Immunoaffinity chromatography of abscisic acid combined with electrospray liquid chromatography-mass spectrometry

Polyclonal antibodies with high specificity for C1-immobilised (+)-cis,trans-abscisic acid (ABA) were raised, characterised by enzyme-linked immunosorbent assay (ELISA) and used for preparation of an immunoaffinity chromatography (IAC) gel. The detection limit of the ELISA was approximately 4.6x10(-10)mol/L. Sensitive electrospray liquid chromatography-mass spectrometry (LC-ESI-MS) methods were also developed with detection limits below 0.1x10(-12)mol. The IAC allowed quick, single-step processing of samples prior to the analyses. The LC-ESI-MS and LC-ELISA techniques were used for comparative estimation of endogenous ABA levels in immunoaffinity purified extracts of normal and water-stressed Nicotiana tabacum L. leaves. The analytical approaches were validated using deuterium- and tritium-labelled internal standards, respectively. The IAC method was found to be highly effective, sensitive and convenient for isolating the target analyte from plant material.     

Immunoaffinity Purification of Indole-3-acetamide Using Monoclonal Antibodies

A single-step immunoaffinity purification procedure using monoclonalantibodies was developed to isolate indole-3-acetamide fromplant extracts. Antibodies from a selected clone, raised againstIAA-Cl'-BSA, with pronounced ability to recognize indole-3-acetamide(IAM) were used to prepare an immunoaffinity absorbent. Antibodiespurified by thiophilic interaction chromatography were immobilizedon divinylsulfoneactivated agarose. This column shows a veryhigh selectivity towards IAM compared to IAA. This single stepof immunoaffinity purification gave plant extracts of sufficientpurity for direct quantification by on-line spectrofluorimetryafter an analytical ionsuppression-HPLC run. Successive approximationby a second analytical ion-pairing-HPLC run confirmed the validityof this analytical technique. (Received November 15, 1986; Accepted May 7, 1987)     

High-level expression, single-step immunoaffinity purification and characterization of human tetraspanin membrane protein CD81

The study of membrane protein structure and function requires their high-level expression and purification in fully functional form. We previously used a tetracycline-inducible stable mammalian cell line, HEK293S-TetR, for regulated high-level expression of G-protein coupled receptors. We here report successfully using this method for high-level expression of de novo oligo-DNA assembled human CD81 gene. CD81 is a member of the vital tetraspanin membrane protein family. It has recently been identified as the putative receptor for the Hepatitis C Virus envelope E2 glycoprotein (HCV-E2). In this study we used a single-step rho-1D4-affinity purification method to obtain >95% purity from HEK293S-TetR-inducible stable cell lines. Using ELISA assay we determined that the affinity of the purified CD81 receptor for HCV-E2 protein is 3.8+/-1.2 nM. Using fluorescent confocal microscopy we showed that the inducibly overexpressed CD81 receptor in HEK293S-TetR cells is correctly located on the plasma membrane. We demonstrated that the combination of high-level expression of CD81 with efficient single-step immunoaffinity purification is a useful method for obtaining large quantities of CD81 membrane receptor suitable for detailed structural analyses of this elusive tetraspanin protein. Furthermore, this simple single-step immunoaffinity purification to high purity of membrane protein could be useful broadly for other membrane protein purifications, thus accelerating the determination of structures for large numbers of difficult-to-obtain membrane proteins.     

Determination of beta-sympathomimetics in liver and urine by immunoaffinity chromatography and gas chromatography—mass-selective detection

A specific and sensitive method for the determination of several β-agonistic drugs in liver and urine is described. Following clean-up by immunoaffinity chromatography and two different derivatizations, gas chromatography—mass spectrometry with electron-impact ionization is performed. The immunoaffinity chromatography columns were packed with Sepharose-immobilized polyclonal antibodies raised against the β-agonist clenbuterol. Owing to the high clean-up efficiency of the immunoaffinity column large sample volumes can be used (up to 100 ml urine or 25 gram liver). The immunoaffinity sample pretreatment is highly specific and no further sample pretreatment was necessary. Due to the combination of two different derivatizations only GC—MS with electron-impact ionization is necessary to fulfil legal requirements. The first confirmation step consists of a derivatization reaction between the hydroxyl group of the parent compound and trimethylsilane. The second confirmation method is a derivatization to a cyclic derivative with the hydroxyl group and the aliphatic nitrogen group. Limits of determination in liver as well in urine are at the 10 ng/kg or ng/l (ppt) level with acceptable signal-to-noise ratio. The method is suitable for identification and quantification of trace amounts of several similar β-agonistic drugs either used separately or in combination and can be used also for quantification of clenbuterol in liver with regard to levels exceeding the maximum residue limit (MRL) of 1 μg/kg (ppb).     

One-step purification of nisin A by immunoaffinity chromatography.

The lantibiotic nisin A was purified to homogeneity by a single-step immunoaffinity chromatography method. An immunoadsorption matrix was developed by direct binding of anti-nisin A monoclonal antibodies to N-hydroxysuccinimide-activated Sepharose. The purification procedure was rapid and reproducible and rendered much higher final yields of nisin than any other described method.     

Beneficial properties of single-domain antibody fragments for application in immunoaffinity purification and immuno-perfusion chromatography

We explored the possibility to apply single-domain antibodies from Camelidae for immunoaffinity purification of the ice structuring protein (ISP) from Lolium perenne, which modifies ice crystal growth and therefore has potential application in medicine, biotechnology, agriculture and (frozen) foods. Using phage display together with an appropriate selection method, a group of candidate fragments was isolated from a llama-derived immune library. Affinity chromatography using a purposely selected antibody coupled to a matrix yielded a completely pure and functional ISP. Due to the extreme refolding capabilities and physical stability of single-domain antibodies, the affinity matrix could be regenerated more than 2000 times without loss of capacity, while the fragment's monomeric nature permitted an efficient elution of antigen. The results of this study show that highly pure proteins can be recovered from biological material in a single-step process.     

弓形虫（Toxoplasma gondii）的PCR检测方法在笼养猕猴群中的应用

目的建立快速、灵敏、特异及检测结果易判断的PCR方法,并应用于大规模猕猴种群的弓形虫常规检测中。同时比较巢式PCR和单一PCR的一致性。方法根据弓形虫保守基因p30（SAG1）设计了内、外两对进行巢式PCR扩增以及B1基因设计一对引物进行单一PCR扩增,将DNA样本进行10倍倍比稀释,以检测巢式PCR反应的灵敏度;并对医学生物学研究所自繁猕猴共150只进行了弓形虫检测。结果巢式PCR检测法检测限度可达10^-3ng/uL,而且方法特异。两种PCR法检测结果基本一致,其中巢式PCR检测阳性率（10％）稍高于单一PCR检测阳性率（8．67％）。结论巢式PCR和一次PCR方法都可应用于猕猴弓形虫的常规检测中,并提示巢式PCR比单一PCR更敏感、检出率更高。     

用免疫亲和层析法纯化萝卜 PHGPx 天然蛋白

萝卜磷脂氢谷胱甘肽过氧化物酶 (RsPHGPx) 是一个定位于线粒体的蛋白质 . 为了阐明该蛋白质线粒体定位信号的准确切割位点,采用了免疫亲和层析方法纯化天然的 RsPHGPx. 用重组 RsPHGPx 蛋白免疫兔子获得了抗 RsPHGPx 的多克隆抗血清,以重组 RsPHGPx 蛋白为配体,采用亲和层析技术对抗血清进行了纯化,得到了单特异性的抗 RsPHGPx 的抗体 . 将纯化好的抗体偶联到一个 N- 羟基琥珀酰亚胺 (NHS) 预先激活的琼脂糖柱子上,装配成一个以单特异性的抗 RsPHGPx 抗体为配体的免疫亲和层析柱 . 经过对纯化条件的摸索和优化,形成了一个简单、特异的一步法纯化方案 . 按照该方案,从萝卜幼苗线粒体总蛋白质提取物中纯化到一个分子质量与预期值相一致的特异蛋白质 . 免疫印迹分析表明,该蛋白质被抗 RsPHGPx 的抗血清特异识别 . 酶活性分析表明,该蛋白质具有显著的 PHGPx 活性 . 这些结果表明,纯化到的特异蛋白质是萝卜的 RsPHGPx 天然蛋白 . 这是首个关于定位于植物细胞器的 PHGPx 蛋白纯化的报道 . 这一结果为准确测定 RsPHGPx 信号肽的切割位点奠定了基础,并将有助于对植物 PHGPx 的亚细胞定位机制及其生理功能的深入研究 .     

Selective isolation of human plasma low-density lipoprotein particles containing apolipoproteins B and E by use of a monoclonal antibody to apolipoprotein B

A monoclonal antibody to human plasma apolipoprotein B was used in a single-step immunoaffinity chromatography procedure to isolate a subpopulation of low-density lipoprotein particles from normolipidemic human plasma. The isolated particles were homogeneous in terms of size (20 nm), flotation coefficient (Sf = 9.5), and electrophoretic mobility (beta band). Their protein moiety consisted of apolipoproteins B and E in a molar ratio close to 2. The lipid moiety consisted of 47.3% cholesterol, 4.7% triglycerides, and 48.0% phospholipids. To indicate its characteristic apolipoprotein composition and hydrated density properties, this family of particles was named LP-B:EL2. In most normolipidemic subjects, LP-B:EL2 particles accounted for less than 10% of the total plasma apolipoprotein B content. The LP-B:EL2 particles bound to the membranes of the human hepatoma HepG2 cells in a specific and saturable manner indicative of receptor-mediated binding. Their binding was significantly higher than that of low-density lipoprotein particles containing only apolipoprotein B.     

Comparison on genomic predictions using three GBLUP methods and two single-step blending methods in the Nordic Holstein population

Background

A single-step blending approach allows genomic prediction using information of genotyped and non-genotyped animals simultaneously. However, the combined relationship matrix in a single-step method may need to be adjusted because marker-based and pedigree-based relationship matrices may not be on the same scale. The same may apply when a GBLUP model includes both genomic breeding values and residual polygenic effects. The objective of this study was to compare single-step blending methods and GBLUP methods with and without adjustment of the genomic relationship matrix for genomic prediction of 16 traits in the Nordic Holstein population.

Methods

The data consisted of de-regressed proofs (DRP) for 5 214 genotyped and 9 374 non-genotyped bulls. The bulls were divided into a training and a validation population by birth date, October 1, 2001. Five approaches for genomic prediction were used: 1) a simple GBLUP method, 2) a GBLUP method with a polygenic effect, 3) an adjusted GBLUP method with a polygenic effect, 4) a single-step blending method, and 5) an adjusted single-step blending method. In the adjusted GBLUP and single-step methods, the genomic relationship matrix was adjusted for the difference of scale between the genomic and the pedigree relationship matrices. A set of weights on the pedigree relationship matrix (ranging from 0.05 to 0.40) was used to build the combined relationship matrix in the single-step blending method and the GBLUP method with a polygenetic effect.

Results

Averaged over the 16 traits, reliabilities of genomic breeding values predicted using the GBLUP method with a polygenic effect (relative weight of 0.20) were 0.3% higher than reliabilities from the simple GBLUP method (without a polygenic effect). The adjusted single-step blending and original single-step blending methods (relative weight of 0.20) had average reliabilities that were 2.1% and 1.8% higher than the simple GBLUP method, respectively. In addition, the GBLUP method with a polygenic effect led to less bias of genomic predictions than the simple GBLUP method, and both single-step blending methods yielded less bias of predictions than all GBLUP methods.

Conclusions

The single-step blending method is an appealing approach for practical genomic prediction in dairy cattle. Genomic prediction from the single-step blending method can be improved by adjusting the scale of the genomic relationship matrix.     

Immunoaffinity extraction and tandem mass spectrometric analysis of human chorionic gonadotropin in doping analysis

A confirmatory and quantitative HPLC-tandem mass spectrometry (MS-MS) method for human chorionic gonadotropin hormone (hCG) at concentrations as low as 5 IU/l following immunoaffinity extraction of the glycoprotein from urine was developed. The extraction method involved retention of urinary hCG in the immunoaffinity column via specific antigen-antibody interaction. A variety of eluents were then used to quantitatively elute hCG from the immunoaffinity column. Qualitative and quantitative analysis of hCG were undertaken using MS-MS by identifying the amino acid sequence of the marker peptide betaT5 obtained from hCG by tryptic digestion and the peak areas of three product ions b(6)(+), b(9)(+) and y(11)(+), respectively.     

Sensitive and specific method for the determination of josamycin in human plasma by liquid chromatography–mass spectrometry

A highly sensitive and specific method for the determination of josamycin in human plasma by LC–MS was developed and validated. Josamycin was extracted from human plasma by a single-step liquid–liquid extraction and analyzed by LC–MS via an electrospray ionization interface. Selected ion monitoring was used to detect josamycin and its internal standard. The intra-day precision and accuracy, expressed as C.V. and R.E., ranged from 2.8% to 13.5% and −10.3% to 7.6%, respectively. The lower limit of detection was 0.1 ng/ml and the lower limit of quantitation was set at 1 ng/ml when 0.5 ml of plasma was used. No endogenous interference was observed in human plasma obtained from drug-free volunteers.     

Immunoaffinity purification of polyepitope proteins against Plasmodium falciparum with chicken IgY specific to their C-terminal epitope tag

Developing a polyepitope vaccine, a chimeric protein which contains diverse types of antigenic epitopes, is a promising strategy to prevent malaria. Previously, we had constructed a library of polyeptitope chimeric genes against Plasmodium falciparum without any protein tags. In an attempt to develop an efficient and universal procedure for purification of polyepitope chimeric proteins, we assembled an immunoaffinity chromatography (IAC) column with affinity-purified specific polyclonal IgY (mpIgY) antibodies that recognized the same C-terminal epitope tag of chimeric proteins in the library. A single-step and universal protocol was established and successfully applied for the purification of chimeric proteins. Using this protocol, chimeric proteins were specifically purified from an Escherichia coli expression system, and the purity and authenticity were verified by gel electrophoresis and Western blot analysis. Moreover, the comparison between this IAC method and the conventional chromatography, using two anion exchange columns followed by a step of gel filtration, showed that the new method was more efficient with an 8-fold greater yield. The results suggest that this IAC method will be an efficient approach for the purifications of polyepitope vaccine candidates against P. falciparum in our future study, and also be valuable for other similar applications.     

Immunoaffinity Nanoprobe-Based MALDI-TOF MS for Detection of Fragments of N-Telopeptides of Type I Collagen

Osteoporosis is one of the major bone-related diseases. Among the biomarkers of bone resorption, type I collagen cross-linked N-telopeptides (NTx) are terminal metabolites specifically derived from the degradation of bone collagen, thus, the level of NTx in urine has been regarded as a highly specific index for bone resorption. Our previous studies have identified a synthetic peptide fragment in the N-terminal of NTx (peptide P2) with highest affinity for anti-NTx antibodies by using enzyme-linked immunosorbent assay and surface plasma resonance-based methods. The objective of this study is to prepare the mouse anti-P2 polyclonal antibodies (anti-P2 Ab) and develop an immunonanoprobe-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method for detection of fragment of NTx of type I collagen. Anti-P2 Ab were prepared, purified, characterized, and used to produce Ab-conjugated magnetic nanoparticles (Ab@MNPs) for specifically isolation of different concentrations of Ab-bound P2 standards. The profile of P2 standards which were bound to Ab–NPs was analyzed by combining immunoaffinity MNPs with MALDI-TOF MS. We demonstrated that an immunoaffinity nanoprobe-based MALDI-TOF MS method for detecting the fragment of NTx, and might provide a tool to discover a promising biomarker of osteoporosis. The potential of this method for early diagnosis of osteoporosis will be further investigated by recruiting osteoporosis patients from our collaborative hospitals.     

One-step immunopurification and lectinochemical characterization of the Duffy atypical chemokine receptor from human erythrocytes

Duffy antigen/receptor for chemokines (DARC) is a glycosylated seven-transmembrane protein acting as a blood group antigen, a chemokine binding protein and a receptor for Plasmodium vivax malaria parasite. It is present on erythrocytes and endothelial cells of postcapillary venules. The N-terminal extracellular domain of the Duffy glycoprotein carries Fy(a)/Fy(b) blood group antigens and Fy6 linear epitope recognized by monoclonal antibodies. Previously, we have shown that recombinant Duffy protein expressed in K562 cells has three N-linked oligosaccharide chains, which are mainly of complex-type. Here we report a one-step purification method of Duffy protein from human erythrocytes. DARC was extracted from erythrocyte membranes in the presence of 1% n-dodecyl-β-D-maltoside (DDM) and 0.05% cholesteryl hemisuccinate (CHS) and purified by affinity chromatography using immobilized anti-Fy6 2C3 mouse monoclonal antibody. Duffy glycoprotein was eluted from the column with synthetic DFEDVWN peptide containing epitope for 2C3 monoclonal antibody. In this single-step immunoaffinity purification method we obtained highly purified DARC, which migrates in SDS-polyacrylamide gel as a major diffuse band corresponding to a molecular mass of 40-47?kDa. In ELISA purified Duffy glycoprotein binds anti-Duffy antibodies recognizing epitopes located on distinct regions of the molecule. Results of circular dichroism measurement indicate that purified DARC has a high content of α-helical secondary structure typical for chemokine receptors. Analysis of DARC glycans performed by means of lectin blotting and glycosidase digestion suggests that native Duffy N-glycans are mostly triantennary complex-type, terminated with α2-3- and α2-6-linked sialic acid residues with bisecting GlcNAc and α1-6-linked fucose at the core.     

Mass spectrometry-based immunoassays for the next phase of clinical applications

Recent applications of affinity mass spectrometry into clinical laboratories brought a renewed interest in immunoaffinity mass spectrometry as a more specific affinity method capable of selectively targeting and studying protein biomarkers. In mass spectrometry-based immunoassays, proteins are affinity retrieved from biological samples via surface-immobilized antibodies, and are then detected via mass spectrometric analysis. The assays benefit from dual specificity, which is brought about by the affinity of the antibody and the protein mass readout. The mass spectrometry aspect of the assays enables single-step detection of protein isoforms and their individual quantification. This review offers a comprehensive review of mass spectrometry-based immunoassays, from historical perspectives in the development of the immunoaffinity mass spectrometry, to current applications of the assays in clinical and population proteomic endeavors. Described in more detail are two types of mass spectrometry-based immunoassays, one of which incorporates surface plasmon resonance detection for protein quantification. All mass spectrometry-based immunoassays offer high-throughput targeted protein investigation, with clear implications in clinical research, encompassing biomarker discovery and validation, and in diagnostic settings as the next-generation immunoassays.     

Mass spectrometry-based immunoassays for the next phase of clinical applications

Recent applications of affinity mass spectrometry into clinical laboratories brought a renewed interest in immunoaffinity mass spectrometry as a more specific affinity method capable of selectively targeting and studying protein biomarkers. In mass spectrometry-based immunoassays, proteins are affinity retrieved from biological samples via surface-immobilized antibodies, and are then detected via mass spectrometric analysis. The assays benefit from dual specificity, which is brought about by the affinity of the antibody and the protein mass readout. The mass spectrometry aspect of the assays enables single-step detection of protein isoforms and their individual quantification. This review offers a comprehensive review of mass spectrometry-based immunoassays, from historical perspectives in the development of the immunoaffinity mass spectrometry, to current applications of the assays in clinical and population proteomic endeavors. Described in more detail are two types of mass spectrometry-based immunoassays, one of which incorporates surface plasmon resonance detection for protein quantification. All mass spectrometry-based immunoassays offer high-throughput targeted protein investigation, with clear implications in clinical research, encompassing biomarker discovery and validation, and in diagnostic settings as the next-generation immunoassays.     

Determination of Diniconazole in Agricultural Samples by Sol-Gel Immunoaffinity Extraction Procedure Coupled with HPLC and ELISA

Background

In the European Union (EU), the use of diniconazole-M is no longer authorized. However, residues of diniconazole-M occur in various plant commodities.

Methodology/Principal Findings

A selective and simple analytical method for the trace level determination of diniconazole in soil, fruit, vegetables and water samples was developed based on immunoaffinity extraction followed by Enzyme-linked immunosorbent assay (ELISA) and the high-performance liquid chromatography (HPLC) analysis. The ELISA was based on monoclonal antibodies highly specific to diniconazole and was a fast, cost-effective, and selective screening method for the detection of diniconazole. The results of the ELISA correlated well with gas chromatography (GC) results, with the correlation coefficient of 0.9879 (n = 19). A simple gel permeation chromato- graphy clean-up method was developed to purify extracts from matrices containing high amounts of fat and natural pigments, without the need for a large dilution of the sample. The immunoaffinity column (IAC) capacity was 0.180 mg g⁻¹. The columns could be re-used approximately 20 times with no significant alteration in capacity. The recoveries from complex samples were in the range of 89.2% to 96.1% with a relative standard deviation (RSD) of 0.770%–6.11% by ELISA. The results were in good agreement with those obtained by HPLC method.

Conclusion/Significance

The IAC extraction procedure coupled with HPLC and ELISA analysis could be also used as alternative effective analytical methods for the determination of diniconazole concentrations in complex samples.     

Colorimetric Assay System for Screening Antiviral Compounds against Hepatitis B Virus

A highly sensitive, rapid, and accurate assay system was developed for the in vitro evaluation of anti-hepatitis B virus (anti-HBV) agents. Chronic HBV-producing HB611 cells were used in combination with immunoaffinity purification, polymerase chain reaction (PCR), and hybrid capture detection. HB611 cells were incubated with putative anti-HBV agents for 7 days in 96-well microtiter plates. HBV was purified from HB611 cell culture media using immunoaffinity purification. The HBV DNA was extracted, amplified with PCR, and assayed using a hybrid capture colorimetric method. This assay provided quantitative detection of extracellular HBV DNA from 25 μl of cell culture media. Using the colorimetric method, we found that 50% effective concentration levels of several known anti-HBV agents (HPMPA, PMEDAP, PMEA and others) were similar to those reported in studies using Southern blot analysis. These results demonstrate that this new and easily automated colorimetric assay system can be used for the rapid and accurate assessment of anti-HBV compound selectivity.     

Selective removal of immunoglobulin E from rat blood by membrane-immobilized antibody

We examined the suitability of an immunoaffinity membrane [rabbit IgG specific for rat immunoglobulin E (IgE) immobilized on a cellulose membrane] for removing IgE from rat blood passed through a simple extracorporeal circulatory system. To determine the concentration of IgE in the blood, we also developed a highly sensitive chemiluminescence enzyme immunoassay for rat IgE. The IgE levels in the outlet blood from the immunoaffinity membrane module decreased to 30% of the initial concentration within 30 min.