Immobilization strategies for single-chain antibody microarrays

Sandwich ELISA microarrays have great potential for validating disease biomarkers. Each ELISA relies on robust-affinity reagents that retain activity when immobilized on a solid surface or when labeled for detection. Single-chain antibodies (scFv) are affinity reagents that have greater potential for high-throughput production than traditional IgG. Unfortunately, scFv are typically less active than IgG following immobilization on a solid surface and not always suitable for use in sandwich ELISAs. We therefore investigated different immobilization strategies and scFv constructs to determine a more robust strategy for using scFv as ELISA reagents. Two promising strategies emerged from these studies: (i) the precapture of epitope-tagged scFv using an antiepitope antibody and (ii) the direct printing of a thioredoxin (TRX)/scFv fusion protein on glass slides. Both strategies improved the stability of immobilized scFv and increased the sensitivity of the scFv ELISA microarray assays, although the antiepitope precapture method introduced a risk of reagent transfer. Using the direct printing method, we show that scFv against prostate-specific antigen (PSA) are highly specific when tested against 21 different IgG-based assays. In addition, the scFv microarray PSA assay gave comparable quantitative results (R(2) = 0.95) to a commercial 96-well ELISA when tested using human serum samples. In addition, we find that TRX-scFv fusions against epidermal growth factor and toxin X have good LOD. Overall, these results suggest that minor modifications of the scFv construct are sufficient to produce reagents that are suitable for use in multiplex assay systems.     

Design of recombinant antibody microarrays for complex proteome analysis: choice of sample labeling-tag and solid support

Antibody-based microarray is a novel technology with great potential within high-throughput proteomics. The process of designing high-performing antibody (protein) microarrays has, however, turned out to be a challenging process. In this study, we have developed further our human recombinant single-chain variable-fragment (scFv) antibody microarray methodology by addressing two crucial technological issues, choice of sample labeling-tag and solid support. We examined the performance of a range of dyes in a one- or two-color approach on a selection of solid supports providing different surface and coupling chemistries, and surface structures. The set-ups were evaluated in terms of sensitivity, specificity, and selectivity. The results showed that a one-color approach, based on NHS-biotin (or ULS-biotin) labeling, on black polymer Maxisorb slides (or Nexterion slide H) was the superior approach for targeting low-abundant (pg/mL) analytes in nonfractionated, complex proteomes, such as human serum or crude cell supernatants. Notably, microarrays displaying adequate spot morphologies, high S/Ns, minimized nonspecific binding, and most importantly a high selectivity, specificity, and sensitivity (>or=fM range) were obtained. Taken together, we have designed the first generation of a high-performing recombinant scFv antibody microarray technology platform on black polymer Maxisorb slides for sensitive profiling of low-abundant analytes in nonfractionated biotinylated complex proteomes.     

Biocompatibility of surfaces for antibody microarrays: design of macroporous silicon substrates

Major efforts to develop antibody microarray technology to enable global proteome analysis to be performed in a facile manner are under way. In this process, the design and the properties of the substrate will play crucial roles. In the present study, we have developed novel, highly biocompatible solid supports for microarrays, using adsorbed recombinant human single-framework antibody fragments as probes. Several silicon-based supports, including planar silicon, micro- and macroporous silicon, and nitrocellulose-coated variants thereof, were designed and evaluated in a stepwise procedure. The surfaces were scored based on biocompatibility and probe binding capacity as judged by spot morphology, signal intensities, signal to noise ratios, dynamic range, sensitivity, and reproducibility. A set of five commercially available substrates, selected to represent a set of supports providing different surface and coupling chemistries, was used as reference surfaces. The results showed that several well-performing silicon-based supports could be designed; in particular, a nitrocellulose-coated macroporous variant, MAP3-NC7, received the highest scores. In comparison, MAP3-NC7 displayed properties equal to or better than those of the reference substrates. Taken together, designed surfaces based on silicon can undoubtedly meet the requirements of the next generation of solid supports for antibody microarrays.     

Systematic comparison of surface coatings for protein microarrays

To process large numbers of samples in parallel is one potential of protein microarrays for research and diagnostics. However, the application of protein arrays is currently hampered by the lack of comprehensive technological knowledge about the suitability of 2-D and 3-D slide surface coatings. We have performed a systematic study to analyze how both surface types perform in combination with different fluorescent dyes to generate significant and reproducible data. In total, we analyzed more than 100 slides containing 1152 spots each. Slides were probed against different monoclonal antibodies (mAbs) and recombinant fusion proteins. We found two surface coatings to be most suitable for protein and antibody (Ab) immobilization. These were further subjected to quantitative analyses by evaluating intraslide and slide-to-slide reproducibilities, and the linear range of target detection. In summary, we demonstrate that only suitable combinations of surface and fluorescent dyes allow the generation of highly reproducible data.     

Lithographic techniques and surface chemistries for the fabrication of PEG-passivated protein microarrays

This article presents a new technique to fabricate patterns of functional molecules surrounded by a coating of the inert poly(ethylene glycol) (PEG) on glass slides for applications in protein microarray technology. The chief advantages of this technique are that it is based entirely on standard lithography processes, makes use of glass slides employing surface chemistries that are standard in the microarray community, and has the potential to massively scale up the density of microarray spots. It is shown that proteins and antibodies can be made to self-assemble on the functional patterns in a microarray format, with the PEG coating acting as an effective passivating agent to prevent non-specific protein adsorption. Various standard surface chemistries such as aldehyde, epoxy and amine are explored for the functional layer, and it is conclusively demonstrated that only an amine-terminated surface satisfies all the process constraints imposed by the lithography process sequence. The effectiveness of this microarray technology is demonstrated by patterning fluorescent streptavidin and a fluorescent secondary antibody using the well-known and highly specific interaction between biotin and streptavidin.     

Optimizing antibody immobilization strategies for the construction of protein microarrays

Antibody microarrays have the potential to revolutionize protein expression profiling. The intensity of specific signal produced on a feature of such an array is related to the amount of analyte that is captured from the biological mixture by the immobilized antibody (the "capture agent"). This in turn is a function of the surface density and fractional activity of the capture agents. Here we investigate how these two factors are affected by the orientation of the capture agents on the surface. We compare randomly versus specifically oriented capture agents based on both full-sized antibodies and Fab' fragments. Each comparison was performed using three different antibodies and two types of streptavidin-coated monolayer surfaces. The specific orientation of capture agents consistently increases the analyte-binding capacity of the surfaces, with up to 10-fold improvements over surfaces with randomly oriented capture agents. Surface plasmon resonance revealed a dense monolayer of Fab' fragments that are on average 90% active when specifically oriented. Randomly attached Fab's could not be packed at such a high density and generally also had a lower specific activity. These results emphasize the importance of attaching proteins to surfaces such that their binding sites are oriented toward the solution phase.     

High-throughput affinity ranking of antibodies using surface plasmon resonance microarrays

A method was developed to rapidly identify high-affinity human antibodies from phage display library selection outputs. It combines high-throughput Fab fragment expression and purification with surface plasmon resonance (SPR) microarrays to determine kinetic constants (kon and koff) for 96 different Fab fragments in a single experiment. Fabs against human tissue kallikrein 1 (hK1, KLK1 gene product) were discovered by phage display, expressed in Escherichia coli in batches of 96, and purified using protein A PhyTip columns. Kinetic constants were obtained for 191 unique anti-hK1 Fabs using the Flexchip SPR microarray device. The highest affinity Fabs discovered had dissociation constants of less than 1 nM. The described SPR method was also used to categorize Fabs according to their ability to recognize an apparent active site epitope. The ability to rapidly determine the affinities of hundreds of antibodies significantly accelerates the discovery of high-affinity antibody leads.     

狂犬病毒抗体ELISA检测试剂盒的改进研究

为了提高狂犬病毒抗体检测的灵敏度和特异性,采用狂犬病毒单克隆抗体包被酶标板,再分别加入重组的狂犬病毒糖蛋白或细胞培养抗原做固相层的方法(抗体捕捉法),用传统的间接ELISA法做对照,按常规方法检测抗狂犬病毒抗体。结果显示,抗体捕捉法的非特异性反应低于间接法,而灵敏度达到0．51U水平,高于间接法。在800份临床标本检测中,检出率明显高于间接法。用15份阳性血清作小鼠中和试验,并和抗体捕捉ELISA法比较具有高度的一致性。试验结果充分表明,该方法优于传统的ELISA间接法。因此可作为临床注射狂犬疫苗后检测血清中狂犬病毒抗体的常规方法。     

Functional peptide microarrays for specific and sensitive antibody diagnostics

Peptide microarrays displaying biologically active small synthetic peptides in a high-density format provide an attractive technology to probe complex samples for the presence and/or function of protein analytes. We present a new approach for manufacturing functional peptide microarrays for molecular immune diagnostics. Our method relies on the efficiency of site-specific solution-phase coupling of biotinylated synthetic peptides to NeutrAvidin (NA) and localized microdispensing of peptide-NA-complexes onto activated glass surfaces. Antibodies are captured in a sandwich manner between surface immobilized peptide probes and fluorescence-labeled secondary antibodies. Our work includes a total of 54 peptides derived from immunodominant linear epitopes of the T7 phage capsid protein, Herpes simplex virus glycoprotein D, c-myc protein, and three domains of the Human coronavirus polymerase polyprotein and their cognate mAbs. By using spacer molecules of different type and length for NA-mediated peptide presentation, we show that the incorporation of a minimum spacer length is imperative for antibody binding, whereas the peptide immobilization direction has only secondary importance for antibody affinity and binding. We further demonstrate that the peptide array is capable of detecting low-picomolar concentrations of mAbs in buffered solutions and diluted human serum with high specificity.     

Design of high-density antibody microarrays for disease proteomics: Key technological issues

Antibody-based microarray is a novel proteomic technology setting a new standard for molecular profiling of non-fractionated complex proteomes. The first generation of antibody microarrays has already demonstrated its potential for generating detailed protein expression profiles, or protein atlases, of human body fluids in health and disease, paving the way for new discoveries within the field of disease proteomics. The process of designing highly miniaturized, high-density and high-performing antibody microarray set-ups have, however, proven to be challenging. In this mini-review we discuss key technological issues that must be addressed in a cross-disciplinary manner before true global proteome analysis can be performed using antibody microarrays.     

Analysis conditions for proteomic profiling of mammalian tissue and cell extracts with antibody microarrays

Antibody microarrays are a developing tool for global proteomic profiling. A protocol was established that permits robust analyses of protein extracts from mammalian tissues and cells rather than body fluids. The factors optimized were buffer composition for surface blocking, blocking duration, protein handling and processing, labeling parameters like type of dye, molar ratio of label versus protein, and dye removal, as well as incubation parameters such as duration, temperature, buffer, and sample agitation.     

Blocking and detection chemistries affect antibody performance on reverse phase protein arrays

Antibody specificity is critical for RP protein arrays (RPA). The effects of blocking and detection chemistries on antibody specificity were evaluated for Western blots and RPA. Blocking buffers significantly affected nonspecific banding on Western blots, with corresponding effects on arrays. Tyramide signal amplification (TSA) increased both specific and nonspecific signals on Westerns and arrays, masking the expected gradations in signal intensity. These results suggest that consistent blocking and detection conditions should be used for antibody validation and subsequent RPA experiments.     

Design of atto-vial based recombinant antibody arrays combined with a planar wave-guide detection system

Antibody microarray is a rapidly emerging, powerful approach with great promise within high-throughput proteomics. However, before a truly proteome-wide analysis can be performed, the antibody array format needs to be miniaturized even further in order to enable ultradense arrays to be fabricated. To this end, we have designed and generated proof-of-concept for the first generation of an atto-vial based recombinant antibody array platform. Briefly, we have designed a novel nanostructured substrate using electron beam lithography. Vials, ranging in volume/size from 6 (200 nm in diameter) to 4000 aL (5 microm in diameter), were fabricated. Human recombinant single-chain Fv antibody fragments, microarray adopted by design, were used as probes. The set-up was interfaced with planar wave-guide technology for evanescant field fluorescence detection. The results showed that protein analytes could be specifically detected in the subzeptomole range for pure systems, using vials down to 57 aL. Further, low-abundant (pg/mL) protein analytes could be detected in directly labeled complex proteomes, such as human whole serum, using 157 aL-vials. Taken together, these results outline the potential of the atto-vial array set-up for miniaturized affinity proteomics-based approaches.     

Design of recombinant antibody microarrays for cell surface membrane proteomics

Generating proteomic maps of membrane proteins, common targets for therapeutic interventions and disease diagnostics, has turned out to be a major challenge. Antibody-based microarrays are among the novel rapidly evolving proteomic technologies that may enable global proteome analysis to be performed. Here, we have designed the first generation of a scaleable human recombinant scFv antibody microarray technology platform for cell surface membrane proteomics as well as glycomics targeting intact cells. The results showed that rapid and multiplexed profiling of the cell surface proteome (and glycome) could be performed in a highly specific and sensitive manner and that differential expression patterns due to external stimuli could be monitored.     

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.     

ELISA方法测定血清中Hib多糖抗体含量的条件比较研究

分别以牛血清白蛋白和人血清白蛋白作为封闭液的主要成分测定7份血清中抗b型流感嗜血杆菌荚膜多糖的抗体含量,结果显示,两组数据之间没有显著性差异(P>0.05)。分别用HRP标记的羊抗人IgG和HRP标记的鼠抗人IgG测定该7份血清,结果显示两组结果无显著性差异(P>0.05)。同时,将血清反应的温度和时间由37℃1.5h改为4℃16h(过夜),结果显示两者无显著性差异(P>0.05)。从而优化了该方法。     

Investigating copper-regulated protein expression in Menkes fibroblasts using antibody microarrays

Neurodegenerative illnesses are characterized by aberrant metabolism of biometals such as copper (Cu), zinc (Zn) and iron (Fe). However, little is known about the metabolic effects associated with altered metal homeostasis. In this study, we used an in vitro model of altered Cu homeostasis to investigate how Cu regulates cellular protein expression. Human fibroblasts containing a natural deletion mutation of the Menkes (MNK) ATP7A Cu transporter (MNK deleted) were compared to fibroblasts overexpressing ATP7A (MNK transfected). Cultures of MNK-transfected (Low-Cu) cells exhibited 95% less intracellular Cu than MNK-deleted (High-Cu) cells. Comparative proteomic analysis of the two cell-lines was performed using antibody microarrays, and significant differential protein expression was observed between Low-Cu and High-Cu cell-lines. Western blot analysis confirmed the altered protein expression of Ku80, nexilin, L-caldesmon, MAP4, Inhibitor 2 and DNA topoisomerase I. The top 50 altered proteins were analysed using the software program Pathway Studio (Ariadne Genomics) and revealed a significant over-representation of proteins involved in DNA repair and maintenance. Further analysis confirmed that expression of the DNA repair protein Ku80 was dependent on cellular Cu homeostasis and that Low-Cu levels in fibroblasts resulted in elevated susceptibility to DNA oxidation.     

Toward optimized antibody microarrays: a comparison of current microarray support materials

With the advent of protein and antibody microarray technology several different coatings and protocols have been published, which may be broadly divided into two types: gel-coated surfaces and plain non-gel-coated glass or plastic surfaces, some with chemical groups attached. We have screened 11 different array surfaces of both types and compared them with respect to their detection limit, inter- and intrachip variation, and storage characteristics. Five different antibodies were immobilized onto each type of microarray support, with total protein concentrations ranging from 40 fmol to 25 amol per spot. From these results, it was seen that some antibodies were more suited for use on antibody arrays. All measurements were performed in quadruplicate, and the results revealed high signal uniformity and reproducibility of most plain glass and plastic slides. Lower detection limits were obtained with polyacrylamide-coated slides, making them more suitable for the detection of very low concentrations of antigen. All microarray coatings could be stored for a period of 8 weeks; however, improved results were seen after 2 weeks of storage. In conclusion, the results indicate the need to test each antibody to be used on an antibody array and to select the microarray coating based on experimental requirements.     

Design of recombinant antibody microarrays for membrane protein profiling of cell lysates and tissue extracts

Generating global protein expression profiles, including also membrane proteins, will be crucial for our understanding of biological processes in health and disease. In this study, we have expanded our antibody microarray technology platform and designed the first human recombinant antibody microarray for membrane proteins targeting crude cell lysates and tissue extracts. We have optimized all key technological parameters and successfully developed a setup for extracting, labeling and analyzing non-fractionated membrane proteomes under non-denaturing conditions. Finally, the platform was also extended and shown to be compatible with simultaneous profiling of both membrane proteins and water-soluble proteins.     

High-throughput optimization of surfaces for antibody immobilization using metal complexes

Using a high-throughput surface discovery approach, we have generated a 1600-member library of metal-containing surfaces and screened them for antibody binding potential. The surface library assembly involved graft modification of argon plasma-treated polyvinylidenedifluoride (PVDF) membranes with alternating maleic anhydride-styrene copolymer followed by anhydride ring opening with a range of secondary amines and microarray contact printing of transition metal complexes. The microarrays of metal-containing surfaces were then tested for their antibody binding capacity by incubation with a biotinylated mouse antibody in a chemiluminescence assay. A total of 11 leads were identified from the first screen, constituting a "hit" rate of 0.7%. A smaller 135-member surface library was then synthesized and screened to optimize existing hits and generate additional leads. To demonstrate the applicability of these surfaces to other formats, high-binding surface leads were then transferred onto Luminex beads for use in a bead flow cytometric immunoassay. The novel one-step antibody coupling process increased assay sensitivity of a Luminex tumor necrosis factor immunoassay. These high-binding surfaces do not require prior incorporation of polyhistidine tags or posttreatments such as oxidation to achieve essentially irreversible binding of immunoglobulin G.