Simplification and standardization of dot-ELISA for human schistosomiasis mansoni

Dot-ELISA, a technique that shares the same principles as the enzyme immunoassay, is useful for detection of anti-Schistosoma mansoni antibodies in the sera of patients with Schistosoma mansoni infections. The antigens were fixed to the nitrocellulose strips, blocked with 1% bovine serum albumin in 0.05% Tween 20. Patient sera (40) and normal laboratory personnel sera (9) were applied to the sheet directly, without cutting the strips into small discs. The nitrocellulose sheets are kept in a humid chamber for 30 min and then washed. After incubation with peroxidase-conjugated goat anti-human antibody, washing, and addition of substrate, positive reactions appear as brown dots against the white background. The room temperature assay takes about 2 hr. The optimum antigen concentration is 20-80 ng per dot and the optimum serum dilution is 1:100-1:400. The sensitivity and specificity of the assay are 90-95% and 90%, respectively. The level of positivity of the dot-ELISA by an arbitrary scale compares with standard micro-ELISA. The single positive reaction in a normal serum sample in dot-ELISA is also positive in micro-ELISA. Cross-reactivity between the S. mansoni antigen and human fascioliasis sera was noticed in 2 out of 8 patient sera. Good correlation between the arbitrary level of dot-ELISA and the absorbance of standardized micro-ELISA shows that the dot-ELISA is useful both for laboratory and field studies.     

General detection of proteins after electroblotting by trinitrobenzene sulphonic acid derivatization and immunochemical staining with a monoclonal antibody against the trinitrophenyl group

A sensitive method for the general detection of proteins electroblotted onto nitrocellulose sheets after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is described. The proteins on the blots were reacted with 2,4,6-trinitrobenzene sulphonic acid. The resulting trinitrophenyl groups on the proteins were rendered visible by immunochemical staining with a monoclonal anti-trinitrophenyl antibody, and a peroxidase-conjugated second antibody. Using various proteins, the method was compared to the amidoblack method for staining of protein blots. The method was 10-100-fold more sensitive than the amidoblack method. Amounts as low as 1 ng of human serum albumin could be detected.     

Sensitivity and efficiency of four immunohistochemical methods as defined by staining of artificial sections

Direct (DIF) and indirect (IIF) immunofluorescence, indirect immunoperoxidase conjugate (IPC) and unlabelled antibody peroxidase antiperoxidase (PAP) staining was performed on sections of artificial substrate containing different concentrations of human immunoglobulin (Ig)A or IgG. Detection sensitivity, in terms of the lowest amount of discernible antigen, was evaluated by direct microscopy and by microphotometry. Staining efficiency (signal-to-noise ratio) was evaluated by microphotometry. Only minor differences in antigen detection sensitivity were found when IPC and PAP were compared with DIF and IIF under appropriate conditions. The sensitivity of DIF was only marginally improved by raised conjugate concentration and prolonged incubation time. Microphotometry of DIF on ethanol-fixed IgA substrate revealed that the staining intensity increased proportionally with the antigen concentration whereas on formaldehyde-fixed substrate a progressive masking of the antigen was indicated which, however, could be overcome by applying raised conjugate concentration and prolonged incubation time. Such antigenic self masking was of relatively little importance to IPC and PAP staining, probably because of the inherent amplification in these methods. An additional masking effect due to extraneous protein was revealed by DIF when ethanol-fixed sections had been soaked in bovine serum albumin and postfixed with formaldehyde; unmasking was achieved by proteolytic treatment of the sections.     

A direct antigen-binding assay to screen hybridoma supernatants

A simple and convenient method of directly assaying hybridoma supernatants for the desired monoclonal antibodies is described which obviates the need for labeled second or third antibody conjugates. Culture supernatants (1-5 microliters) were directly spotted onto a nitrocellulose sheet, and additional protein binding sites blocked with bovine serum albumin and incubated with enzyme-labeled, radioactive, or fluorescent antigen. Positive hybridoma supernatants were identified after washing and detection of bound antigen by appropriate means.     

Avidin- or streptavidin-biotin as a highly sensitive method to stain total protein on membranes

A sensitive method for staining proteins after transfer from polyacrylamide gels to nitrocellulose paper is described. Transferred proteins are first derivatized by reaction of the nitrocellulose replica with sulfosuccinimidobiotin and are then reacted sequentially with streptavidin, rabbit anti-streptavidin, and horseradish peroxidase-conjugated goat anti-rabbit IgG antibody. Incubation with the enzyme substrate α-chloronaphthol, produces dark protein bands against a white background. The binding of streptavidin to the proteins is dependent on biotin derivatization as demonstrated by competition with biotinylated bovine serum albumin or 10 nM biotin. The procedure detects less than 5ng of transferred protein in a single band and is thus 5–10 times more sensitive than horseradish peroxidase-conjugated avidin alone. For bovine serum albumin, the method is comparable in sensitivity to silver staining of protein in polyacrylamide gels.     

Applying phage antibodies to proteomics: selecting single chain Fv antibodies to antigens blotted on nitrocellulose

Two-dimensional gel electrophoresis is a powerful tool for identification of proteins that differ between patients with qualitatively or quantitatively different disease states. Further characterization of these protein differences would be greatly facilitated by the availability of antibodies that could be used to detect and quantitate the temporo-spatial pattern and cellular and tissue location of the different proteins. To generate such antibodies, methods were developed which permit the successful selection of monoclonal phage antibodies from phage display libraries against antigens blotted from SDS-PAGE gels onto nitrocellulose. First, it was determined that nitrocellulose and PVDF membranes gave significantly lower levels of background phage binding than two other membranes studied. Next, it was determined that blocking with fish gelatin and binding in the presence of 0.5 M NaCl could reduce nonspecific binding 10,000-fold and result in enrichment ratios greater than 500-fold with antigen concentrations as low as 1 ng/mm(2). When optimized conditions were applied to phage antibody libraries, panels of monoclonal phage antibodies were generated against the proteins ErbB2 and bovine serum albumin electroblotted from SDS-PAGE gels onto nitrocellulose. Antibodies were obtained with as little as 10 to 1 ng of antigen, depending on whether the libraries displayed single or multiple copies of antibody per phage. The antibodies worked as reagents in both ELISA and Western blotting.     

新型冠状病毒胶体金抗原快速检测试剂的研制及性能评价*

目的:建立新型冠状病毒(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)胶体金抗原快速检测试剂的制备方法,并对检测试剂的性能指标进行评价。方法:采用柠檬酸三钠还原法制备胶体金溶液,用鼠抗核衣壳蛋白(nucleocapsid protein, NP)单克隆抗体及二硝基苯酚-牛血清白蛋白(DNP-BSA)作为标记抗体,硝酸纤维素膜上分别包被鼠抗核衣壳蛋白单克隆抗体和兔抗DNP多抗作为检测线和质控线制备免疫胶体金试纸条;对试剂最低检出限、交叉反应性、加速稳定性及临床诊断特异性和灵敏度进行性能评价。结果:检测热灭活培养物的最低检出限为2.0×10² TCID₅₀/mL;测试16种常见呼吸道病原体高浓度样本均无交叉反应;试剂盒50℃加速破坏8周稳定。临床及健康人群鼻咽拭子样本测试,诊断灵敏度为96.67%(29/30),特异性为99.23%(129/130),总符合率为98.75%(158/160);一致性检验Kappa值为0.959 0,P<0.05。结论:SARS-CoV-2胶体金抗原快速检测试剂检测灵敏度和特异性高,检测速度快,操作便携,无需设备,肉眼观察,可作为现有核酸检测法的补充手段,用于新型冠状病毒的早期筛查。     

一种基于量子点检测抗CCP抗体的免疫荧光层析法

抗环瓜氨酸多肽 (cyclic citrullinated peptide, CCP) 抗体是类风湿关节炎 (rheumatoid arthritis, RA) 早期诊断的重要生物标志物. 为了实现对RA的早期诊断,本研究建立了一种基于CdTe量子点标记技术检测抗CCP抗体的免疫荧光层析法. 将CCP多肽与小牛血清白蛋白 (bovine serum albumin, BSA) 连接,再将CCP-BSA和 羊抗鼠IgG分别在硝酸纤维素膜 (nitrocellulose membrane, NC膜) 上划线,作为检测线 (test line, T线) 和质控线 (control line, C线). 制备量子点并在量子点上标记鼠抗人IgG,喷在玻璃纤维上并烘干,最后组装大卡、切割并封装制成检测试纸条. 应用该试纸条检测了RA患者及健康人血清临床样本200份,以酶联免疫吸附 测定法 (enzyme-linked immunosorbent assay, ELISA) 为对照,计算免疫荧光层析法的检测灵敏度和特异性. 结果显示,建立的量子点免疫荧光层析试纸条检测抗 CCP抗体的灵敏度为97.5 %,特异性为95.8%. 该方法操作简单、快速,可实现床旁检测 (point-of-care testing, POCT),能应用于RA的早期诊断.     

A rapid, simple immunofluorometric assay: development and characterization

A novel two-site immunofluorometric assay, which includes only one incubation step and one separation step, is described. The assay is based on using small-sized beads (0.5 mum diameter) as a soild support and measuring the unbound fraction of labeled antibody in the liquid. The use of a mixture of the solid-phase and labeled antibodies at different concentrations makes it possible to determine antigen concentration over a wide range, without an initial sample dilution. Two assays were developed: one using anti-IgG polyclonal antibodies and the other using antibovine serum albumin monoclonal antibodies. The detection range of the polyclonal antibody assay using 30-minute incubation was 0.2 to 40 mug/mL for human IgG standard. The detection range of the monoclonal antibody assay was 0.2 to 14 mug/mL for bovine serum albumin (BSA) standard with 2 minutes required for the incubation. The interassay variability for the BSA measurement was 1.9% at 4.0 mug/mL of BSA and intra-assay variability was 2.3% at 3.2 mug/mL of BSA. The principles of this assay can be applied in the measurement of any protein in a rapid and reproducible way. (c) 1992 John Wiley & Sons, Inc.     

Immunosensors

The current trends and future aspects of the research and development of immunosensors are overviewed. A non-labelled immunosensor, whose selectivity depends on immunochemical affinity of an antigen for its corresponding antibody, has been developed as the basis for the potentiometric determination of an antigen, with an antibody-bound membrane or electrode. Non-labelled immunosensors for syphilis antibody, blood typing, human chorionic gonadotropin (HCG), and human serum albumin have been investigated. In contrast with non-labelled immunosensors, labelled immunosensors may be characterized by marked enhancement of sensitivity. Of these labelled immunosensors, enzyme immunosensors that use the chemical amplification of a labelling enzyme for sensitivity are promising. Enzyme immunosensors with an oxygen electrode have been developed to determine AFP, HCG, IgG and toxin. Bioaffinity sensors with a preformed metastable ligand-receptor complex, which are similar to the enzyme immunosensor have been found effective for the determination of thyroxine (T4), biotin, and insulin.     

Rat sulfite oxidase antibodies cross-react with two gene family-related proteins: albumin and vitamin D-binding protein.

Screening lambda cDNA libraries from rat liver with antibody to native rat liver sulfite oxidase (RLSO) showed cross-reaction with two proteins that belong to the same gene family: serum albumin and vitamin D-binding protein. Antibodies raised against native RLSO or sodium dodecyl sulfate-denatured protein cross-reacted with these proteins by Western blot analysis. The relative effectiveness of RLSO antibody binding was estimated to be 1/5 for rat serum albumin and 1/10 for rat vitamin D-binding protein. This result was not caused by contaminating proteins in the RLSO used for immunization as the RLSO preparation did not react with rat serum albumin antibody. RLSO antibodies, selected for their ability to bind rat serum albumin immobilized on nitrocellulose, recognized both rat serum albumin and RLSO. RLSO antibody, with albumin-reactive antibody removed, still recognized vitamin D-binding protein, suggesting that multiple determinants specific to each protein are involved in the cross-reaction. Comparison of RLSO antibody binding to the rat and human proteins indicated that the determinants were species-specific. cDNA clones identified by screening cDNA libraries with RLSO antibody demonstrated that these determinants reside in the C-terminal domain of these proteins. These results suggest that these proteins contain some common immunological features and may be evolutionarily related.     

Enhanced chemiluminescence ELISA for the detection of antibody to hepatitis B virus surface antigen

A chemiluminescent enzyme linked immunosorbent assay (ELISA) for the detection of antibody to hepatitis B virus surface antigen (anti-HBs) in human serum has been developed. Polystyrene microtitre plates were coated with recombinant, yeast-derived hepatitis B surface antigen (rec-HBsAg). Patient serum samples and appropriate controls were added to the rec-HBsAg-coated wells and incubated to bind anti-HBs. The wells were then washed and a fluorescein isothiocyanate (FITC) conjugate of a human plasma-derived hepatitis B surface antigen (HBsAg) was added. Following incubation and further washing the bound FITC-labelled HBsAg was detected after addition of a horseradish peroxidase (HRP) conjugate of a monoclonal anti-FITC antibody and assaying for the enzyme. The activity of the HRP was measured using luminol and hydrogen peroxide as substrates and iodophenol as a chemiluminescence enhancer. The luminescence was recorded using a camera luminometer. Preliminary tests have shown the assay to be suitable for the detection of antibody in sera from both vaccinees and also from individuals with a past hepatitis B virus infection. The use of the FITC-anti-FITC system together with the measurement of a chemiluminescence signal makes possible the completion of this assay in a few hours. The assay has been shown to be both specific and sensitive and provides a permanent photographic record.     

Western immunoblotting: temperature-dependent reduction in background staining

The effect of incubation temperature on the background staining of Western blots with monoclonal antibodies to a human milk protein, alpha-lactalbumin (Mr 14,500), is presented. Human milk proteins were electrophoretically separated and transferred to nitrocellulose membranes which were then blocked with bovine serum albumin, "BLOTTO", casein, or Tween 20. They were subsequently incubated with mouse monoclonal antibody to human alpha-lactalbumin, biotinylated anti-mouse antibody, strepavidin-biotinylated horseradish peroxidase complexes and a substrate containing diaminobenzidine and nickel chloride. Reduction of incubation temperature from 37 degrees C to 22 degrees C and 4 degrees C was found to decrease the extent of non-specific background staining independent of the type of blocking reagent used. Good specific staining with minimal background was found using 0.1% Tween 20 in phosphate-buffered saline, pH 7.2, as blocking agent and incubation temperatures of 4 degrees C.     

Potential application of dot-immunobinding assay as a rapid diagnostic test in tuberculous meningitis

A simple dot-immunobinding assay (Dot-Iba) in nitrocellulose paper was developed for the detection of specific IgG antibody to Mycobacterium tuberculosis antigen 5 and mycobacterial antigen in cerebrospinal fluid of patients with tuberculous meningitis (TBM). The assay gave 77.1% sensitivity for the detection of IgG antibody to M. tuberculosis antigen 5 and 48.6% sensitivity for the detection of mycobacterial antigen in patients with TBM.     

The use of gold reagents to quantitate antibodies eluted from nitrocellulose blots: application to electron microscopic immunocytochemistry

An assay is described in which gold reagents were used to quantitate nanogram amounts of antibody that had been eluted from antigens immobilized on nitrocellulose paper. Standard curves were generated by the application of rabbit immunoglobulin G (IgG) to nitrocellulose sheets assembled in a dot blot matrix apparatus. Blots were stained using either colloidal gold or immunogold, enabling quantitation of IgG concentration by scanning densitometry. Linear and reproducible standard curves were obtained. As little as 1 ng IgG/dot could be quantified using either gold reagent. In contrast to colloidal gold, immunogold could be used specifically to quantitate rabbit IgG regardless of the presence of bovine serum albumin or antigen coeluted from the nitrocellulose blot. The applicability of the immunogold assay was demonstrated by fractionating a complex rabbit antiserum raised against the RIM protein of frog retinal rod outer segments. Anti-RIM antibody was affinity-purified, quantitated by the immunogold assay, and subsequently employed in immunocytochemical studies using thin sections of retina embedded in a hydrophilic plastic, LR-Gold.     

Development of a new sensitive immunostrip assay based on mesoporous silica and colloidal Au nanoparticles

A new competitive immunostrip assay was developed to detect human serum albumin (HSA) in urine sample with use of conjugated monoclonal antibody gold nanoparticles (mAb–AuNPs) and mobile crystalline material (MCM)-41–HSA bioconjugate. To prepare the immunostrip, the colloidal AuNPs with an average particle diameter of 20 nm, was synthesized, labeled with antibody and applied on the conjugate pad as the detection reagent. Then, HSA was attached to the MCM-41 mesoporous nanoparticles and immobilized to a nitrocellulose membrane as the test line. In the optimized investigational conditions, the immunostrip could detect HSA in a high linear range (from 1 to 200 μg/ml) and low detection limit (ng/ml). The reliability of the testing procedure was examined by performing the immunostrip test with 30 urine samples and comparing the results with those obtained via immunoturbidimetry. The immunostrip was adequately sensitive and accurate for a rapid screening of HSA in the urine. This new strategy for competitive immunostrip design can be used and developed for other antigen based immunostrip assay.     

Functional properties of proteins immobilized on albumin microspheres

Bovine serum albumin (BSA) microspheres with an average diameter of 12.5 micron were prepared by crosslinking of BSA molecules with glutaraldehyde in the presence of polymethylmethacrylate dissolved in chloroform-toluene. Trypsin and anti-human IgG antibody were immobilized onto their surfaces by the glutaraldehyde-activation method. The catalytic activity and storage stability of the immobilized trypsin were satisfactorily high. The enzyme immunoassay (EIA) method using BSA-microspheres as a solid phase has a high sensitivity (the minimum concentration of detectable antigen in the sample: 0.2 ng/ml) and a wide concentration range (final concentration 0.027-3000 ng/ml) for the detection of human IgG.     

Trophoblast/leukocyte-common antigen is expressed by human testicular germ cells and appears on the surface of acrosome-reacted sperm

The murine monoclonal antibody H316 reacts with a cell-surface antigen of human trophoblast, leukocytes, certain epithelia, and several malignant cell types. We have found that the H316 antibody also recognizes an antigen synthesized by pre- and post-meiotic human testicular germ cells and is expressed in the acrosomal region of methanol-fixed testicular, epididymal, and ejaculated sperm. The antigen is poorly expressed on the surface of fresh ejaculated motile sperm, but is detectable on most viable sperm after a 6-h incubation in medium containing human serum albumin (HSA), or 60-min incubation with the calcium ionophore A23187 (both treatments induce sperm acrosomal changes termed capacitation and acrosome reaction). We found that antigen recognized by H316 is immunoprecipitated as a single, broad 50 kDa band from radiolabeled ionophore-treated sperm extracts and that preincubation of HSA-capacitated sperm with this antibody causes a moderate, but significant, inhibition of hamster egg penetration. These data indicate that the antigen recognized by the H316 monoclonal antibody is synthesized by testicular germ cells and is surface-expressed on capacitated/acrosome-reacted sperm populations. Its potential as a human sperm acrosome reaction marker, and possible biological role in sperm-egg or sperm-lymphocyte interactions, warrants further investigation.     

Sensitivity and efficiency of four immunohistochemical methods as defined by staining of artificial sections

Summary Direct (DIF) and indirect (IIF) immunofluorescence, indirect immunoperoxidase conjugate (IPC) and unlabelled antibody peroxidase antiperoxidase (PAP) staining was performed on sections of artificial substrate containing different concentrations of human immunoglobulin (Ig)A or IgG. Detection sensitivity, in terms of the lowest amount of discernible antigen, was evaluated by direct microscopy and by microphotometry. Staining efficiency (signal-to-noise ratio) was evaluated by microphotometry. Only minor differences in antigen detection sensitivity were found when IPC and PAP were compared with DIF and IIF under appropriate conditions. The sensitivity of DIF was only marignally improved by raised conjugate concentration and prolonged incubation time. Microphotometry of DIF on ethanol-fixed IgA substrate revealed that the staining intensity increased proportionally with the antigen concentration whereas on formaldehyde-fixed substrate a progressive masking of the antigen was indicated which, however, could be overcome by applying raised conjugate concentration and prolonged incubation time. Such antigenic self masking was of relatively little importance to IPC and PAP staining, probably because of the inherent amplification in these methods. An additional masking effect due to extraneous protein was revealed by DIF when ethanol-fixed sections had been soaked in bovine serum albumin and postfixed with formaldehyde; unmasking was achieved by proteolytic treatment of the sections.This work was supported by the Norwegian Cancer Society, the Norwegian Research Council for Science and the Humanities, and Anders Jahres Foundation