ELISA-based quantification of cibacron blue F3GA used as ligand in affinity chromatography.

The development of an enzyme-linked immunosorbent assay (ELISA) for Cibacron Blue F3GA is reported. It quantifies a trace amount of blue dye used as an affinity chromatography ligand, rendering possible the measurement of traces of leached dye. Polyclonal antibodies were prepared after conjugation on the dye on KLH and injection into rabbits. The ELISA test was based on the competitive inhibition between a hemoglobin-dye complex adsorbed on the wells of a titration microplate and a free dye. The sensitivity of the assay was about 1000 times higher than a classical spectrophotometric assay and was modulated by some chemical substituents to the dye. The described ELISA assay was also successfully applied to the quantification of dye traces in the presence of human albumin.     

Identification of the human beta-casein C-terminal fragments that specifically bind to purified antibodies to bovine beta-lactoglobulin

The presence of foreign proteins in human milk after the ingestion of bovine dairy products is thought to be one of the possible causes of allergic sensitization in exclusively breast-fed predisposed infants. The immunologic determination of bovine beta-lactoglobulin (LG) concentration in human milk has been reported by several researchers, but the results are conflicting. Moreover, a strong cross-reactivity between antibodies to bovine beta-LG and human milk proteins and peptides was reported, throwing doubt on the reliability of radioimmunoassay and enzyme-linked immunosorbent assay detection and quantification assays for bovine beta-LG in human milk. Thus, the goal of this study was to isolate human milk peptides with a molecular mass >or= 1,000 Da cross-reactive with antibodies to bovine beta-LG in order to identify possible common epitopes between human and bovine milk proteins. The proteins were first isolated by affinity chromatography with purified polyclonal antibodies to bovine beta-LG, followed by gel filtration fast phase liquid chromatography and reverse phase-high performance liquid chromatography purification of the components specifically bound in the affinity separation step. Affinity-bound peptides were identified by determining their amino acid sequence. All the sequenced peptides belonged to the C-terminal part of human beta-casein, which confirms the cross-reactivity of human milk proteins and peptides with antibodies to bovine beta-LG and allows the identification of possible common epitopes between the two proteins. No bovine beta-LG peptides with a molecular mass >or= 1,000 Da were found in our milk samples from healthy mothers on a diet rich in bovine milk and dairy products.     

Direct dye binding--a quantitative assay for solid-phase immobilized protein.

A direct dye-binding procedure was established for the quantification of protein after its immobilization on a solid phase, using IgG and BSA as model proteins. The assay, which in the range 0-5 mg protein/ml gel correlates well with indirect protein determination by A280 as well as determination of protein hydrolyzed from the gel, is based on a modified Bradford dye-binding assay. As the protein coupled to the gel binds the dye, a decrease in A465 of the supernatant is measured. Three solid supports commonly used for protein immobilization (Sepharose, Sephadex, Sephacryl) were found to be compatible with the dye-binding assay while nonspecific dye binding was found to HEMA gels. Protein was coupled to Sephacryl S-1000 using three different activation methods (aldehyde, hydrazine, and adipic acid dihydrazide). Artifactual dye-binding was not observed using any of the three different "linkers." The assay is easily carried out and represents a useful tool, e.g., when optimizing procedures for protein immobilization.     

Towards a human proteome atlas: high-throughput generation of mono-specific antibodies for tissue profiling

A great need exists for the systematic generation of specific antibodies to explore the human proteome. Here, we show that antibodies specific to human proteins can be generated in a high-throughput manner involving stringent affinity purification using recombinant protein epitope signature tags (PrESTs) as immunogens and affinity-ligands. The specificity of the generated affinity reagents, here called mono-specific antibodies (msAb), were validated with a novel protein microarray assay. The success rate for 464 antibodies generated towards human proteins was more than 90% as judged by the protein array assay. The antibodies were used for parallel profiling of patient biopsies using tissue microarrays generated from 48 human tissues. Comparative analysis with well-characterized monoclonal antibodies showed identical or similar specificity and expression patterns. The results suggest that a comprehensive atlas containing extensive protein expression and subcellular localization data of the human proteome can be generated in an efficient manner with mono-specific antibodies.     

Microplate fluorescence assay for the quantification of double stranded DNA using SYBR Green I dye

A high-throughput, 96-well microplate fluorescence assay (MFA) was developed for DNA quantification using the double-stranded DNA-binding dye SYBR Green I. Samples mixed with SYBR Green I in the wells of a microtiter plate produced fluorescence in proportion with DNA concentration which was measured using a fluorescence plate reader. The performance characteristics of the assay were compared with spectrophotometric quantification based on ultraviolet absorption and the Hoefer DyNA Quant assay utilizing the fluorescent dye, Hoechst 33258. The MFA accurately quantified different types of DNA over a broad linear dynamic range of concentrations (0.25–2,500 pg/μl), and was not affected by a variety of contaminants in the assay mixture.     

Microfluidic analysis of antibody specificity in a compact disk format

A new and flexible technology for high throughput analysis of antibody specificity and affinity is presented. The method is based on microfluidics and takes advantage of compact disks (CDs) in which the centrifugal force moves fluids through microstructures containing immobilized metal affinity chromatography columns. Analyses are performed as a sandwich assay, where antigen is captured to the column via a genetically attached His6-tag. The antibodies to be analyzed are applied onto the columns. Thereafter, fluorescently labeled secondary antibodies recognize the bound primary antibodies, and detection is carried out by laser-induced fluorescence. The CDs contain 104 microstructures enabling analysis of antibodies against more than 100 different proteins using a single CD. Importantly, through the three-dimensional visualization of the binding patterns in a column it is possible to separate high affinity from low affinity binding. The method presented here is shown to be very sensitive, flexible and reproducible.     

基于SYBR Green I的双链DNA定量方法

摘要 基于SYBR Green I荧光染料与双链DNA（dsDNA）结合产生荧光的原理,建立一种高精度、高通量的双链DNA 定量方法。将梯度稀释后的基因组DNA及已知浓度的?DNA与等体积的SYBR Green I（4×）充分混合后,利用荧光定量PCR仪采集荧光信号,以ROX（1×）作为校正染料进行定量分析;同时利用紫外分光光度计对样品进行平行测定,比较该方法与紫外分光光度法的检测限与准确度。紫外分光光度法的检测限为2 ng/?l,而SYBR Green I荧光定量法的检测限可达到0.015 ng/?l,并且在0.015~2 ng/?l范围内,SYBR Green I荧光强度与?DNA浓度呈线性关系（R2=0.9999）,比紫外分光光度法灵敏100倍以上,并可准确定量低纯度的DNA样品。此方法具有重复性好、高通量的特点,仅需少量的生物样本即可满足定量要求,为分子生物学研究及临床检验等多个领域提供了一种可靠的dsDNA定量方法。     

ELISA-Mimic Screen for Synthetic Polymer Nanoparticles with High Affinity to Target Proteins

Synthetic polymer nanoparticles (NPs) that display high affinity to protein targets have significant potential for medical and biotechnological applications as protein capture agents or functional replacements of antibodies ("plastic antibodies"). In this study, we modified an immunological assay (enzyme-linked immunosorbent assay: ELISA) into a high-throughput screening method to select nanoparticles with high affinity to target proteins. Histone and fibrinogen were chosen as target proteins to demonstrate this concept. The selection process utilized a biotinylated NP library constructed with combinations of functional monomers. The screen identified NPs with distinctive functional group compositions that exhibited high affinity to either histone or fibrinogen. The variation of protein affinity with changes in the nature and amount of functional groups in the NP provided chemical insight into the principle determinants of protein-NP binding. The NP affinity was semiquantified using the ELISA-mimic assay by varying the NP concentrations. The screening results were found to correlate with solution-based assay results. This screening system utilizing a biotinylated NP is a general approach to optimize functional monomer compositions and can be used to rapidly search for synthetic polymers with high (or low) affinity for target biological macromolecules.     

Protein microarrays for antibody profiling: specificity and affinity determination on a chip

Protein microarray technology facilitates the detection and quantification of hundreds of binding reactions in one reaction from a minute amount of sample. Proof-of-concept studies have shown that the set-up of sensitive assay systems based on protein arrays is possible, however, the lack of specific capture reagents limits their use. Therefore, the generation and characterisation of capture molecules is one of the key topics for the development of protein array based systems. Recombinant antibody technologies, such as HuCAL (human combinatorial antibody library; MorphoSys, Munich, Germany), allow the fast generation of highly specific binders to nearly any given target molecule. Although antibody libraries comprise billions of members, it is not the selection process, but the detailed characterisation of the pre-selected monoclonal antibodies that presents the bottleneck for the production of high numbers of specific binders. In order to obtain detailed information on the properties of such antibodies, a microarray-based method has been developed. We show that it is possible to define the specificity of recombinant Fab fragments by protein and peptide microarrays and that antibodies can be classified by binding patterns. Since the assay uses a miniaturised system for the detection of antibody-antigen interactions, the observed binding occurs under ambient analyte conditions as defined by Ekins (J. Pharm. Biomed. Anal. 1989, 7, 155-168). This allows the determination of a relative affinity value for each binding event, and a ranking according to affinity is possible. The new microarray based approach has an extraordinary potential to speed up the screening process for the generation of recombinant antibodies with pre-defined selection criteria, since it is intrinsically a high-throughput technology.     

Monitoring protein expression in whole-cell extracts by targeted label- and standard-free LC-MS/MS

Targeted quantification of proteins is a daily task in biological research but often relies on techniques such as western blotting that are only barely quantitative. Here we present a broadly applicable workflow for protein quantification from unpurified whole-cell extracts that can be completed in less than 3 d. Without prefractionation or affinity enrichment, a whole-cell extract is trypsin-digested in an acetonitrile-containing ammonium carbonate buffer and high-molecular-weight compounds are removed by filtration. A normalization strategy, which involves endogenous reference proteins, facilitates the determination of relative changes in protein expression without requiring isotope labeling or standard addition. On a triple-quadrupole mass spectrometer, we demonstrate standard-free quantification of yeast proteins present over five orders of magnitude and present at ≥500 copies per cell. Liquid chromatography/multiple reaction monitoring (LC-MRM)-based proteomics is therefore a next-generation alternative to western blotting, as it allows simultaneous and reliable quantification of multiple endogenous proteins without the need for enrichment, isotope labeling or use of antibodies.     

抗欧亚活血丹凝集素特异性多克隆抗体的制备

欧亚活血丹外源凝集素(Gleheda)是分离自欧亚活血丹 (Glechoma hederacea) 叶片中的一种糖基化植物新蛋白. 如同其他糖基化蛋白,通过免疫学方法探测 Gleheda 的过程中通常受到一些不相干糖蛋白的妨碍,为此制定了抗 Gleheda 特异性多克隆抗体的纯化方案. 免疫血清蛋白经硫酸铵选择性沉淀后,分别以 Gleheda 和刺槐外源凝集蛋白 (RPA) 结合在 Sepharose 4B作为亲和配体,采用亲和层析法连续纯化 2 次,然后进一步采用离子交换层析 Q Fast Flow 提纯. 经每一步骤提纯得到的抗体组分对 Gleheda 的特异性,均同时采用双向免疫扩散检验和 Western blot 分析. 结果表明,以 Gleheda 为配体,亲和纯化制备得到的抗体组分对叶片粗提物中的许多植物 (糖) 蛋白仍然表现交叉反应. 为除去由植物糖蛋白中的聚糖所引起这些非特异性交叉反应抗体,接着以 RPA 为配体再次进行亲和纯化,Western blot 分析显示,抗体的特异性得到提高但并非除去了所有非特异性交叉反应的抗体. 最后进一步采用离子交换层析制备得到仅抗 Gleheda 蛋白的特异性抗体组分,此抗体组分适用于免疫探测研究. 该抗体纯化制备程序简易而高效,而且不需要昂贵的设备.     

Quantification by affinity perfusion chromatography of phosphorylated BRCAl and BRCA2 proteins from tumor cells after lycopene treatment

A new procedure for the quantification of phosphorylated BRCA1 (P-BRCA1) and BRCA2 (P-BRCA2) proteins in breast cell lines after different treatments was carried out. Cells were cultivated with [35S]-methionine and extracts subjected to three perfusion chromatographies. First heparin affinity chromatography purified cellular DNA-binding proteins. Subsequent specific immunoprecipitation of BRCA1 and BRCA2 proteins was performed with antibodies raised against BRCA1 or BRCA2. The immune complexes were isolated by protein A affinity chromatography. Phosphorylated BRCA1 or BRCA2 proteins were then purified with a Poros 20 AL column where anti-phosphothreonine and anti-phosphoserine antibodies were previously bound. The percentage of phosphorylated BRCA1 or BRCA2 proteins was calculated as follows: 100 x dpm of P-BRCA1 or P-BRCA2 eluted from the POROS 20AL column/total dpm eluted from POROS 20AL column. Treatment with 10 microM lycopene increased P-BRCA1 and P-BRCA2 in the breast tumor cell line MCF7 but not in MDA-MB-231 or MCF-10a, breast tumor or fibrocystic cell lines, respectively.     

Protein-protein interaction analysis by C-terminally specific fluorescence labeling and fluorescence cross-correlation spectroscopy

Here, we describe novel puromycin derivatives conjugated with iminobiotin and a fluorescent dye that can be linked covalently to the C-terminus of full-length proteins during cell-free translation. The iminobiotin-labeled proteins can be highly purified by affinity purification with streptavidin beads. We confirmed that the purified fluorescence-labeled proteins are useful for quantitative protein-protein interaction analysis based on fluorescence cross-correlation spectroscopy (FCCS). The apparent dissociation constants of model protein pairs such as proto-oncogenes c-Fos/c-Jun and archetypes of the family of Ca2+-modulated calmodulin/related binding proteins were in accordance with the reported values. Further, detailed analysis of the interactions of the components of polycomb group complex, Bmi1, M33, Ring1A and RYBP, was successfully conducted by means of interaction assay for all combinatorial pairs. The results indicate that FCCS analysis with puromycin-based labeling and purification of proteins is effective and convenient for in vitro protein-protein interaction assay, and the method should contribute to a better understanding of protein functions by using the resource of available nucleotide sequences.     

Analysis of host-cell proteins in biotherapeutic proteins by comprehensive online two-dimensional liquid chromatography/mass spectrometry

Assays for identification and quantification of host-cell proteins (HCPs) in biotherapeutic proteins over 5 orders of magnitude in concentration are presented. The HCP assays consist of two types: HCP identification using comprehensive online two-dimensional liquid chromatography coupled with high resolution mass spectrometry (2D-LC/MS), followed by high-throughput HCP quantification by liquid chromatography, multiple reaction monitoring (LC-MRM). The former is described as a “discovery” assay, the latter as a “monitoring” assay. Purified biotherapeutic proteins (e.g., monoclonal antibodies) were digested with trypsin after reduction and alkylation, and the digests were fractionated using reversed-phase (RP) chromatography at high pH (pH 10) by a step gradient in the first dimension, followed by a high-resolution separation at low pH (pH 2.5) in the second dimension. As peptides eluted from the second dimension, a quadrupole time-of-flight mass spectrometer was used to detect the peptides and their fragments simultaneously by alternating the collision cell energy between a low and an elevated energy (MSE methodology). The MSE data was used to identify and quantify the proteins in the mixture using a proven label-free quantification technique (“Hi3” method). The same data set was mined to subsequently develop target peptides and transitions for monitoring the concentration of selected HCPs on a triple quadrupole mass spectrometer in a high-throughput manner (20 min LC-MRM analysis). This analytical methodology was applied to the identification and quantification of low-abundance HCPs in six samples of PTG1, a recombinant chimeric anti-phosphotyrosine monoclonal antibody (mAb). Thirty three HCPs were identified in total from the PTG1 samples among which 21 HCP isoforms were selected for MRM monitoring. The absolute quantification of three selected HCPs was undertaken on two different LC-MRM platforms after spiking isotopically labeled peptides in the samples. Finally, the MRM quantitation results were compared with TOF-based quantification based on the Hi3 peptides, and the TOF and MRM data sets correlated reasonably well. The results show that the assays provide detailed valuable information to understand the relative contributions of purification schemes to the nature and concentrations of HCP impurities in biopharmaceutical samples, and the assays can be used as generic methods for HCP analysis in the biopharmaceutical industry.     

蛋白质微阵列检测抗原-抗体相互作用

为了制备蛋白质微阵列和研究芯片表面抗原-抗体的相互作用,研究了如何在玻片表面固化蛋白质和用荧光染料（Cy3,Cy5）对蛋白质进行标记.结果表明,在醛基修饰的玻璃表面,通过共价偶联的方法将抗原或抗体固定到芯片表面,能使二者保持其特异性结合能力.同时,荧光标记后的抗原或抗体仍然具有特异性结合能力.蛋白质微阵列是通过机械手在玻片表面排阵制作的.芯片上的荧光信号获取采用了激光共焦荧光扫描系统.用不同浓度的抗原探针阵列,对其相应的抗体靶分子的特异性结合进行了分析和研究.此外,还通过在玻片表面固定兔IgG和固定鼠IgG,对羊抗兔和羊抗鼠抗体与其相应抗原的特异性相互作用进行了检测.     

Toward optimal clearance: A universal affinity-based mass spectrometry approach for comprehensive ELISA reagent coverage evaluation and HCP hitchhiker analysis

In the control strategy for process related impurities in biopharmaceuticals, the enzyme linked immunosorbent assay (ELISA) is the method of choice for the quantification of host cell proteins (HCPs). Besides two dimensional-western blots (2D-WB), the coverage of ELISA antibodies is increasingly evaluated by affinity purification-based liquid chromatography–tandem mass spectrometry (AP-MS) methods. However, all these methods face the problem of unspecific binding issues between antibodies and the matrix, involving the application of arbitrarily defined thresholds during data evaluation. To solve this, a new approach (optimized AP-MS) was developed in this study, for which a cleavable linker was conjugated to the ELISA antibodies enabling the subsequent isolation of specifically interacting HCPs. By comparing both approaches in terms of method variability and the number of false positive or negative hits, we could demonstrate that the optimized AP-MS method is very reproducible and superior in the identification of antibody detection gaps, while previously described strategies suffered from over- or underestimating the coverage. As only antibody associated HCPs were identified, we demonstrated that the method is beneficial for hitchhiker analysis. Overall, the method described herein has proven as a powerful tool for reliable coverage determination of ELISA antibodies, without the need to arbitrarily exclude HCPs during the coverage evaluation.     

Protein–protein interaction analysis by C-terminally specific fluorescence labeling and fluorescence cross-correlation spectroscopy

Here, we describe novel puromycin derivatives conjugated with iminobiotin and a fluorescent dye that can be linked covalently to the C-terminus of full-length proteins during cell-free translation. The iminobiotin-labeled proteins can be highly purified by affinity purification with streptavidin beads. We confirmed that the purified fluorescence-labeled proteins are useful for quantitative protein–protein interaction analysis based on fluorescence cross-correlation spectroscopy (FCCS). The apparent dissociation constants of model protein pairs such as proto-oncogenes c-Fos/c-Jun and archetypes of the family of Ca²⁺-modulated calmodulin/related binding proteins were in accordance with the reported values. Further, detailed analysis of the interactions of the components of polycomb group complex, Bmi1, M33, Ring1A and RYBP, was successfully conducted by means of interaction assay for all combinatorial pairs. The results indicate that FCCS analysis with puromycin-based labeling and purification of proteins is effective and convenient for in vitro protein–protein interaction assay, and the method should contribute to a better understanding of protein functions by using the resource of available nucleotide sequences.     

Quantification of dye-mediated photodamage during single-molecule DNA imaging

Single-molecule fluorescence imaging of DNA-binding proteins has enabled detailed investigations of their interactions. However, the intercalating dyes used to visually locate DNA molecules have the undesirable effect of photochemically damaging the DNA through radical intermediaries. Unfortunately, this damage occurs as single-strand breaks (SSBs), which are visually undetectable but can heavily influence protein behavior. We investigated the formation of SSBs on DNA molecules by the dye YOYO-1 using complementary single-molecule imaging and gel electrophoresis-based damage assays. The single-molecule assay imaged hydrodynamically elongated lambda DNA, enabling the real-time detection of double-strand breaks (DSBs). The gel assay, which used supercoiled plasmid DNA, was sensitive to both SSBs and DSBs. This enabled the quantification of SSBs that precede DSB formation. Using the parameters determined from the gel damage assay, we applied a model of stochastic DNA damage to the time-resolved DNA breakage data, extracting the rates of single-strand breakage at two dye staining ratios and measuring the damage reduction from the radical scavengers ascorbic acid and β-mercaptoethanol. These results enable the estimation of the number of SSBs that occur during imaging and are scalable over a wide range of laser intensities used in fluorescence microscopy.     

Immunofluorescent labeling of centromeres for flow cytometric analysis.

A procedure to stain the centromeric region of chromosomes for dual beam flow cytometric analysis is described. Serum from a CREST (Scleroderma syndrome) patient presenting a high titer of anticentromeric antibodies was chosen on the basis of specificity of labeling of cells on slides. The high affinity of the antibodies to centromeres and low binding to chromosomal arms allowed the development of an indirect immunofluorescent labeling procedure using isolated and unfixed chromosomes stabilized by Mg++ ions. Discontinuous Ficoll gradients were used to separate chromosomes from unbound antibodies. With this procedure, chromosome clumping and degradation were minimal. The chromosomes were then stained with the DNA dyes Hoechst 33258 and chromomycin A3, before dual beam flow cytometric analysis. Flow karyotypes, with good chromosome peak resolution, were obtained for both human and hamster chromosomes subjected to the immunolabeling procedure. For quantification of FITC fluorescence due to bound antibody, chromosomes were counterstained with Hoechst only. The FITC intensity of antibody-labeled human and hamster chromosomes were 4-10 and 20 times greater than control chromosomes, respectively. These results suggest that the staining procedure may be suitable for immunolabeling of chromosomes with antibodies recognizing other nuclear proteins and their subsequent quantification by flow cytometry.     

An accurate proteomic quantification method: Fluorescence labeling absolute quantification (FLAQ) using multidimensional liquid chromatography and tandem mass spectrometry

A facile proteomic quantification method, fluorescent labeling absolute quantification (FLAQ), was developed. Instead of using MS for quantification, the FLAQ method is a chromatography-based quantification in combination with MS for identification. Multidimensional liquid chromatography (MDLC) with laser-induced fluorescence (LIF) detection with high accuracy and tandem MS system were employed for FLAQ. Several requirements should be met for fluorescent labeling in MS identification: Labeling completeness, minimum side-reactions, simple MS spectra, and no extra tandem MS fragmentations for structure elucidations. A fluorescence dye, 5-iodoacetamidofluorescein, was finally chosen to label proteins on all cysteine residues. The fluorescent dye was compatible with the process of the trypsin digestion and MALDI MS identification. Quantitative labeling was achieved with optimization of reacting conditions. A synthesized peptide and model proteins, BSA (35 cysteines), OVA (five cysteines), were used for verifying the completeness of labeling. Proteins were separated through MDLC and quantified based on fluorescent intensities, followed by MS identification. High accuracy (RSD% < 1.58) and wide linearity of quantification (1-10(5) ) were achieved by LIF detection. The limit of quantitation for the model protein was as low as 0.34 amol. Parts of proteins in human liver proteome were quantified and demonstrated using FLAQ.