Systematic validation of antibody binding and protein subcellular localization using siRNA and confocal microscopy

We have developed a platform for validation of antibody binding and protein subcellular localization data obtained from immunofluorescence using siRNA technology combined with automated confocal microscopy and image analysis. By combining the siRNA technology with automated sample preparation, automated imaging and quantitative image analysis, a high-throughput assay has been set-up to enable confirmation of accurate protein binding and localization in a systematic manner. Here, we describe the analysis and validation of the subcellular location of 65 human proteins, targeted by 75 antibodies and silenced by 130 siRNAs. A large fraction of (80%) the subcellular locations, including locations of several previously uncharacterized proteins, could be confirmed by the significant down-regulation of the antibody signal after the siRNA silencing. A quantitative analysis was set-up using automated image analysis to facilitate studies of targets found in more than one compartment. The results obtained using the platform demonstrate that siRNA silencing in combination with quantitative image analysis of antibody signals in different compartments of the cells is an attractive approach for ensuring accurate protein localization as well as antibody binding using immunofluorescence. With a large fraction of the human proteome still unexplored, we suggest this approach to be of great importance under the continued work of mapping the human proteome on a subcellular level.     

Comparison and critical analysis of robotized technology for monoclonal antibody high-throughput production

We have previously demonstrated how to transform the conventional method of hybridoma production and screening into a fast, high-throughput technology. Nevertheless, there were still open questions related to automated procedures and immunization protocols that we address now by comparing the hybridoma production work-flow in automated and manually executed processes. In addition, since the animals' antibody responses to single or multiple antigen challenge affect monoclonal antibody throughput, different immunization and fusion strategies were tested. Specifically, the results obtained with multiplexing (multiple target antigens injected into a single animal) and single antigen immunization followed by splenocyte pooling immediately before fusion were compared with conventional methods. The results presented here demonstrate that the optimal protocol consists of automated somatic-cell fusion and hybridoma dilution followed by manual plating of hybridoma cells. Additionally, more specific and productive hybridoma clones were obtained with multiplexed immunization in a single animal with respect to the splenocyte pooling from single antigen immunized animals. However, in terms of overall antibody yield, the conventional method consisting of single immunization for each single animal assured ten times more specific hybridoma cell lines than the strategy based on the multiple antigen immunization followed by separate fusion step. In conclusion, the most productive approach for recovering a large number of suitable antibodies relies on single antigen immunization followed by automated fusion and dilution steps and manual plating.     

Automated high throughput microscale antibody purification workflows for accelerating antibody discovery

To rapidly find “best-in-class” antibody therapeutics, it has become essential to develop high throughput (HTP) processes that allow rapid assessment of antibodies for functional and molecular properties. Consequently, it is critical to have access to sufficient amounts of high quality antibody, to carry out accurate and quantitative characterization. We have developed automated workflows using liquid handling systems to conduct affinity-based purification either in batch or tip column mode. Here, we demonstrate the capability to purify >2000 antibodies per day from microscale (1 mL) cultures. Our optimized, automated process for human IgG1 purification using MabSelect SuRe resin achieves ～70% recovery over a wide range of antibody loads, up to 500 µg. This HTP process works well for hybridoma-derived antibodies that can be purified by MabSelect SuRe resin. For rat IgG2a, which is often encountered in hybridoma cultures and is challenging to purify via an HTP process, we established automated purification with GammaBind Plus resin. Using these HTP purification processes, we can efficiently recover sufficient amounts of antibodies from mammalian transient or hybridoma cultures with quality comparable to conventional column purification.     

Regions of poliovirus protein VP1 produced in Escherichia coli induce neutralizing antibodies.

Poliovirus type 1 cDNA was prepared from viral RNA encoding the VP1 capsid region of the virus by using a specific DNA primer and was cloned in Escherichia coli. DNA fragments corresponding to VP1 amino acid positions 129 to 302 (pPM5k3), 52 to 302 (pPMhae3), and 24 to 129 (pPMDxba) were incorporated into plasmid vectors designed to express Trp LE-poliovirus VP1 fusion proteins under the control of the inducible tryptophan promoter-operator system. Induction of bacterial cultures containing the plasmids resulted in the production of fusion proteins which accounted for 21% (pPMhae3), 68% (pPM5k3), and 27% (pPMDxba) of the total cell protein. The proteins were purified, and each reacted with polyclonal antibodies raised against intact virions as measured by an enzyme-linked immunosorbent assay. The sera from rabbits immunized with the bacterially produced fusion proteins pPMDxba and pPMhae3 contained poliovirus-neutralizing antibodies.     

Automation of a Hemagglutination-Inhibition Test for Parainfluenza 3 Antibodies in Bovine Sera

An automated hemagglutination-inhibition (HI) test for the "shipping fever" strain (SF-4) of parainfluenza 3 antibody in bovine sera was developed and compared to manual tube and microtiter test procedures. The automated system operating at 60 samples per hr provided the most test results per specified time period, and the manual tube test provided the least. The manual microtiter test and the automated system at 40 samples per hr, falling between the two above procedures, were comparable in the number of sera that could be titrated in 1 day by one technician. There was little difference between automated and manual test reproducibility when measured at the twofold titer one-dilution difference level. However, the automated system titrated a higher number of sera at the same titer on repeat runs than either of the manual test procedures. The automated one-quartile difference reproducibility (each twofold dilution subdivided into 4 units-"quartiles") was equal to the manual test one-dilution difference reproducibility. The standard deviation of the per cent variation from the mean of paired serum titers for 40-sample-per-hr runs ranged from +/-3.49 to +/-5.36%. The manual and automated systems were of comparable sensitivity in their detection of negative sera.     

An immunocytochemical approach to cell kinetics automation.

Antibodies specific for 5-bromodeoxyuridine (BrdUrd) can be used to measure labeling indices in an automated system by image analysis. The antibody, used with an indirect immunoperoxidase technique, will detect de novo DNA synthesis subsequent to growing the cells for various time intervals in 5-bromodeoxyuridine-containing medium. Asynchronously growing CHO cells were pulsed with 3H-5-bromodeoxyuridine, fixed, denatured and then stained with anti-bromouridine antiserum. Peroxidase-coupled goat anti-rabbit IgG was used as the secondary antibody, and slides were stained with diaminobenzidine. Cells which are positive display a reticular pattern indicative of replicating chromatin. "Labeling indices" were generated by scanning the nuclei by TV image analysis. The percentage of labeled cells by the immunocytochemical technique correlates well with that found by autoradiography. Some of the applications of this automated method include cell kinetics and analysis of S-phase by pattern recognition technique.     

Automated Complement Fixation with Low Reagent Consumption

A microvolume automated (continuous-flow) system for the detection of complement-fixing antigen or antibody was evaluated. The model complement fixation test used was the detection of deoxyribonucleic acid antibody, and comparisons were made with a manual procedure. An "automatic two-dilution" feature of the apparatus was not used because of the clinical need for antibody titers. The system proved useful for the detection and estimation of anti-deoxyribonucleic acid antibody in patients with systemic lupus erythematosus and, indeed, accomplished this with low reagent consumption, a useful feature not available in "older" continuous-flow complement fixation systems.     

Micro isoelectric point heterogeneity of a murine monoclonal antibody (L6) originating from cell cultivation conditions

Summary Isoelectric point (pI) microheterogeneity of an IgG monoclonal antibody (L6) was studied by rapid affinity chromatography, isoelectric focusing on polyacrylamide gels, and rapid ion-exchange chromatography. An abiotic or strictly chemical basis for observed changes is postulated. The described methods are useful for monoclonal antibody process monitoring since they are rapid, automated assay procedures.     

Development and application of an automated, low-volume chromatography system for resin and condition screening

A small-volume chromatography system was developed for rapid resin and parameter screening and applied to the purification of a therapeutic monoclonal antibody from a key product-related impurity. Accounting for constraints in peripheral volume, gradient formation, column integrity, and fraction collection in microtiter plates, the resulting system employed 2-mL columns and was successfully integrated with plate-based methods for rapid sample analysis (e. g., use of automated liquid handlers, plate readers, and HPLC). Several cation-exchange chromatography resins were screened using automated programs and tailored gradients for the combination of a particular resin and a given antibody feedstock produced during Phase 1 development. Results from the tailored gradient runs were used to select a resin, and to arrive at efficient stepwise elution schedules for the chosen resin. By maintaining a constant residence time, final operating parameters were successfully scaled to representative bed heights and column diameters up to 2.6 cm (106 mL). This approach significantly improved throughput while reducing development time and material consumption.     

噬菌体抗体库技术与高通量筛选抗体

噬菌体抗体库技术是组合技术与基因工程抗体技术相结合的产物 ,为快速筛选特异性抗体提供了简便而高效的操作系统 ,随着蛋白质组学的飞速发展 ,对抗体的大规模制备的需求日益增加 ,迫切需要发展高质量的抗体库和与之相整合的高通量筛选技术。近年来 ,以上技术的发展和自动化设备的引入为大规模抗体制备的实现提供了条件 ,对这一领域的研究进展做一概述。     

Automatic pre-transfusion serology]

This paper describes an automated apparatus combining Rosenfield's and Lalezari's antibody screening and identification basic technics. PVP bromelin and low ionic strength acid polybren channels are used; agglutinates are decanded; the remaining cells are hemolyzed and the optical density is then measured through a colorimeter and recorded on a chart; speed is of 40 samples an hour. This machine was also used for irregular antibody screening and identification. Sensitivity is shown to be equal to that of manual technics for ABO, Lewis, Lutheran as well as K, S, M, Kpb, Xga, U and Vel antibodies detection. Nevertheless, a much greater sensitivity is achieved (titers 3 to 10 times higher) than by manual technics for Rh, -k, S, Fya antibodies detection. Polybren channel is suitable for anti-Rh, Duffy, I and M (human detection; bromelin channel however, has a greater sensitivity for other specificities. Anti-M and anti-N sera from rabbits were shown to be non specific when using this machine. Over almost 15 000 sera tested, no antibody (detected by manual techniques) escaped the automated screening. This antibody detection machine was applied to compatibility tests prior to transfusion. (21 480 units were tested. aimed to be transfused to 5 611 patients). A third, PVP without bromelin, was set in parallel in order not to let escape any anti-M, even a weak one. The sera distributor was slaved to the cells distributor so that the whole procedure was automated. Furthermore, each serum was tested against red cells to be transfused, but also against the patient's own red cells to be transfused, but also against the patient's own red cells and against two selected red cells panels, so as to ensure irregular antibody detection at the same time. Using this machine, 3 to 4% of the cell samples were rejected, i.e. more than with usual techniques. All manually detected antibodies were identified, but also some others, which showed only weak reactions by classical techniques. Total results can be obtained within 20 to 30 minutes, which is quite rapid, compared to techniques using for example antiglobulin tests.     

Virus detection using filament-coupled antibodies

Two attractive features of ELISA are the specificity of antibody-antigen recognition and the sensitivity achieved by enzymatic amplification. This report describes the development of a non-enzymatic molecular recognition platform adaptable to point-of-care clinical settings and field detection of biohazardous materials. This filament-antibody recognition assay (FARA) is based on circumferential bands of antibody probes coupled to a 120 microm diameter polyester filament. One advantage of this design is that automated processing is achieved by sequential positioning of filament-coupled probes through a series of 25-60 microL liquid filled microcapillary chambers. This approach was evaluated by testing for the presence of M13KO7 bacterial virus using anti-M13KO7 IgG(1) monoclonal antibody coupled to a filament. Filament motion first positioned the antibodies within a microcapillary tube containing a solution of M13KO7 virus before moving the probes through subsequent chambers, where the filament-coupled probes were washed, exposed to a fluorescently labeled anti-M13K07 antibody, and washed again. Filament fluorescence was then measured using a flatbed microarray scanner. The presence of virus in solution produced a characteristic increase in filament fluorescence only in regions containing coupled antibody probes. Even without the enzymatic amplification of a typical ELISA, the presence of 8.3 x 10(8) virus particles produced a 30-fold increase in fluorescence over an immobilized negative control antibody. In an ELISA comparison study, the filament-based approach had a similar lower limit of sensitivity of approximately 1.7 x 10(7) virus particles. This platform may prove attractive for point-of-care settings, the detection of biohazardous materials, or other applications where sensitive, rapid, and automated molecular recognition is desired.     

Kinetic screening of antibodies from crude hybridoma samples using Biacore

Experimental and data analysis protocols were developed to screen antibodies from hybridoma culture supernatants using Biacore surface plasmon resonance biosensor platforms. The screening methods involved capturing antibodies from crude supernatants using Fc-specific antibody surfaces and monitoring antigen binding at a single concentration. After normalizing the antigen responses for the amount of antibody present, a simple interaction model was fit to all of the binding responses simultaneously. As a result, the kinetic rate constants (k(a) and k(d)) and affinity (K(D)) could be determined for each antibody interaction under identical conditions. Higher-resolution studies involving multiple concentrations of antigen were performed to validate the reliability of single-concentration measurements. The screening protocols can be used to characterize antigen binding kinetics to approximately 200 antibody supernatants per day using automated Biacore 2000 and 3000 instruments.     

Automated in-solution protein digestion using a commonly available high-performance liquid chromatography autosampler

A completely automated peptide mapping liquid chromatography/mass spectrometry (LC/MS) system for characterization of therapeutic proteins in which a common high-performance liquid chromatography (HPLC) autosampler is used for automated sample preparation, including protein denaturation, reduction, alkylation, and enzymatic digestion, is described. The digested protein samples are then automatically subjected to LC/MS analysis using the same HPLC system. The system was used for peptide mapping of monoclonal antibodies (mAbs), known as a challenging group of therapeutic proteins for achieving complete coverage and quantitative representation of all peptides. Detailed sample preparation protocols, using an Agilent HPLC system, are described for Lys-C digestion of mAbs with intact disulfide bonds and tryptic digestion of mAbs after reduction and alkylation. The automated procedure of Lys-C digestion of nonreduced antibody, followed by postdigestion disulfide reduction, produces both the nonreduced and reduced digests that facilitate disulfide linkage analysis. The automated peptide mapping LC/MS system has great utility in preparing and analyzing multiple samples for protein characterization, identification, and quantification of posttranslational modifications during process and formulation development as well as for protein identity and quality control.     

Automated screening procedure for high-throughput generation of antibody fragments

In the emerging field of proteomics, there is an urgent need for catcher molecules such as antibodies for detecting the proteome or parts of the proteome in a microarray format. A suitable source for providing a large diversity of binders is obtained by combinatorial libraries, such as phage display libraries of single chain antibody fragments (scFv) or Fab fragments. To find novel binders from the n-CoDeR libraries with a high throughput, we have automated the screening process with robotics. The automated system is configured to screen tens of thousands of clones per day to target antigens in various formats, including peptides and soluble proteins, as well as cell-bound targets; thus, it is well designed to meet demands from the proteomics area.     

At-line quantification of bioactive antibody in bioreactor by surface plasmon resonance using epitope detection

We report an innovative at-line method to monitor concentration of bioactive antibody (i.e., antibody with conserved antigen-binding activity) secreted during bioreactor culture by the use of surface plasmon resonance (SPR)-based biosensor technology. In a first series of experiments, conditions for SPR-based measurements were validated off-line by monitoring bioactive antibody concentration in conditioned medium from 500-ml baffled flask hybridoma cell cultures. A fully automated experimental setup in which the SPR-based biosensor was harnessed to a bioreactor was then used at-line to monitor the concentration of bioactive antibody produced in a 3.5-L bioreactor. Quantitative SPR measurements performed both at-line and off-line were in excellent agreement with quantitative Western blotting followed by densitometry analyses. Thus, our experimental study confirms that SPR biosensors can be applied to at-line quantification of correctly folded proteins that are secreted by cells cultured in a bioreactor. Our experimental approach represents a novel and robust analytical strategy to be applied to the control and optimization of the production of bioactive secreted proteins.     

High-throughput screening in the diagnostics industry

The diagnostics industry is constantly under pressure to bring innovation quicker to market and so the impetus to speed up product-development cycle times becomes greater. There are a number of steps in the product-development cycle where the application of high-throughput screening can help. In the case of lateral-flow immuno-diagnostics the selection of antibody reagents is paramount. In particular, rapid identification of antibody pairs that are able to 'sandwich' around the target antigen is required. One screen that has been applied successfully is the use of surface plasmon resonance biosensors like Biacore. Using such a system one can evaluate over 400 antibody pairings in under 5 days. Conventional approaches to screen this number of antibody pairs would take many months. Other automated screening systems like DELFIA can be used in processing the vast amount of tests required for clinical trials. In addition, the use of robotics to automate routine product testing can be used to shorten the product-development cycle.     

Spatially addressed combinatorial protein libraries for recombinant antibody discovery and optimization

Antibody discovery typically uses hybridoma- or display-based selection approaches, which lack the advantages of directly screening spatially addressed compound libraries as in small-molecule discovery. Here we apply the latter strategy to antibody discovery, using a library of ～10,000 human germline antibody Fabs created by de novo DNA synthesis and automated protein expression and purification. In multiplexed screening assays, we obtained specific hits against seven of nine antigens. Using sequence-activity relationships and iterative mutagenesis, we optimized the binding affinities of two hits to the low nanomolar range. The matured Fabs showed full and partial antagonism activities in cell-based assays. Thus, protein drug leads can be discovered using surprisingly small libraries of proteins with known sequences, questioning the requirement for billions of members in an antibody discovery library. This methodology also provides sequence, expression and specificity information at the first step of the discovery process, and could enable novel antibody discovery in functional screens.     

Structure of human erythrocyte spectrin. I. Isolation of the alpha-I domain and its cyanogen bromide peptides

The alpha-I domain of human erythrocyte spectrin was produced by a mild tryptic digestion of the intact molecule and purified by a single step affinity chromatography procedure using a monoclonal antibody. A tryptic peptide representing the alpha-I domain, which migrated on polyacrylamide gels as an 80,000-dalton peptide, was subjected to automated Edman-Begg degradation. Products from automated sequencing were identified by reverse-phase high performance liquid chromatography. Two smaller proteolytic products of the alpha-I domain (T74 and T50) were also subjected to automated sequence analysis. CNBr cleavage of the alpha-I domain produced nine unique peptides which were separated by gel filtration on a high performance liquid chromatograph. Peptides were further purified by reverse-phase chromatography and characterized by amino acid analysis. Partial sequences were determined by automated NH2-terminal sequence analysis. A single aspartate-proline bond, which was partially hydrolyzed during the cyanogen bromide cleavage reaction, was also identified. These sequence data include the first 86 residues of the alpha-I domain, and the spectrin oligomer binding site has been tentatively localized within the first 39 residues. The sequence of 293 residues of a total 633 residues in the alpha-I domain is presented and represents the first structural information for this protein.     

Automation of immunohematologic testing activities at French blood transfusion centers

In May 1982, a questionnaire was sent to all of the 170 French Blood Transfusion Services (BTS), on behalf of the French Society of Blood Transfusion. The purpose was to determine the types of automated equipment used for immunohematological controls, the way in which they are used and the result of automation and computerization in daily laboratory operations. We received 135 replies (80%). A generalized conclusion can be drawn from the collected information. 50% of the respondents are neither automated nor computerized. 30% are both automated and computerized. 10% are automated but not computerized and 8% are not automated but are computerized. In the field of automated serology there is an increased tendency to complete the ABO/Rh testing by Cc D Ee and Kell phenotyping. The use of computers allows the current test determination to be compared with previous donation data. However, no fully automated equipment, which can conduct antibody screening, exists, cost effectively, in small or average BTS. In France, there has been a significant increase in automation between 1970 and 1980 but only the most important BTS have carried out automation at the same time as computerization. The smaller BTS have usually become automated without becoming computerized. In 1978, Codabar was first used. This has been one of the principal advances of the last 10 years, allowing all the users of automation to start moving towards complete computerization. This advance was assisted by the use of prepackaged software. This questionnaire also determined that the current emphasis is now to computerize administrative and management activities before laboratory activities. This survey has been conducted during a turning point of the automation of French BTS. It shows that they are, on the whole, satisfied with their automation. As far as the safety and the efficiency of the service are concerned, it is only fair to consider that the main purposes of the automation have been achieved. But in terms of cost, and serological accuracy for antibody screening, a new generation of automated equipment should appear to satisfy the users in the nineties.