An evaluation of elution techniques in the study of immune complex glomerulonephritis.

Antigen and antibody from glomerular immune complex deposits in rabbits with experimental bovine serum albumin-(BSA) induced chronic serum sickness (CSS) were quantitated in elutes from kidneys in which a portion of the antigen and antibody had been radiolabeled. The largest quantities of 125I BSA eluted with 1 M roprionic acid at pH 2.7 (86%) and 0.1 M borate buffer at pH 11.25 (80%). However, these buffers yielded less functional anti-BSA antibody than 0.02 M citrate buffer at pH 3.2 (344 mug/g kidney). Citrate buffer-eluted anti-BSA antibody was reactive in immunodiffusion, immunofluorescence, and radiolabeled BSA binding test systems, but complement fixation was impaired relative to chaotropic ion-eluted antibody. It was found that up to 75% of the eluted antibody was lost to further study by recombination with eluted BSA. This could be prevented by fractionation of the dissociated eluate before neutralization. IgG fractionated eluates were successfully fluorescein conjugated or radiolabeled for use as reagents. Elution of cryostat sections of CSS kidney was also studied; BSA, IgG, and complement (C3) eluted in parallel, and sub-microgram quantities of anti-BSA antibody were recovered.     

Enzyme-linked immunosorbent assay for rat hepatic triglyceride lipase

A noncompetitive enzyme-linked immunosorbent assay to measure rat hepatic triglyceride lipase (H-TGL) was developed. Antibodies to rat H-TGL were purified from goat antisera by immunoadsorption on an H-TGL-Sepharose 4B column. Routinely, Immulon 2 Removawell strips were coated with the purified antibody overnight at 4 degrees C. After blocking the wells with bovine serum albumin (BSA) for 2 hr at room temperature, standards (0.85 ng/ml-13.1 ng/ml) or samples were added to the wells and were incubated with the bound anti-rat H-TGL overnight at 4 degrees C. The standards and samples had been pretreated with 5-20 mM SDS for 30 min at room temperature and were then diluted so that the final SDS concentration in the assay was 1 mM or less. The pretreatment with SDS was necessary to achieve maximal immunoreactivity. The sample incubation was followed by an overnight incubation at 4 degrees C with an anti-rat H-TGL-horseradish peroxidase conjugate. Rat H-TGL was detected by the color development after the addition of 0.4 mg/ml of o-phenylenediamine in 0.01% H2O2, 0.1 M citrate phosphate, pH 5.0. A linear relationship was obtained between absorbance at 490 nm and the amount of highly purified rat H-TGL used as a standard. Inclusion of 1 M NaCl in the assay buffer (1% BSA, 0.05% Tween 20, 10 mM phosphate, pH 7.4) during the sample and conjugate incubations minimized non-specific interactions. Recoveries of purified rat H-TGL added to a rat liver perfusate sample ranged from 98.6% to 103%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Generation and evaluation of anti-mouse IgG IgY as secondary antibody

Abstract

In order to evaluate the possibility of using IgY as the secondary antibody in immunoassay, specific IgY (1: 128,000) was generated by immunizing hens with mouse serum IgG purified by protein A column. IgY was extracted from egg yolk by polyethylene glycol 6000 (PEG-6000), and further purified using protein M affinity chromatography column. The purified IgY was conjugated with horseradish peroxidase (HRP) and fluorescein?isothiocyanate (FITC), in that order. The reactivity of conjugated antibodies was evaluated by ELISA, Western blot and Immunofluorescence, demonstrating that the obtained IgY was able to conjugate with enzymes, react with mouse primary IgG antibody, and subsequently amplify the antigen-antibody signals in different immune reaction conditions, in a comparable secondary effect to conventional goat anti-mouse IgG antibody. The obtained conjugated antibodies showed high stability in broad pH ranges (4–10; >70%) and high thermostability at 37?°C for 84?h (>85%). Despite the need to further consider and evaluate the industrial standardization and production process, our data provided the primary evidence that conjugated IgY antibodies can be used as a secondary antibody for broad immunological analysis.     

Lysozyme for capture of microorganisms on protein biochips

Lysozyme placed on the SiO₂ surfaces that have previously been derivatized with C₁₈ coating will capture both Escherichia coli and Listeria monocytogenes cells from PBS buffer at pH 7.2. This phenomenon is of significance for the design and fabrication of protein biochips that are designed to capture bacteria from buffer or water so that these can be further interrogated with respect to possible pathogenicity. Fluorescent microscopy shows that two types of bacteria (gram-negative E. coli and gram-positive Listeria spp.) will be adsorbed by lysozyme placed on the surface of the biochip but that strong adsorption of the bacteria is reduced but not eliminated when Tween 20 is present (at 0.5%) in the PBS buffer in which the cells are suspended. In comparison, Tween 20 and Bovine Serum Albumin (BSA) almost completely block adsorption of these bacteria on C₁₈ coated surfaces. The combination of a lysozyme surface with Tween 20 gives a greater degree of adsorption of L. monocytogenes than E. coli, and hence suggests selectivity for the more hydrophobic E. coli may be reduced by the Tween 20. This paper presents protocols for preparing protein-coated, SiO₂ surfaces and the effect of buffer containing Tween 20 on adsorption of bacteria by SiO₂ surfaces coated with C₁₈ to which BSA, lysozyme or C11E9 antibody is immobilized at pH 7.2 and ambient temperature.     

Stabilization of diluted aqueous solutions of horseradish peroxidase

Effects of pH, enzyme concentration, and various supplements on the catalytic activity, temperature stability, and secondary structure of horseradish peroxidase (HRP) were studied in diluted aqueous solutions. In 5.0 mM citrate-phosphate buffer (pH 4.2) at 55 degrees C and infinite dilution, HRP was inactivated with a rate constant of 2.86 x 10(-3) s-1. CaCl2, BSA, and glycerol caused protective effects, whereas KCl, LiCl, maltose, PEG-6000 (at a concentration above 3%), Triton X-100, ethanol, and Kathon CG had an opposite effect and altered the secondary structure of HRP. Two HRP-stabilizing media: the "glycerol-based" one containing 10% ethanol and 20% glycerol, or the "protein-based" one containing 0.1% Kathon CG and 0.2 g/l of BSA in 50.0 mM Tris-HCl buffer (pH 7.2) supplemented with 50 mM CaCl2 were developed, and the stability of HRP (0.36 nM) and its immunoglobulin, cortisol, and progesterone conjugates were compared in these two media. The protein-based medium displayed a greater stabilizing effect particularly on HRP-steroid conjugates.     

Development of enzyme-linked immunosorbent assay for determination of polybrominated diphenyl ether BDE-121

Our interests are in the development of immunoassay-based fast scanning methods for persistent organic pollutants. To develop the immunoassay method of polybrominated diphenyl ether (PBDE), a model compound of PBDE, 2,3′,4,5′,6-pentabromodiphenylether (BDE-121), has been chosen to develop its antibody and the competitive indirect enzyme-linked immunosorbent assay (ELISA) is developed. The hapten of BDE-121 containing reactive carboxylic acid was synthesized and conjugated to carrier proteins (bovine serum albumin [BSA] and ovalbumin [OVA]). Anti-BDE-121 polyclonal antibody was then developed in rabbits as a result of immunization with the BDE-121–BSA conjugate. The optimal amount of coating antigen BDE-121–OVA conjugate and the dilution of antiserum needed in the ELISA were determined with the checkerboard method, and the effects of the properties of PBST (phosphate-buffered saline and Tween 20) buffer (pH and salt concentration) and chemical solvent (types and concentrations) on the ELISA were investigated to achieve a rapid robust assay with high sensitivity. Under the optimized conditions, the developed indirect ELISA shows a linear detection range from 1.74 to 84.1 ng/ml, with an IC₅₀ value of 8.07 ng/ml and a detection limit of 0.644 ng/ml. In total, 11 kinds of compounds were tested for calculating the cross-reactivity, which was less than 8% for nearly all of them. Real samples were analyzed by the proposed immunoassay and gas chromatography/mass spectrometry (GC/MS).     

Direct and indirect competitive monoclonal antibody-based ELISA of Aflatoxin B1 in groundnut

Direct and indirect competitive enzyme-linked immunosorbent assays were optimized for the determination of aflatoxin B₁ in groundnut utilizing a specific monoclonal antibody developed at the University of Strathclyde, UK. The monoclonal antibody was conjugated to horseradish peroxidase (HRP) for direct competitive assay, while a commercially available goat-antimouse IgG-HRP conjugate was employed for indirect competitive ELISA. Both ELISAs detected aflatoxin B₁ as low as 20 pg/well. Methanol-water-KCl (70 + 30 v/v, 0.5 %) extracts of groundnut were assayed by ELISA after diluting 1: 10 with PBS-Tween buffer or subjected to simple cleanup for 5:1 concentration prior to assay. The mean recoveries from groundnut spiked with 10 to 200/ig/kg of pure aflatoxin B₁ were >90% in either ELISA, but the toxin recoveries at concentrations of 1–5μg/kg were only 65–67 % when subjected to cleanup and concentration before assay. The mean within-assay, inter-assay, and sub-sample coefficients of variation by ELISA of aflatoxin B₁ in naturally contaminated groundnuts were, respectively, 8.9%, 11.1%, and 7.9% for direct competitive assay and 4.6%, 11.2%, and 8% for indirect competitive assay. Both ELISA methods are useful for routine analysis of aflatoxin B₁ in groundnuts.     

Quantitative assessment of factors affecting the sensitivity of a competitive immunomicroarray for pesticide detection

Analytical protein microarrays offering highly parallel analysis can become an invaluable tool for a wide range of immunodiagnostic applications. Here we describe factors that influence the sensitivity of a competitive immunomicroarray that quantifies small molecules; in this case, the pesticides dichlobenil metabolite 2,6-dichlorobenzamide (BAM) and atrazine. Free pesticide concentrations in solution are quantified by the competitive binding of fluorescence-conjugated monoclonal antibodies to either surface-immobilized pesticide hapten-protein conjugates or pesticides in solution. We investigated the influence of antibody labeling techniques, microarray substrates, and spotting and incubation buffers. The results showed that microarrays immobilized on EasySpot or in-house fabricated agarose substrates printed with Genetix Amine Spotting Solution resulted in optimum results when the arrays were incubated with the sample/antibodies diluted in a Tris buffer supplemented with 0.05% each bovine serum albumin (BSA) and Tween 20. Furthermore, the application of directly labeled primary antibodies allowed for better sensitivity compared to secondary polyclonal antibody quantification.     

成人腹泻轮状病毒ELISA方法的建立和应用

本文通过特异性试验、阻断试验、交叉试验、敏感度试验和重复性试验,建立了成人腹泻轮状病毒一酶联免疫吸附试验法(ADRV—ELISA)。应用此法检测了全国20多个省区202份病人腹泻标本,检出率为91％。采用本ELISA、核酸电泳、电镜三种方法对48份病人腹泻标本进行了双盲法检测比较,结果三种方法的阳性检出率分别为100％、85.4％、56.25％(P<0.05)。实验结果表明,本ELISA应用于检测成人腹泻轮状病毒(ADRV),具有敏感度高。特异性强等优点。     

Selective immunoprecipitate identification using monoclonal anti-rabbit IgG

A monoclonal mouse antibody directed against rabbit IgG has been conjugated with horseradish peroxidase and used to identify immunoprecipitates which contain rabbit antibodies. By combining a specific rabbit antisera with a general antiserum from another species (e.g., goat antiserum against human serum), immunoprecipitates containing the antigen(s) recognized by the rabbit antibodies have been selectively identified by colorimetric development of peroxidase activity. Since the monoclonal antibody is specific for rabbit IgG and nonprecipitating, the peroxidase conjugate can be included in the agarose with the primary antisera.     

Propyl gallate inhibition of iodide and tetramethylbenzidine oxidation catalyzed by human thyroid peroxidase

The kinetic characteristics (kcat, Km, and their ratio) for oxidation of iodide (I-) at 25 degrees C in 0.2 M acetate buffer, pH 5.2, and tetramethylbenzidine (TMB) at 20 degrees C in 0.05 M phosphate buffer, pH 6.0, with 10% DMF catalyzed by human thyroid peroxidase (HTP) and horseradish peroxidase (HRP) were determined. The catalytic activity of HRP in I- oxidation was about 20-fold higher than that of HTP. The kcat/Km ratio reflecting HTP efficiency was 35-fold higher in TMB oxidation than that in I- oxidation. Propyl gallate (PG) effectively inhibited all four peroxidase processes and its effects were characterized in terms of inhibition constants Ki and the inhibitor stoichiometric coefficient f. For both peroxidases, inhibition of I- oxidation by PG was characterized by mixed-type inhibition; Ki for HTP was 0.93 microM at 25 degrees C. However, in the case of TMB oxidation the mixed-type inhibition by PG was observed only with HTP (Ki = 3.9 microM at 20 degrees C), whereas for HRP it acted as a competitive inhibitor (Ki = 42 microM at 20 degrees C). A general scheme of inhibition of iodide peroxidation containing both enzymatic and non-enzymatic stages is proposed and discussed.     

Measurement of apolipoprotein A-I concentration in nonhuman primate serum by enzyme-linked immunosorbent assay (ELISA)

A sensitive and specific enzyme-linked immunosorbent assay (ELISA) for nonhuman primate serum apolipoprotein A-I (apoA-I) is described. The assay is a noncompetitive, sandwich ELISA in which polystyrene microtiter plates were used with purified, monospecific goat anti-monkey apoA-I antibodies adsorbed on the wells. The serum samples were added to the coated wells, incubated, and after washing, antibodies conjugated to horseradish peroxidase were added. After further washing, the bound label was assayed. A heat treatment step, 52 degrees C for 3 hr, was used to maximize the apoA-I immunoreactive sites in diluted serum. Serum samples extracted with chloroform-methanol, delipidated with tetramethylurea, or denatured by heating gave essentially equivalent results. The working range of the apoA-I standards was 0.5 to 5 ng and parallel responses were observed for apoA-I in serum, in isolated HDL, and in buffer as a purified apoprotein. Recovery of apoA-I added to serum was quantitative (106 +/- 3%). The intra- and interassay coefficients of variation were 6.2 and 6.9%, respectively. The enzyme immunoassay yielded values that compared favorably with those obtained by radial immunodiffusion (r = 0.84). ApoA-I concentration in African green monkey serum was highly correlated with the HDL cholesterol concentration (r = 0.86). It is concluded that this ELISA is an accurate and precise method for determination of apoA-I concentrations in primate serum.     

The standardization of a 'spot-test' ELISA for the rapid screening of sera and hybridoma cell products I. The determination of the optimum buffering system

Using the different commercially available enzyme-linked immunosorbent assay (ELISA) plates, several sources of albumin were tested along with Tween 20 as supplements to the diluting buffer in ELISA for their ability to minimize non-specific reactions. There was an obligate requirement for Tween 20 (0.05%) but the different albumin sources had varied effectiveness on each of the different ELISA plates. In general, however, the optimum buffering system was concluded to be phosphate-buffered saline supplemented with 0.05% (v/v) Tween 20 and 3% (w/v) lactalbumin yeast hydrolysate or bovine serum (plasma) albumin fraction V.     

免疫检测信号放大抗体偶联物的制备及应用*

目的: 以聚赖氨酸(polylysine,PL)为骨架提高辣根过氧化物酶(horseradish peroxidase,HRP)与羊抗兔IgG的连接数量,比较几种化学偶联剂的偶联效果,通过免疫检测技术对其灵敏度进行检测和比较。方法: 对HRP与PL、聚合物HRP-PL与N-琥珀酰-S-乙酰乙酸(N-succinimidyl-S-acetylthioacetate,SATA)和2-亚氨基硫烷(Traut’s)两种试剂、羊抗兔IgG与琥珀酰亚胺基4-(N-马来酰亚胺甲基)环己烷-1-羧酸酯[succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate,Sulfo-SMCC]、活化后羊抗兔IgG及HRP-PL进行摩尔比的优化;对偶联物IgG-PL-HRP及商品化二抗分别进行斑点免疫印迹、ELISA和免疫组化,并计算偶联物IgG-PL-HRP的检测放大倍数。结果: 当PL与HRP摩尔比为1∶5,HRP-PL与Traut’s摩尔比为1∶15,羊抗兔IgG与Sulfo-SMCC摩尔比为1∶30,羊抗兔IgG与HRP-PL摩尔比为1∶10时,反应效率较高;商品化二抗及偶联物IgG-PL-HRP在斑点免疫印迹实验中的最低检测限分别为2.5 μg和312.5 ng,最大稀释倍数分别为50和100倍;在ELISA实验中的最大稀释倍数分别为5 000和20 000倍;在免疫组化实验中偶联物IgG-PL-HRP的检测特异性及强度均大于商品化二抗。结论: 成功合成抗体偶联物IgG-PL-HRP,且免疫检测信号放大倍数为商品化二抗的3~7倍,这对后续免疫诊断及生物学的研究具有重要意义。     

Detection of Listeria monocytogenes using polyclonal antibody

Polyclonal antibody sensitive to Listeria was assayed for the detection of Listeria using two different methods, direct and sandwich enzyme linked immunosorbent assays (ELISAs). The direct ELISA uses anti-goat IgG antibody conjugated with horse-radish peroxidase, while the sandwich ELISA uses two antibodies both specific to Listeria antigens, one coated onto the microtitre plate and the other conjugated to horse-radish peroxidase. The results obtained show that the direct ELISA is superior to the sandwich ELISA in two distinct ways: (i) with direct ELISA the non- Listeria gave readings <0.2, whereas with sandwich ELISA it gave readings of 0.3–0.4; (ii) the direct ELISA is more cost-effective than the sandwich ELISA.     

应用异型双功能交联剂制备酶-抗体结合物

酶标免疫测定法(ELISA)中最关键的化合物是酶-抗体结合物,将酶和抗体交联起来需用交联剂。本文作者使用了N-琥珀酰亚胺基3-(2-吡啶基二硫)丙酸酯(简称SPDP)将辣根过氧化物酶(HRP)和兔抗小鼠IgG(兔IgG)交联起来。我们试验了SPDP/HRP,SPDP/IgG和HRP/IgG的不同比例,以期获得活性高的酶-抗体结合物。此外还研究了从结合物中去除自由HRP和自由IgG的方法。用SDS-PAGE及硝酸纤维膜电泳转移法证明本法制备的结合物不含HRP及IgG的自身聚合物。用ELISA法鉴定结合物制品时,一般稀释度可达到1:10,000以上,有的可达到1:20,000(当结合物浓度A_(280nm)=1.0,底物显色A_(492nm)=1.0时)。     

Development of a microtitre plate indirect ELISA for measuring cortisol in teleosts, and evaluation of stress responses in rainbow trout and gilthead sea bream

A microtitre plate indirect enzyme‐linked immunoassay (ELISA) was developed for measuring plasma cortisol levels in rainbow trout Oncorhynchus mykiss, gilthead sea bream Sparus auratus sea bass Dicentrarchus labrax and Senegalese sole Solea senegalensis. Covalink microplates pretreated with disuccinimidyl suberate were coated with bovine serum albumin (BSA) conjugated to cortisol‐3‐carboxymethyl oxime. After blocking with BSA, competition was started by addition of plasma samples and anti‐cortisol antibody raised in rabbit. Goat anti‐rabbit IgG conjugated‐peroxidase was added as second antibody and then incubated with orthophenylenediamine as substrate. Reaction was stopped with 0·1 M HCl and absorbance was read at 450 nm in an automatic plate reader. The standard curve was linear from the lower limit of sensitivity of the assay (c. 0·3 ng ml^?1) to c. 3000 ng ml^?1. Dose‐response inhibition curves using serially diluted plasma samples of four species consistently showed parallelism with the standard curve using cortisol. The ELISA satisfied the strictest criteria of specificity (cross‐reactivity of anti‐cortisol antibody with testosterone, progesterone and 17ß‐oestradiol was negligible, cross‐reactivity with cortisone, corticosterone and 11‐deoxycortisol, was 1·5, 1 and 0·1%, respectively), reproducibility (interassay CV <6%), precision (intra‐assay CV <4%), and accuracy (average recovery >98%). Plasma cortisol concentration in rested fishes was in the range of 5–30 ng ml^?1. To physiologically validate the technique, changes in plasma cortisol concentrations were also measured in plasma of rainbow trout and gilthead sea bream following an acute 15 min chasing or 3 min air‐exposure stress, respectively. In both species plasma concentrations of cortisol, glucose and lactate rose significantly with respect to controls, showing concentrations similar to those reported previously for these species under similar stress conditions. Furthermore, gilthead sea bream chronically stressed by maintaining for 14 days under increased stocking density conditions also showed increased concentrations of plasma cortisol and glucose. These results validate the indirect ELISA technique developed for use in the evaluation of plasma cortisol concentration of at least four fish species.     

High sensitivity enzyme-linked immunosorbent assay (ELISA) method for measuring protein carbonyl in samples with low amounts of protein

The oxidative modification of proteins has been shown to play a major role in a number of pathological processes. One such modification is the addition of the carbonyl groups to the amino acid residue in proteins. For the measurement of the carbonyl groups in low concentration protein samples, we have modified the ELISA (enzyme-linked immunosorbent assay) method that was developed by Buss et al. [Buss, I. H; Chan, T. P.; Sluis, K. B.; Domigan, N. M.; Winterbourn, C. C. Protein carbonyl measurement by a sensitive ELISA method. Free Radic. Biol. Med.23:361-366; 1997 ]. In the modified method, protein samples diluted in phosphate-buffered saline were adsorbed to wells of an ELISA plate and then reacted with dinitrophenylhydrazine (DNPH). The protein-conjugated DNPH was probed by a commercial anti-DNPH antibody, and then a second antibody conjugated with horseradish peroxidase was added for quantification. The method was calibrated using oxidized albumin, and required only 5 mug protein. This obviated the need to concentrate protein in experimental and clinical samples with low amounts of protein. In addition the effect of TCA on carbonyl measurement is eliminated. The standard curve was linear in the range of 0-3.36 nmol carbonyls/mg protein, which is the range within which clinical samples fell. The results correlated well with the colorimetric carbonyl assay. The method was used to analyze the amount of protein carbonyl in aqueous humor and diluted plasma samples.     

An enzyme-linked immunosorbent assay (ELISA) for measurement of heterophile antibody

An enzyme-linked immunosorbent assay (ELISA) test has been developed for measurement of heterophile antibody. The microtiter test utilizes a bovine erythrocyte monolayer as antigen and anti-human IgM antiserum conjugated with horseradish peroxidase to measure the degree of binding of the heterophile antibody in the test serum with the erythrocytes. A single serum dilution yields quantitative results when read in a spectrophotometer. The ELISA test showed a sensitivity comparable with the immune adherence hemagglutination assay (IAHA) and other heterophile tests, good reproducibility, and high specificity.