolysis in the circulation by capturing endogenous tPA.     

Expression and purification of monospecific and bispecific recombinant antibody fragments derived from antibodies that block the CD80/CD86-CD28 costimulatory pathway

The development of recombinant techniques for rapid cloning, expression, and characterization of cDNAs encoding antibody (Ab) subunits has revolutionized the field of antibody engineering. By fusion to heterologous protein domains, chain shuffling, or inclusion of self-assembly motifs, novel molecules such as bispecific Abs can be generated that possess the subset of functional properties designed to fit the intended application. We describe the engineering of Ab fragments produced in bacteria for blocking the CD28-CD80/CD86 costimulatory interaction in order to induce tolerance against transplanted organs. We designed single-chain Fv antibodies, monospecific and bispecific diabodies, and a bispecific tetravalent antibody (BiTAb) molecule directed against the CD80 and/or CD86 costimulatory molecules. These recombinant Ab molecules were expressed in Escherichia coli, followed by purification and evaluation for specific interaction with their respective antigen in an enzyme-linked immunosorbent assay (ELISA). A specific sandwich ELISA confirmed the bispecificity of the bispecific diabodies and the BiTAb.     

Miniantibodies: use of amphipathic helices to produce functional, flexibly linked dimeric FV fragments with high avidity in Escherichia coli.

We have designed dimeric antibody fragments that assemble in Escherichia coli. They are based on single-chain FV fragments, with a flexible hinge region from mouse IgG3 and an amphiphilic helix fused to the C-terminus of the antibody fragment. The sequence of the helix was taken either from that of a previously reported four-helix bundle design or from a leucine zipper, optionally extended with a short cysteine-containing peptide. The bivalent fragments associate in vivo, either with covalent linkage or with a monomer-dimer equilibrium, and results from ultracentrifugation sedimentation studies and SDS-PAGE are consistent with dimers. All constructs are able to bind to surface-bound antigen under conditions in which only bivalent but not monovalent antibody fragments bind. The covalent bundle helix construct shows binding characteristics nearly identical to those of the much larger whole mouse antibody, resulting in substantially more stable immunoglobulin-antigen complexes than in the case of monovalent fragments. This modular design of natural and engineered protein domains directly leads to a boost of avidity, and it allows the construction of bispecific antibody fragments in functional form in E. coli.     

神经纤毛蛋白-1的原核表达及兔抗小鼠神经纤毛蛋白-1抗体免疫学活性的研究

目的:在原核系统中分段表达神经纤毛蛋白-1(Nrp1);将纯化的蛋白免疫家兔后获得特异的抗体,并将其应用于检测组织和细胞中Nrp1分子的表达。方法:提取BALB/c胎鼠脑组织总RNA,通过RT-PCR分段扩增获得Nrp1基因片段,将PCR产物插入表达载体pET28a( ),获得含5个Nrp1基因片段的重组质粒,在大肠杆菌BL21(DE3)中诱导蛋白表达并纯化;将纯化的重组蛋白免疫新西兰大白兔获得针对目的蛋白的特异性多克隆抗体;利用Nrp1特异性多抗检测胎鼠脑组织和HeLa细胞中Nrp1的表达。结果:在原核系统中分段表达了Nrp1蛋白,通过Ni-NTA纯化了Nrp1蛋白片段;纯化的Nrp1蛋白免疫新西兰大白兔获得了具有免疫活性的多抗;兔抗小鼠Nrp1特异性多抗可用于检测组织、真核细胞中Nrp1的表达。结论:应用原核系统成功地表达了Nrp1蛋白,兔抗小鼠Nrp1特异性多抗可用于免疫学检测Nrp1分子的表达。     

Single chain Fab (scFab) fragment

Background  

The connection of the variable part of the heavy chain (VH) and and the variable part of the light chain (VL) by a peptide linker to form a consecutive polypeptide chain (single chain antibody, scFv) was a breakthrough for the functional production of antibody fragments in Escherichia coli. Being double the size of fragment variable (Fv) fragments and requiring assembly of two independent polypeptide chains, functional Fab fragments are usually produced with significantly lower yields in E. coli. An antibody design combining stability and assay compatibility of the fragment antigen binding (Fab) with high level bacterial expression of single chain Fv fragments would be desirable. The desired antibody fragment should be both suitable for expression as soluble antibody in E. coli and antibody phage display.     

Generating bispecific human IgG1 and IgG2 antibodies from any antibody pair

Bispecific antibodies and antibody fragments are a new class of therapeutics increasingly utilized in the clinic for T cell recruitment (catumaxomab anti-EpCAM/CD3 and blinatumomab anti-CD19/CD3), increase in the selectivity of targeting, or simultaneous modulation of multiple cellular pathways. While the clinical potential for certain bispecific antibody formats is clear, progress has been hindered because they are often difficult to manufacture, may suffer from suboptimal pharmacokinetic properties, and may be limited due to potential immunogenicity issues. Current state-of-the-art human IgG-like bispecific technologies require co-expression of two heavy chains with a single light chain, use crossover domains to segregate light chains, or utilize scFv (single-chain fragment variable)-Fc fusion. We have engineered both human IgG1 and IgG2 subtypes, with minimal point mutations, to form full-length bispecific human antibodies with high efficiency and in high purity. In our system, the two antibodies of interest can be expressed and purified separately, mixed together under appropriate redox conditions, resulting in a formation of a stable bispecific antibody with high yields. With this approach, it is not necessary to generate new antibodies that share a common light chain, therefore allowing the immediate use of an existing antibody regardless of whether it has been generated via standard hybridoma or display methods. We demonstrate the generality of the approach and show that these bispecific antibodies have properties similar to those of wild-type IgGs, and we further demonstrate the utility of the technology with an example of a CD3/CD20 bispecific antibody that effectively depletes B cells in vitro and in vivo.     

Efficient construction of a diabody using a refolding system: anti-carcinoembryonic antigen recombinant antibody fragment

Recombinant fragments of the variable region of antibodies are useful in many experimental and clinical applications. However, it can be difficult to obtain these materials in soluble form after their expression in bacteria. Here, we report an efficient procedure for preparing several variable-domain fragments (Fv), single-chain Fv (scFv), and a diabody (the smallest functional bispecific antibody) of anti-carcinoembryonic antigen (CEA) antibody by overexpression in Escherichia coli in inclusion bodies, using a refolding system to obtain renatured proteins. Two types of refolded Fv were prepared: (i) Heavy and light chains of the immunoglobulin variable regions (VH and VL, respectively) were coexpressed with a dicistronic expression vector (designated Fv(co)); (ii) VH and VL were expressed separately, mixed stoichiometrically, and refolded (designated Fv(mix)). All samples refolded with high efficiency; Fv(co), Fv(mix), scFv, and the bispecific diabody bound to several CEA-positive cell lines, exactly as did soluble Fv fragments secreted by E. coli (Fv(sol)) and the parent IgG. The refolded fragments inhibited binding of the parent IgG to CEA-positive cell lines, indicating that their epitope is identical to that of IgG. The bispecific diabody, which combined variable-region fragments of anti-CEA antibody with variable-region fragments of anti-CD3 antibody, was also prepared using the refolding system. This refolded diabody could bind to lymphokine-activated killer cells. In addition, its cytotoxicity toward human bile duct carcinoma TFK-1 and other several other CEA-positive cell lines was concentration-dependent. Taken together, our results suggest that a refolding procedure can be used to prepare various functional antibody fragments (Fv, scFv, and diabody).     

Constant domain-exchanged Fab enables specific light chain pairing in heterodimeric bispecific SEED-antibodies

BackgroundBispecific antibodies promise to broadly expand the clinical utility of monoclonal antibody technology. Several approaches for heterodimerization of heavy chains have been established to produce antibodies with two different Fab arms, but promiscuous pairing of heavy and light chains remains a challenge for their manufacturing.MethodsWe have designed a solution in which the C_H1 and C_L domain pair in one of the Fab fragments is replaced with a C_H3-domain pair and heterodimerized to facilitate correct modified Fab-chain pairing in bispecific heterodimeric antibodies based on a strand-exchange engineered domain (SEED) scaffold with specificity for epithelial growth factor receptor and either CD3 or CD16 (FcγRIII).ResultsBispecific antibodies retained binding to their target antigens and redirected primary T cells or NK cells to induce potent killing of target cells. All antibodies were expressed at a high yield in Expi293F cells, were detected as single sharp symmetrical peaks in size exclusion chromatography and retained high thermostability. Mass spectrometric analysis revealed specific heavy-to-light chain pairing for the bispecific SEED antibodies as well as for one-armed SEED antibodies co-expressed with two different competing light chains.ConclusionIncorporation of a constant domain-exchanged Fab fragment into a SEED antibody yields functional molecules with favorable biophysical properties.General significanceOur results show that the novel engineered bispecific SEED antibody scaffold with an incorporated Fab fragment with C_H3-exchanged constant domains is a promising tool for the generation of complete heterodimeric bispecific antibodies with correct light chain pairing.     

A mixed self-assembled monolayer-based surface plasmon immunosensor for detection of E. coli O157:H7

The sensitivity and specificity of a polyethylene glycol terminated alkanethiol mixed self-assembled monolayers (SAM) on surface plasmon resonance (SPR) immunosensor to detect Escherichia coli O157:H7 is demonstrated. Purified monoclonal (Mabs) or polyclonal antibodies (PAbs) against E. coli O157:H7 were immobilized on an activated sensor chip and direct and sandwich assays were carried to detect E. coli O157:H7. Effect of Protein G based detection and effect of concentrations of primary and secondary antibodies in sandwich assay were investigated. The sensor surface was observed under an optical microscope at various stages of the detection process. The sensor could detect as low as 10(3)CFU/ml of E. coli O157:H7 in a sandwich assay, with high specificity against Salmonella Enteritidis. The detection limit using direct assay and Protein G were 10(6)CFU/ml and 10(4)CFU/ml, respectively. Results indicate that an alkanethiol SAM based SPR biosensor has the potential for rapid and specific detection of E. coli O157:H7, using a sandwich assay.     

A bispecific antibody effectively neutralizes all four serotypes of dengue virus by simultaneous blocking virus attachment and fusion

Although dengue virus (DENV) infection severely threatens the health of humans, no specific antiviral drugs are currently approved for clinical use against DENV infection. Attachment and fusion are 2 critical steps for the flavivirus infection, and the corresponding functional epitopes are located at E protein domain III (E-DIII) and domain II (E-DII), respectively. Here, we constructed a bispecific antibody (DVD-1A1D-2A10) based on the 2 well-characterized anti-DENV monoclonal antibodies 1A1D-2 (1A1D) and 2A10G6 (2A10). The 1A1D antibody binds E-DIII and can block the virus attaching to the cell surface, while the 2A10 antibody binds E-DII and is able to prevent the virus from fusing with the endosomal membrane. Our data showed that DVD-1A1D-2A10 retained the antigen-binding activity of both parental antibodies. Importantly, it was demonstrated to be significantly more effective at neutralizing DENV than its parental antibodies both in vitro and in vivo, even better than the combination of them. To eliminate the potential antibody-dependent enhancement (ADE) effect, this bispecific antibody was successfully engineered to prevent Fc-γ-R interaction. Overall, we generated a bispecific anti-DENV antibody targeting both attachment and fusion stages, and this bispecific antibody broadly neutralized all 4 serotypes of DENV without risk of ADE, suggesting that it has great potential as a novel antiviral strategy against DENV.     

Conjugation and evaluation of 7E3 x P4B6, a chemically cross-linked bispecific F(ab')2 antibody which inhibits platelet aggregation and localizes tissue plasminogen activator to the platelet surface.

A bispecific F(ab')2 monoclonal antibody which recognizes both the platelet GPIIb/IIIa receptor and human tissue plasminogen activator was produced to target tPA to platelets for enhancement of thrombolysis. A stable, thioether-cross-linked bispecific F(ab')2 (7E3 X P4B6) combining the GPIIb/IIIa-specific monoclonal antibody 7E3, which inhibits platelet aggregation, and a nonneutralizing anti-tPA monoclonal antibody (P4B6) was produced. This was performed by coupling each of the parental Fab' moieties with the homobifunctional cross-linker bis(maleimido methyl) ether (BMME). 7E3 X P4B6 was sequentially purified using gel-filtration chromatography and hydrophobic interaction (HIC) HPLC. HIC was shown to completely resolve each of the parental F(ab')2 species from the bispecific one. 7E3 X P4B6 was shown to retain completely each of the parental immunoreactivities in GPIIb/IIIa and tPA binding EIA's. The bispecific antibody inhibited platelet aggregation in vitro at levels comparable to those for 7E3 Fab. Recruitment of tPA activity to washed human platelets was demonstrated using the S-2251 chromogenic substrate assay. 7E3 X P4B6 recruited 12-fold more tPA to the washed platelets than a mixture of the parental F(ab')2 molecules used as controls.     

Fabrication of an antibody microwell array with self-adhering antibody binding protein

One of the promising methods of preparing antibody arrays is immobilizing antibodies with protein A or protein G, each of which binds specifically to the heavy chain constant (Fc) region of immunoglobulin G (IgG). In this system, antibody immobilization efficiency depends on the number of active Fc binding proteins that need to be immobilized on the surface. Here we have designed and constructed an Fc binding protein with a self-adhering ability that can be immobilized on the hydrophobic surface by simple adsorption. It consists of an Fc binding domain of protein G (G3) and hydrophobic domain of elastin (E72). Direct observation revealed its self-adhering ability on the hydrophobic surface. The enzyme-linked immunosorbent assay (ELISA) showed that it retained antibody binding ability on the surface. The antibody array model was prepared on a hydrophobic microwell glass slide with E72G3, which specifically detect the antigen with a sevenfold greater sensitivity than the G3-treated slide. These results suggest that the E72G3 is useful for simple and effective immobilization of antibodies and can be used to fabricate any immuno devices.     

Optimizing recombinant antibody function in SPR immunosensing. The influence of antibody structural format and chip surface chemistry on assay sensitivity

BACKGROUND: Recombinant antibody fragments are valuable tools for SPR-based detection of small molecules such as illicit drugs. However, the multiple structural formats of recombinant antibody fragments are largely uncharacterised with respect to their respective performance in SPR sensing. We have expressed a model anti-M3G antibody in both scFv and chimeric Fab formats to examine its sensitivity and binding profiles in a microplate immunoassay format and Biacore. We have further examined the influence of scFv multimerisation, Fab constant region stability and SPR chip surface coating chemistry, on anti-hapten SPR assay development. RESULTS: Under optimised competition ELISA conditions, the anti-M3G scFv was found to have an IC(50) value of 30 ng/ml, while the most stable Fab construct exhibited an IC(50) value of 2.4 ng/ml. In SPR competition assay on an M3G-OVA-coated SPR chip surface, the two constructs again differed in sensitivity, with IC(50) values of 117 and 19 ng/ml for the scFv and Fab, respectively (the scFv also exhibiting poor linearity of response). However, when the SPR chip surface was directly coated with M3G, both antibody constructs exhibited good linearity of response, similar high sensitivity IC(50) values (scFv 30 ng/ml, Fab 14 ng/ml) and high reproducibility (50 effective regenerations for M3G-OVA, 200 for M3G direct). During SPR assay development it was noticed that scFv and Fab constructs gave differing off-rate profiles. Subsequent HPLC, ELISA and electrophoretic analyses then confirmed that a portion of the scFv population multimerises. Bivalent scFv was found to profoundly affect the dissociation curve for scFv in stringent SPR kinetic analyses, leading to a 40-fold difference in calculated off-rate values (Fab off rate 4.7 x 10(-3)S(-1), scFv off rate 1.03 x 10(-2)S(-1)). CONCLUSION: The structural format of recombinant antibody fragments and chip functionalisation methodology can both profoundly affect the function of anti-M3G SPR assay, with direct coating and Fab format proving to be optimal. The confirmation of scFv multimerisation and resulting changes in SPR kinetics profile, in comparison with a Fab, further suggest that caution must be taken in the interpretation of SPR sensorgrams, which are commonly used in the 'affinity ranking' of scFv panels in which the extent of dimerisation in each sample is unknown.     

抗CD3基因工程嵌合抗体IgG在哺乳动物细胞中的表达和活性测定

利用基因工程方法将鼠源性抗CD3抗体HIT3a的可变区和人源抗体(IgG)的完整的恒定区连接起来,构建全抗型抗CD3嵌合抗体,该型抗体具有较低的免疫源性可作为免疫抑制剂应用于器官移植,减少受体产生免疫排斥,提高移植器官的存活率。利用PCR方法从抗CD3 ScFv重组噬菌体表达载体pCANTAB 5E上扩增抗CD₃抗体的轻链和重链可变区,将轻链和重链可变区组装到含有人抗体（IgG）恒定区的表达载体中,构建抗CD3嵌合抗体IgG的轻链和重链表达载体PKN100和PG1D105,并用脂质体法共转染CHO细胞。结果证明,抗CD₃嵌合抗体的V_L和V_H与HIT3a抗体的V_L和V_H完全相符,ELISA和Western blot检测结果证实转染细胞的培养上清中含有抗CD₃嵌合抗体IgG的表达,表达产物能与Jurkat细胞结合,并能竞争性抑制HIT3a抗体和Jurkat细胞结合活性,³H-TdR掺入实验表明, 抗CD3嵌合抗体与亲代抗体HIT3a一样,具有促进外周血单核细胞增殖的作用。我室构建的全抗型抗CD3嵌合抗体分子表达载体可在CHO细胞中稳定表达,表达产物有较好生物活性,具有潜在的临床应用价值。     

Expression of a single-chain antibody against indole-3-acetic acid in Escherichia coli

A hybridoma cell line that produces a monoclonal antibody specific for indole-3-acetic acid (IAA) was prepared. The DNA fragments coding the variable regions of the light and the heavy chains of the antibody were prepared by PCR using the cDNA of the antibody as a template. A chimera DNA for a single chain variable fragment (scFv) was constructed, and expressed in Escherichia coli. The scFv antibody expressed in E. coli as well as the original monoclonal antibody showed a specific binding to IAA.     

A trivalent anti-erbB2/anti-CD16 bispecific antibody retargeting NK cells against human breast cancer cells

Bispecific antibody (BsAb) can physically cross-link immune cells to tumor cells, circumventing the proper structures for tumor cell-immune cell interactions and activating the cellular cytotoxic mechanisms. The optimal BsAb should target tumor cells with high affinity, but activate trigger molecules on cytotoxic cells by monovalent binding of Fab fragments. In the present study, a trivalent anti-erbB2/anti-CD16 BsAb was produced. This BsAb possesses bivalent arms specifically binding to the extracellular domain of erbB2 and monovalent Fab fragment redirecting NK cells. The recombinant protein could be expressed and purified from Escherichia coli as native proteins without refolding. It was fully functional in bispecific binding to SKBR3 and NK cells. The molecular size of this trivalent BsAb protein is larger than diabody and smaller than whole antibody and expected to have advantages for both high penetration of small antibody fragments and the slow circulation clearance of whole antibody. This novel protein may be an attractive target for further improvement and evaluation.     

Cytotoxic enhancement of a bispecific diabody by format conversion to tandem single-chain variable fragment (taFv): the case of the hEx3 diabody

Diabodies (Dbs) and tandem single-chain variable fragments (taFv) are the most widely used recombinant formats for constructing small bispecific antibodies. However, only a few studies have compared these formats, and none have discussed their binding kinetics and cross-linking ability. We previously reported the usefulness for cancer immunotherapy of a humanized bispecific Db (hEx3-Db) and its single-chain format (hEx3-scDb) that target epidermal growth factor receptor and CD3. Here, we converted hEx3-Db into a taFv format to investigate how format affects the function of a small bispecific antibody; our investigation included a cytotoxicity assay, surface plasmon resonance spectroscopy, thermodynamic analysis, and flow cytometry. The prepared taFv (hEx3-taFv) showed an enhanced cytotoxicity, which may be attributable to a structural superiority to the diabody format in cross-linking target cells but not to differences in the binding affinities of the formats. Comparable cross-linking ability for soluble antigens was observed among hEx3-Db, hEx3-scDb, and hEx3-taFv with surface plasmon resonance spectroscopy. Furthermore, drastic increases in cytotoxicity were found in the dimeric form of hEx3-taFv, especially when the two hEx3-taFv were covalently linked. Our results show that converting the format of small bispecific antibodies can improve their function. In particular, for small bispecific antibodies that target tumor and immune cells, a functional orientation that avoids steric hindrance in cross-linking two target cells may be important in enhancing the growth inhibition effect.     

A differential cell capture assay for evaluating antibody interactions with cell surface targets

Many biological and biomedical laboratory assays require the use of antibodies and antibody fragments that strongly bind to their cell surface targets. Conventional binding assays, such as the enzyme-linked immunosorbent assay (ELISA) and flow cytometry, have many challenges, including capital equipment requirements, labor intensiveness, and large reagent and sample consumption. Although these techniques are successful in mainstream biology, there is an unmet need for a tool to quickly ascertain the relative binding capabilities of antibodies/antibody fragments to cell surface targets on the benchtop at low cost. We describe a novel cell capture assay that enables several candidate antibodies to be evaluated quickly as to their relative binding efficacies to their cell surface targets. We used chimeric rituximab and murine anti-CD20 monoclonal antibodies as cell capture agents on a functionalized microscope slide surface to assess their relative binding affinities based on how well they capture CD20-expressing mammalian cells. We found that these antibodies’ concentration-dependent cell capture profiles correlate with their relative binding affinities. A key observation of this assay involved understanding how differences in capture surfaces affect the assay results. This approach can find utility when an antibody or antibody fragment against a known cell line needs to be selected for targeting studies.