An immunosensor with potential for the detection of viral antigens in body fluids, based on surface second harmonic generation

Field methods of assessing the immune status of animals are required to optimise vaccination programmes to control bovine viral diarrhoea (BVD) virus. An optoelectronic immunosensor was evaluated for the detection of viral antigens in a crude cell lysate in a pilot study. Binding of (BVD) virus antigen by two monoclonal antibodies immobilised on two different media (ELISA plate wells, and glass coverslips) was detected and quantified using the laser induced surface second harmonic generation (SSHG) technique. The results for both assays were correlated with an enzyme-linked immunoassay (ELISA) used for the diagnosis of BVD virus infection in cattle (ELISA plate; R(2)=0.86, coverslips; Exp. 1; R(2)=0.75, Exp. 2; R(2)=0.67). The method will allow rapid detection of antigens in the body fluids of farm animals.     

Piezoelectric printing and probing of Lectin NanoProbeArrays for glycosylation analysis

Glycans have great potential as disease biomarkers and therapeutic targets. However, the major challenge for glycan biomarker identification from clinical samples is the low abundance of key glycosylated proteins. To demonstrate the potential for glycan analysis with nanoliter amounts of glycoprotein, we have developed a new technology (Lectin NanoProbeArray) based on piezoelectric liquid dispensing for non-contact printing and probing of a lectin array. Instead of flooding the glycoprotein probe on the lectin array surface, as in conventional microarray screening, a piezoelectric printer is used to dispense nanoliters of fluorescently labeled glycoprotein probe over the lectin spots on the array. As a proof-of-concept, the ability of Lectin NanoProbeArrays to precisely identify and reliably distinguish between the closely related glycoforms of fetuin is illustrated here. Sensitivity levels comparable to lectin arrays that use evanescent-field scanners was achieved along with several orders of magnitude reduction in the amount of probe required for glycosylation analysis.     

Non-specific adsorption of fish immunoglobulin M (IgM) to blocking reagents on ELISA plate wells

Enzyme-linked immunosorbent assay (ELISA) is a popular technique for quantifiable detection of specific antibodies in warm-blooded animals, but it has not been accepted for detection of fish antibodies because of its low reproducibility, which is due in part to high background optical density (OD) measurements. In the present study, we report that the high background of a fish antibody-detection ELISA resulted from non-specific adsorption of fish immunoglobulin M (IgM) to blocking reagents on the ELISA plate wells. Four fish sera (from rainbow trout Oncorhynchus mykiss, masu salmon O. masou, Japanese flounder Paralichthys olivaceus and koi Cyprinus carpio) were poured into ELISA plate wells pre-blocked with several blocking reagents (skim milk, soybean milk, bovine serum albumin, fetal bovine serum, gelatin and Roche BlockingReagent) and then washed out in order to measure the remaining fish IgM on the ELISA plate wells. Significant amounts of fish IgMs (OD absorbance at 492 nm: 0.3 to 1.1) remained on the ELISA plate wells with no antigenic protein except blocking reagents. The amount of remaining fish IgMs on the ELISA plate wells decreased significantly following treatment of fish sera with skim milk. However, the specific immuno-reactivity of fish IgM was not reduced by such treatment. Thus, we conclude that treatment of fish sera with skim milk is useful in reducing the high background OD often observed in fish IgM detection ELISA.     

Monoclonal antibodies to human sex hormone-binding globulin (SHBG): characterization and use in a simple enzyme-linked immunosorbent assay (ELISA) of SHBG in plasma.

Four monoclonal antibodies to human sex hormone-binding globulin were raised and characterized. Three of the four antibodies recognised different antigenic determinants on SHBG. Two of the distinct antibodies were useful for Western blotting and recognized a major 48 kDa band in human plasma as well as a 46 kDa minor component. Carbohydrate residues do not form part of the antigenic determinants of these two antibodies, although one of these showed increased signal following removal of N-linked oligosaccharides. Some of the antibodies were selected to form a basis of a same-day, non-competitive, enzyme-linked immunosorbent assay (ELISA) for SHBG in plasma. The assay employs a purified IgG2a SHBG monoclonal antibody adsorbed to the wells of a microtitre plate. After blocking any further adsorption to the plate, standards or diluted patient samples were added for a 5-h incubation at room temperature, after which the plate was washed and antibody-bound SHBG was detected with an anti-SHBG IgG1 monoclonal antibody followed by peroxidase-labeled antimouse-IgG1 and o-phenylenediamine substrate. The assay correlated well with an existing 2-day ELISA for SHBG in plasma using polyclonal antibodies and also correlated with a dihydrosterone (DHT) ligand-binding assay. The monoclonal antibody-based ELISA shows excellent performance characteristics and is unaffected by added testosterone or estradiol.     

High-throughput DNA synthesis in a multichannel format.

We describe an approach to high-throughput parallel DNA synthesis in which a multiwell format is used. The reactions are carried out in open wells using an argon ambient atmosphere to prevent reagent contamination. The controlled-pore glass beads which form the substrate for synthesis are held in individual wells with high-density polyethylene filter bottoms through which reagents are drawn into a vacuum manifold. The synthesis is carried out using direct reagent dispensing into the individual reaction wells. A computer controls the sequence in which reagents are dispensed and the timing of the periodic vacuum pulses required to synthesize the desired sequence. Experiments to date have demonstrated the viability of the approach for a variety of test sequences. Results obtained with HPLC analysis demonstrate coupling efficiencies as high as 99.5% under optimized conditions. Use of the oligomers for DNA sequencing templates and as PCR primers has been demonstrated in production applications. The current instrument design consists of a series of discrete reaction chambers in a 12 channel module which can be multiplexed in a 12 x n format where n can be 1-8, i.e. 96 wells. A projected time interval for 12 parallel syntheses is 2.5 h, with 96 syntheses in 3.5 h. Because of the reduced volume of reagents required in the open well format, significant cost savings are projected.     

Method for printing functional protein microarrays

Piezoelectric dispensing of proteins from borosilicate glass capillaries is a popular method of protein biochip fabrication that offers the advantages of sample recovery and noncontact with the printing substrate. However, little regard has been given to the quantitative aspects of dispensing minute volumes (1 nL or less) at the low protein concentrations (20 micrograms/mL or less) typically used in microprinting. Specifically, loss of protein sample due to nonspecific adsorption to the glass surface of the dispensing capillaries can limit the amount of protein delivered to the substrate. We demonstrate the benefits of a low ionic strength buffer containing the carrier protein BSA that effectively minimizes the ionic strength-dependent phenomenon of nonspecific protein adsorption to borosilicate glass. Over the concentration range of 20-2.5 micrograms/mL, the dispensing of a reference IgG in 10 mM PBS including 0.1% BSA resulted in the deposition of 3.6- to 44-fold more IgG compared to the deposition of IgG in standard 150 mM PBS in the absence of BSA. Furthermore, when the IgG was dispensed with carrier protein, the resulting spots exhibited a more uniform morphology. In a direct immunoassay for cholera toxin, capture antibody spots dispensed in 10 mM PBS containing 0.1% BSA produced fluorescent signals that were 2.8- to 4.3-fold more intense than antibody spots that were dispensed in 150 mM PBS without BSA. Interestingly, no differences were observed in the specific activities of the capture antibodies as a result of printing in the different buffers. The implications of these results on the future development of protein biochips are discussed.     

Protein A immunocapture assay detecting antibodies to fluke cysteine proteinases for immunodiagnosis of human paragonimiasis and fascioliasis

Enzyme-linked immunosorbent assays (ELISAs) which detect specific antibodies to fluke cysteine proteinases have provided good sensitivity and specificity for the immunodiagnosis of trematode diseases. To detect specific antibodies without the need for purified proteinase antigens, an immunocapture assay using Protein A was applied for the immunodiagnosis of paragonimiasis and fascioliasis. ELISA plate wells were coated with Protein A, incubated with diluted patient sera, then incubated with a preparation containing fluke cysteine proteinases, excretory-secretory (ES) products of adult Paragonimus westermani or Fasciola sp. The activity of fluke cysteine proteinases bound on the wells was measured by adding fluorogenic peptidyl substrate, Z-Phe-Arg-MCA or Boc-Val-Leu-Lys-MCA. This assay detected specific immunoglobulin G to cysteine proteinases of P. westermani and Fasciola sp. by measuring proteinase activity on the plate wells. Patient sera showed significant high values of proteinase activity when the wells were treated with the respective homologous ES products, whereas the sera had low values after treatment with the heterologous ES products. The sera of patients with other parasitoses and uninfected healthy individuals also showed low values after treatment with the above fluke ES products. Thus, Protein A immunocapture assay, which detected IgG specific for fluke cysteine proteinases, provided a high sensitivity and specificity for immunodiagnosis of paragonimiasis and fascioliasis.     

A piezoelectric immunoagglutination assay for Toxoplasma gondii antibodies using gold nanoparticles

The serologic detection of anti-Toxoplasma gondii immunoglobulins plays a key role in the clinical diagnosis of Toxoplasmosis. In this paper, a simple, rapid and highly sensitive agglutination-based piezoelectric immunoassay has been firstly developed for directly detecting anti-T. gondii immunoglobulins in infected rabbit serum (IRS) and infected rabbit blood (IRB). The proposed technique is based on that the specific agglutination of antigen-coated gold nanoparticles, averaging 10nm in diameter, in the presence of the corresponding antibody causes a frequency change that is monitored by a piezoelectric device. In contrast to the commonly used piezoelectric assays, it possesses an attractive advantage in that the immobilization of antibody or antigen on the crystal is unnecessary. Use of a newly prepared sensing probe which was modified by a plasma-polymerized film (PPF) of n-butyl amine and further by a heparin layer resulted in a response-enhanced immunoagglutination and a high compatibility of the probe with biological samples. An appropriate reagent consisting of 1% normal rabbit serum (NRS) and 0.1% bovine serum albumin (BSA) for diluting the analytes were verified in counteracting the background interference of assay. Moreover, an optimization of assay medium composition with the addition of poly(ethylene glycol) (PEG) serving as immunoagglutination rate and sensitivity enhancer was investigated in detail. It is found that the developed immunoagglutination assay system is sensitive to dilution ratio of anti-T. gondii antibody as low as 1:5500. Analytical results of several specimens obtained using the developed technique are in satisfactory agreement with those given by the ELISA method, implying a promising alternative approach for detecting anti-T. gondii antibodies in the clinical diagnosis.     

The measurement of staphylococcal protein A by ELISA in immunoglobulin preparations.

Chicken antibodies were used to develop an ELISA for the quantitation of parts-per-million levels of protein A in the purification of immunoglobulins or immunoglobulin-like molecules. Quantitation of protein A in the presence of excess human or murine immunoglobulins in this assay was compared with that obtained in ELISAs developed with rabbit antibodies specific either to protein A or to other molecules. Experiments demonstrate that protein A is bound to the immunoglobulins being purified and that this binding affects subsequent recognition by the antibodies used for the assay. Because of these effects and because fragments of protein A might not be detected in assays which rely on Fc binding of protein A, chicken antibodies that bind protein A specifically are an advantage for the quantitation of this protein by ELISA. In addition, comparison of the effect of different types of immunoglobulins on the protein A standard curve suggests that alternatives to including the immunoglobulin under purification with the standards can be utilized.     

Monoclonal antibodies to pregnanediol-3-glucuronide: application to a direct enzyme-linked immunosorbent assay of urine

Monoclonal antibodies to pregnanediol-3-glucuronide were produced and characterized. One of three clones investigated provided antibody suitable for a direct urinary enzyme-linked immunosorbent assay (ELISA). The ELISA uses a pregnanediol-thyroglobulin conjugate adsorbed onto the wells of a standard 96-well microtiter plate. Pregnanediol-3-glucuronide in standards or diluted urine competes with the immobilized steroid for antibody-binding sites. After washing, mouse monoclonal antibody bound to the plate is probed with antimouse immunoglobulin peroxidase. After further washing, o-phenylenediamine substrate is added and, finally, the absorbance is read at 492 nm. The ELISA shows excellent performance and agreement with the previous gas chromatographic method. The ELISA is ideal for aiding the assessment of ovarian function in the routine laboratory.     

Construction of high-density bacterial colony arrays and patterns by the ink-jet method

We have developed a method for fabricating bacterial colony arrays and complex patterns using commercially available ink-jet printers. Bacterial colony arrays with a density of 100 colonies/cm(2) were obtained by directly ejecting Escherichia coli (E. coli) onto agar-coated substrates at a rapid arraying speed of 880 spots per second. Adjusting the concentration of bacterial suspensions allowed single colonies of viable bacteria to be obtained. In addition, complex patterns of viable bacteria as well as bacteria density gradients were constructed using desktop printers controlled by a simple software program.     

A simple electrically-operated feeder for use in salmon rearing

The construction of a simple, automatic feeder designed to dispense dry food used in salmon rearing is described. The feeder is powered by a 12 V battery and controlled by two electric clocks. Frequency of feeding, the daily duration of the feeding period, and the quantity of food dispensed can be adjusted.     

一种改进的分泌抗半抗原抗体杂交瘤细胞株筛选方法简

目的：研究解决半抗原分子单克隆抗体制备技术路径中遇到的在阳性杂交瘤细胞株筛选时无法排除载体蛋白问交叉反应影响的问题,以半抗原去甲肾上腺素（norepinephrine,NE）为例。方法：在NE完全抗原免疫小鼠实施细胞融合后,分别包被牛血清白蛋白（BSA）、卵清白蛋白（OVA）、BSA-NE、OVA-NE等4种不同抗原的酶标板平行检测细胞培养上清液;挑选BSA、OVA检测结果为阴性,BSA-NE、OVA-NE检测结果为阳性的孔内细胞进行克隆化筛选单克隆细胞。结果：本筛选方法可一次性从8板96孔板中筛选到13个符合要求的阳性孔,经3次克隆化后获得6株特异性强的杂交瘤细胞株。结论：本方法避免了载体蛋白间交叉反应对筛选的影响,改进了传统的单一指标筛选方法,筛选效率更高。     

Solid-phase adsorption method for removing undesired antibodies from polyclonal antiserum

We have developed a novel and simple method, requiring only a small amount of antigen, for removal of undesired antibodies from antiserum. The method was established using a well-characterized antiserum against rat luteinizing hormone (anti-rLH). Wells of polystyrene tissue culture plates were coated with rat LH (rLH). Anti-rLH diluted 1:3000 was added to rLH-coated wells and shaken to remove LH antibodies. Control anti-rLH was treated in a similar manner in non-rLH-coated wells. Both antisera were tested by immunocytochemistry on rat pituitaries. Antiserum from rLH-coated wells stained no cells, whereas the control serum stained cells that were morphologically typical of LH cells. The effectiveness of this antibody removal was also confirmed in a modified ELISA. In another experiment, anti-rLH and anti-hTSH beta sera were mixed. The final dilution of both antisera was 1:10,000. Anti-rLH was removed by the purification method described. Completeness of antibody removal was confirmed by a double-immunohistochemical staining of rat pituitary in which sections were first stained by the PAP method and then stained with an immunofluorescence procedure after elution of the first antigen-antibody complex. The mixed antiserum incubated in rLH-coated wells did not stain LH cells. There was no co-localization between the LH immunopositivity demonstrated by an anti-rLH serum using immunofluorescence and cells immunostained with the purified antiserum using the PAP method. As indicated in ELISA, the titer of the TSH beta antiserum was not decreased compared to that of the untreated, mixed control antiserum, and the LH antibodies were eliminated by the treatment. This new purification method has four distinct advantages: (a) antiserum is not treated chemically; (b) it requires only a small amount of antigen compared with the amount required for affinity chromatography; (c) neither the undesired antigen-antibody complex(es) nor an excess amount of antigen is present in the purified antiserum; and (d) removal of undesired antibodies can be monitored by ELISA.     

A 1-Hour Enzyme-Linked Immunosorbent Assay for Quantitation of Acrolein- and Hydroxynonenal-Modified Proteins by Epitope-Bound Casein Matrix Method

A simple and rapid enzyme-linked immunosorbent assay (ELISA) method for quantitation of acrolein and 4-hydroxy-2-nonenal (HNE)-modified proteins was developed. Microtiter plate wells were precoated and blocked simultaneously with epitope-bound bovine caseins as matrix proteins, and aldehyde-modified proteins were quantitated by a competition assay with a monoclonal antibody specific for acrolein-modified lysine or HNE-modified histidine epitopes. Minimal reaction times required for the coating/blocking; first monoclonal antibody and the peroxidase-conjugated second antibody binding steps were 3, 3, and 7 min, respectively, the former two steps being found to be or akin to diffusion-rate-limiting reactions. The convenient ELISA should find an application for analyses of the intricate processes involved in oxidative stress and carcinogenic insult. The epitope-attachment methodology may also be advantageous for the quantitation of various other biologically important haptenic molecules.     

Picoliter-scale protein microarrays by laser direct write

We demonstrate the accurate picoliter-scale dispensing of active proteins using a novel laser transfer technique. Droplets of protein solution are dispensed onto functionalized glass slides and into plastic microwells, activating as small as 50-microm diameter areas on these surfaces. Protein microarrays fabricated by laser transfer were assayed using standard fluorescent labeling techniques to demonstrate successful protein and antigen binding. These results indicate that laser transfer does not damage the active site of the dispensed protein and that this technique can be used to successfully fabricate a functioning protein microarray. Also, as a result of the efficient nature of the process, material usage is reduced by two to four orders of magnitude compared to conventional pin dispensing methods for protein spotting.     

Miniaturization of intracellular calcium functional assays to 1536-well plate format using a fluorometric imaging plate reader

The measurement of intracellular calcium response transients in living mammalian cells is a popular functional assay for identification of agonists and antagonists to receptors or channels of pharmacological interest. In recent years, advances in fluorescence-based detection techniques and automation technologies have facilitated the adaptation of this assay to 384-well microplate format high-throughput screening (HTS) assays. However, the cost and time required performing the intracellular calcium HTS assays in the 384-well format can be prohibitive for HTS campaigns of greater than 1 x 10(6) wells. For these reasons, it is attractive to miniaturize intracellular calcium functional assays to the 1536-well microplate format, where assay volumes and plate throughput can be decreased by several fold. The focus of the research described in this article is the miniaturization of an intracellular calcium assay to 1536-well plate format. This was accomplished by modifying the hardware and software of a fluorometric imaging plate reader (FLIPR) to enable transfer of nanoliters of test compound directly to a 1536-well assay plate, and measure the resulting calcium response from all 1536 wells simultaneously. An intracellular calcium functional assay against the rat muscarinic acetylcholine receptor subtype 1 (rmAchR1) G-protein coupled receptor (GPCR) was miniaturized and executed on this modified instrument. In experiments measuring the activity of known muscarinic receptor agonists and antagonists, the miniaturized FLIPR assay gave EC(50) and IC(50) values and rank order potency comparable to the 384-well format assays. Calculated Z' factors for the miniaturized agonist and antagonist assays were, respectively, 0.56 +/- 0.21 and 0.53 +/- 0.22, which were slightly higher (Z'(agonist) = 0.55 +/- 0.33) and lower (Z'(antagonist) = 0.70 +/- 0.18) than the corresponding values in the 384-well assays. A mock agonist HTS campaign against the muscarinic receptor in miniaturized format was able to identify all wells spiked with the rmAchR1 agonist carbachol.     

The presence of non-isotype-specific antibodies in polyclonal anti-IgE reagents: demonstration of their binding to specifically selected Epstein-Barr virus-transformed cell lines

Polyclonal anti-human IgE reagents were earlier shown to contain variable amounts of nonisotype-specific antibodies depending on the strategy used for their preparation. The presence of these antibodies in two commercial anti-IgE reagents was demonstrated in this work by (a) their binding to human Ig-surface-positive lymphoblastoid cells specifically selected by one of the polyclonal anti-human IgE reagents and (b) their binding to the non-IgE immunoglobulins secreted by those lymphoblastoid cells. Peripheral blood B lymphocytes from two normal and two atopic patients were immortalized with Epstein-Barr virus (EBV) and then selected for cells that rosette with anti-IgE-coated erythrocytes. Selection was repeated four times and cells were then cloned. The cloned cells formed rosettes and their supernatants agglutinated erythrocytes coated with rabbit anti-IgE. The immunoglobulins of these clones were positive in an ELISA for IgE, using two different polyclonal anti-human IgE reagents. They were shown, however, to be 19 S IgMs. This discrepancy was due apparently to substantial contamination of anti-non-IgE-isotype-specific antibodies in the polyclonal anti-IgE reagents used both in the selection of cells and in the ELISA. The human monoclonal B-cell lines which were applied here as targets amplified the non-IgE-isotype specific antibody contamination present in the polyclonal anti-human IgE reagents. Because of the normally very low frequency of IgE-positive cells, the use of polyclonal anti-IgE reagents to detect these cells has to be carefully evaluated.     

兔抗五种鱼免疫球蛋白抗体的制备和HRP标记

目的制备兔抗鱼类免疫球蛋白抗体并进行辣根过氧化酶标记,为鱼类血清学检测系统的建立提供工具。方法利用proteinA亲和层析的方法纯化鱼血清免疫球蛋白,免疫大耳白兔制备抗血清,利用免疫双扩散来检测抗血清的效价,并用Protein A亲和层析的方法来纯化兔抗鱼免疫球蛋白的抗体,采用简易过碘酸钠法对纯化的兔抗鱼免疫球蛋白的抗体进行HRP标记,通过ELISA方法测定标记抗体的效价,并利用Western blotting来考察标记抗体与其他常见鱼类血清蛋白间的交叉反应。结果纯化了鳙、鲤、乌鳢、黄鳝、鲈五种鱼血清免疫球蛋白,免疫双扩散法测定兔抗这五种鱼免疫球蛋白的抗血清效价均达到1∶32,并对纯化的兔抗鳙、鲤、乌鳢、黄鳝、鲈五种鱼类免疫球蛋白的抗体进行了HRP标记,ELISA测定标记抗体的效价达到1∶10000左右,Western blots显示标记抗体与部分其他鱼类免疫球蛋白之间存在不同程度的交叉反应。结论成功制备了HRP标记的兔抗鱼类免疫球蛋白抗体,为鱼类血清学检测体系的建立提供了工具。