Assay sensitivity and differentiation of monoclonal antibody specificity in ELISA with different coating buffers

Buffers of different pH and ionic strength were employed as coating buffers for antigen adsorption to microtitre plates. Their efficiency for coating plates with rinderpest virus (RPV) and foot-and-mouth disease virus (FMDV) antigens was studied by ELISA with polyclonal and monoclonal antibody preparations. While the adsorption and detection of RPV antigen with polyclonal antiserum was highly dependent on the ionic strength and pH of coating buffer, adsorption of antigenically active FMDV antigen was relatively unaffected by the buffering conditions. Both antigens were adsorbed optimally in 0.01 M phosphate buffer, pH 8.0. When monoclonal antibodies were used to detect antigen, there was a greater degree of dependence on the coating buffer than that found with polyclonal antisera. Moreover, when they were used to detect antigen adsorbed under several buffering conditions, monoclonal antibodies showed a variety of preferred buffers. The usefulness of this differential reactivity in distinguishing epitope specificity is demonstrated.     

Biochemical and serological characteristics of soluble yeast phase antigens of Histoplasma Capsulatum

Soluble antigens of whole yeast-phase cells were extracted with a 0.1 M phosphate buffer containing 0.1 M sodium chloride and 0.02% iodoacetate. After being separated by differential filtration into fractions less than or greater than 50,000 daltons, these antigens were purified by molecular sieve and chromatographic separations on ionic exchange resins. Two high molecular weight fractions obtained from diethylaminoethyl-cellulose (DEAE) at pH 8.0 and 7.0 with tris (hydroxymethyl) aminomethane (Tris) buffer were M antigens; those obtained at pH 4.0 and 4.0 with salt were H antigens. The four fractions had protein to carbohydrate ratios of 7.3, 14.0, 8.4, and 6.5 respectively, and all had essentially the same amino acid composition with no methionine and tyrosine and little histidine, arginine, phenylalanine and lysine. They had high concentrations of glucose, less mannose and traces of galactose. The low molecular weight fractions had the new complex Y antigen, M antigen, and H antigen with protein to carbohydrate ratios of 1.4, 1.4 and 0.3 respectively. The amino acid and sugar composition of Y antigen strongly resembled the composition of the low molecular weight H and M antigens. Unlike the high molecular weight antigens, these low molecular weight antigens had methionine in relatively high concentrations; they had the same sugars as their respective high molecular weight counterparts. The yeast phase antigens differed from their respective mycelial counterparts in the following ways: glucose was the major sugar in the yeast phase with less amounts of mannose and traces of galactose, whereas in the mycelial antigens, mannose was the major sugar, with lesser amounts of galactose, glucose, and hexosamine. The H and M antigens of the yeast phase had high concentrations of glycine and alanine, whereas in the mycelial phase, these antigens had high concentrations of threonine and proline; the H and M antigens of the yeast phase had 5 to 16 times the protein to carbohydrate ratio observed for the same antigens of histoplasmin.     

Detection of Escherichia coli O157:H7 using immunomagnetic separation and absorbance measurement

An assay system for detection of Escherichia coli O157:H7 was developed based on immunomagnetic separation of the target pathogen from samples and absorbance measurement of p-nitrophenol at 400 nm from p-nitrophenyl phosphate hydrolysis by alkaline phosphatase (EC 3.1.3.1) on the "sandwich" structure complexes (antibodies coated onto micromagnetic beads--E. coli O157:H7-antibodies conjugated with the enzyme) formed on the microbead surface. The effects of immunoreaction time, phosphate buffer concentration, pH and temperature on the immunomagnetic separation of E. coli O157:H7 from samples were determined and the conditions used for the separation were 1-h reaction time, 1.0 x 10(-2) M PBS, pH 8.0 and 33 degrees C in this system. The effects of MgCl(2) concentration, Tris buffer concentration, pH and temperature on the activity of alkaline phosphatase conjugated on the immuno-"sandwich" structure complexes were investigated after immunomagnetic separation of the target pathogen and the conditions used for the enzymatic amplification were 1.0 x 10(-4) M MgCl(2), 1.0 M Tris buffer, pH 8.0, 28 degrees C and 30-min reaction time during the assay. The selectivity of the system was examined and no interference from the other pathogens including Salmonella typhimurium, Campylobacter jejuni and Listeria monocytogenes was observed. Its working range was from 3.2 x 10(2) to 3.2 x 10(4) CFU/ml, and the relative standard deviation was 2.5-9.9%. The total detection time was less than 2 h.     

Highly sensitive electroimmunodiffusion method of detecting antigens on cellulose acetate film]

A highly sensitive electro-immunodiffusion test suggested by the authors for antigen detection on cellulose-acetate films consists of three stages: antigen concentration in a discontinuous buffer system on cellulose-acetate films; antigen detection on the same films by immunodiffusion using standard test system; to detect the precipitation bands the washed films are stained with protein dyes in case the reaction takes place in the zone of vision, or subject to further treatment by means of "plating" the precipitates with antiglobulin antibody or by radioautography. The method permits one to reveal the nanogram levels of alfa-fetoprotein and it may be applied for detection of antigens with different molecular weights and electrophoretic mobility.     

Application of heat-induced antigen retrieval to aldehyde-fixed fresh frozen sections.

We applied the heat-induced antigen retrieval (HIAR) to aldehyde-fixed fresh frozen sections based on a new approach (i.e., a rapid and complete immobilization of antigen followed by heating). Frozen sections were fixed with 10% formalin in 0.1 M cacodylate buffer (pH 7.4) containing 25 mM CaCl(2) for 30 min, or with 0.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 1 min at room temperature, and then autoclaved in 20 mM Tris-HCl buffer (pH 9.0) for 10 min at 120 C. Both fixatives yielded good tissue structure after autoclaving. In the sections fixed with formalin containing CaCl(2), 20 of 22 antigens located in the nucleus, cytoplasm, membranes, and extracellular matrix greatly recovered their antigenicity after autoclaving; only two antigens exhibited stronger immunoreaction in acetone-fixed fresh frozen sections than these sections. Heating also retrieved the immunoreactivity of at least 14 antigens in the sections fixed with glutaraldehyde. We used the similar procedures to localize ligand-free estrogen receptor alpha (ERalpha) and glucocorticoid receptors (GR). Mouse uterine cells exhibited almost the same nuclear ERalpha immunostaining regardless of the hormonal status in glutaraldehyde-fixed fresh frozen sections and unliganded GR was localized mainly in the nucleus of mouse hepatocytes in fresh frozen sections fixed with 20% formalin containing 50 or 75 mM CaCl(2) at 40 C, after autoclaving. These results demonstrate that HIAR is useful for the immunohistochemistry of many antigens in aldehyde-fixed fresh frozen sections.     

Enantiomeric separation of tramadol hydrochloride and its metabolites by cyclodextrin-mediated capillary zone electrophoresis

The enantiomeric separation of tramadol hydrochloride and its major metabolites, O-demethyltramadol (M1) and N-demethyltramadol (M2) was studied using cyclodextrin (CD)-mediated capillary zone electrophoresis (CZE). Influence of the choice of type and concentration of CD, capillary temperature, length of capillaries, buffer pH and the addition of polymer modifier on the chiral separation of tramadol and its metabolites was evaluated. It was found that the drug and the metabolites can be baseline-separated simultaneously by using 50 mM phosphate buffer (pH 2.5) containing 75 mM methyl-β-CD, 220 mM urea and 0.05% (w/v) hydroxypropylmethyl cellulose.     

Effect of conditions of monoclonal antibody adsorption on antigen-binding activity

The dependence of the antigen-binding activity of immobilized antibodies on pH of a saturating buffer has been investigated. We analyzed 28 monoclonal antibodies (MCAs) produced by various hybridomas to three virus antigens, i.e., the nuclear p23 protein of hepatitis C virus (C core protein p23), p24 protein of HIV 1, and the surface antigen of hepatitis B virus (HBsAg). Antibodies were adsorbed on the surfaces of immune plates in acidic (pH 2.8), neutral (pH 7.5), and alkaline (pH 9.5) buffers. The binding of labeled antigens, i.e., biotinylated or conjugated with horseradish peroxidase, with immobilized antigens was tested. It was shown that 10 out of 28 analyzed MCAs (36%) considerably better preserved their antigen-binding activity if their passive adsorption was carried out on the surface of polystyrene plates in an acidic buffer (pH 2.8). This approach allowed constructing a highly sensitive sandwich method for HBsAg assay with a minimal reliably determined antigen concentration of 0.013–0.017 ng/ml. The described approach may be recommended for the optimization of sandwich methods and solid-phase competitive methods.     

Capillary electrophoresis method for simultaneous determination of penicillin G, procaine and dihydrostreptomycin in veterinary drugs

Capillary electrophoresis method for identification and simultaneous determination of procaine, dihydrostreptomycin and penicillin G, present in multiantibiotic veterinary preparations, was elaborated. The influence of pH (5.0-9.75) and concentration of disodium tetraborate decahydrate in running buffers (0.02-0.1 M) as well as temperatures (25-40 degrees C) on separation efficacy were analyzed. For quantitative analysis, 0.08 M borate buffer (pH 8.0) at 35 degrees C and 15 kV were chosen. Method was validated, selectivity, precision, linearity, LOD, LOQ, accuracy and specificity of capillary zone electrophoresis (CZE) were evaluated.     

Characterization of Epstein-Barr virus antigens. I. Biochemical analysis of the complement-fixing soluble antigen and relationship with Epstein-Barr virus-associated nuclear antigen.

The Epstein-Barr virus-soluble (S) antigen extracted from RAJI cells was characterized by sucrose gradient centrifugation, gel filtration, and ion-exchange chromatography. The sedimentation coefficient was estimated to be 8.5S corresponding to a molecular weight of 180,000. The S antigen binds to DEAE-A25 ion exchanger from which it can be eluted with 0.3 M NaCl in Tris buffer (pH 7.2). All fractions which contained complement-fixing S antigen also inhibited the anticomplement immunofluorescence reaction as used to detect the Epstein-Barr virus-associated nuclear antigen. These results are consistent with the hypothesis that the S and Epstein-Barr virus-associated nuclear antigens are either a single antigen or that both activities are present on the same molecule.     

Procedures for the production and separation of H and M antigens in histoplasmin: Chemical and serological properties of the isolated products

Two strains of Histoplasma capsulatum were required to prepare maximum yields of H and of M antigen from histoplasmin. The antigens were separated and partially purified by a series of procedures yielding an overall recovery of 70 to 90% of the individual antigens. Stable products suitable for use as reference products were obtained when the final purification step employed DEAE-cellulose with phosphate buffer elution at increasing molarity and decreasing pH. A final step of purification of each antigen with slab acrylamide gel electrophoresis gave products which were highly reactive and specific in a variety of serological tests with sera from persons with proven cases of histoplasmosis and with natural infections of heterologous deep mycoses. These antigens were maximally active at concentrations of 2 to 16 g protein in the complement fixation, capillary precipitin, microimmunodiffusion, or immunoelectrophoresis tests; 0.5 g gave a maximum delayed cutaneous hypersensitivity reaction in homologously infected animals and caused no appreciable reaction in control animals. Although these antigens appeared to be specific when tested with sera from persons with natural infections, the M and H antigens demonstrated the presence of an additional antigen reacting with sera of rabbits immunized with cell membrane and cell particulate fractions of Blastomyces dermatitidis. After purification by electrophoresis, both the H and M antigens of some preparations showed some decomposition and loss of reactivity after storage at 5 C for more than six months. The overall results suggest that the purified H and M antigens of Heiner (12) have multiple serological reactivity and may function in precipitin reactions, complementfixing reactions, hemagglutination of formalin-fixed goose red blood cells, and as antigens for delayed cutaneous tests.     

Evaluation of the use of cyclodextrins in chiral separation of basic drug substances by capillary electrophoresis

The influence of the choice of type and/or concentration of cyclodextrin, other additives, the temperature surrounding the capillary, and buffer pH on the separation of some chiral basic drug substances in capillary zone electrophoresis has been evaluated. It was found that pH of the buffer and type and concentration of cyclodextrin had a major influence on the separation. © 1995 Wiley-Liss, Inc.     

Separation of the eleven priority pollutant phenols by capillary zone electrophoresis

Capillary zone electrophoresis (CZE) provides highly efficient separation of the eleven US EPA priority pollutant phenols. All of these phenols are completely resolved in fewer than 15 min in a 100 cm × 75 μm I.D. uncoated fused-silica capillary at 22.5 kV using a pH 9.8 phosphate-borate buffer. Buffer pH is the most critical parameter controlling resolution and separation time. A simple theoretical treatment greatly simplifies the pH optimization procedure. The effects on the separation of buffer concentration, applied voltage, and sample quantity injected were studied. Good calibration data were obtained for phenol concentrations up to 50 mb/l. Limits of detection for all phenols were less than 1 ppm.     

Bullous pemphigoid antigen: isolation from normal human skin.

A protein moiety from epidermal PBS-soluble products was isolated by gel filtration (Bio-Gel A-1.5m) and ion exchange chromatography (DEAE-cellulose). This protein (A-1-Epid) was not retarded by DEAE-cellulose in Tris-HCl buffer, 15mM, pH 8.1. By IEP against an antiserum to epidermal antigens, it showed a single cathodal arc. On disc electrophoresis, at low pH (4.3) a single band was apparent. On SDS gels this protein demonstrated two bands, one with a molecular weight of 20,000, and the second with a molecular weight of 9,200. This purified antigen was able to block the staining of the basement membrane zone produced by bullous pemphigoid antibodies on monkey esophagus and normal human skin with the use of indirect immunofluorescence. This study also demonstrates that bullous pemphigoid antigen (A-1-Epid) and a second epidermal protein (A-2-Epid) are present in the PBS-soluble products of human esophageal mucosa, saliva, and urine. These antigens appear to be unrelated with the blood group substances or secretor status of the donors.     

Preparative isoelectric focusing in immobilized pH gradients. I. General principles and methodology

A new method for preparative protein purification is described, based on the use of Immobiline matrices. After electrofocusing, the protein zone of interest is recovered by electrophoretic transfer to a hydroxyapatite gel, from which it is eluted with 0.2 M phosphate buffer, pH 6.8, with yields for the proteins studied in the range 76-98%. For six different proteins, the focusing step gives a common upper limit of approximately 45 mg protein/ml gel as mean concentration in a focused protein zone. It is demonstrated that in practical preparative work, components with a pI difference of 0.007 pH units can be completely resolved, and that on a 5-mm-thick gel of dimensions 240 X 110 mm, samples containing as much as 400 mg of the major protein component can be applied. Focusing of large amounts of a salt-containing sample is demonstrated with the aid of human serum. A theoretical expression is given relating the concentration distribution and maximum protein concentration within a focused zone to the applied voltage, the pH slope used and the zone width. Based on this expression and the finding of an upper concentration limit for a protein we shown how to optimize the parameters in preparative work with immobilized pH gradients in relation to the separation power needed. Finally, it is shown that, in comparison with conventional preparative electrofocusing in polyacrylamide gels, immobilized pH gradients allow a ten-fold increase in load, whilst still giving a resolution comparable to that of analytical isoelectric focusing.     

Latex-based thin-layer immunoaffinity chromatography for quantitation of protein analytes.

A rapid immunochromatographic method for qualitative and quantitative analysis of protein antigens is described. The method is based on the "sandwich" assay format using monoclonal antibodies (Mabs) of two distinct specificities. Mabs of one specificity are covalently immobilized to a defined detection zone on a porous membrane while Mabs of the other specificity are covalently coupled to blue latex particles which serve as a label. The sample is mixed with the Mab-coated particles and allowed to react. The mixture is then passed along a porous membrane by capillary action past the Mabs in the detection zone, which will bind the particles which have antigen bound to their surface, giving a blue color within this detection zone with an intensity logarithmetrically proportional to the antigen concentration in the sample. Analysis is complete in less than 10 min, requires a minimum amount of sample (4 microliters), and has a detection limit below the nanomolar range for the antigen we studied, human chorionic gonadotropin.     

Optimization of a capillary zone electrophoresis method by using a central composite factorial design for the determination of codeine and paracetamol in pharmaceuticals

Capillary zone electrophoresis was optimized to quantitatively determine codeine and paracetamol via central composite factorial design. Critical parameters (concentration, buffer, pH, voltage) assessed effects on resolution, analysis time and efficiencies. Optimum separation conditions were achieved using phosphate buffer 20 mM (pH 6.8) and voltage (15 kV). The optimized procedure easily determined codeine and paracetamol with separation in less than 3 min. Calibration curves (R > 0.999) were prepared, with LODs of 13.5 and 340 ng mL(-1) for codeine and paracetamol, respectively, and a good R.S.D.% (<3%). This method was applied to determine codeine and paracetamol in pharmaceutical formulations; recoveries coincided with stated contents.     

Detection of d-Serine in rat brain by capillary electrophoresis with laser induced fluorescence detection

A capillary zone electrophoresis method with laser induced fluorescence detection for the chiral separation of highly fluorescent enantiomeric derivatives of d/l-Serine from 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-d/l-Serine) was developed and optimized. Enantiomeric separation of NBD-d/l-Serine was accomplished by using 40 mM hydroxypropyl-beta-cyclodextrin (HP-beta-CD) contained in 100 mM borate buffer, pH 10.0. A 70 cm (effective length of 50 cm) uncoated fused-silica capillary at a voltage of 15 kV was used for the separation. The optimized electrophoretic conditions were subsequently applied to the analysis of d-Serine in rat brain, and satisfactory analytical results with respect to accuracy were obtained. This assay showed acceptable precision, with linearity in the d-Serine concentration range of 0.2-20.0 microM. The limit of detection for d-Serine was 3.0 x 10(-7)M.     

Antigen retrieval trial for post-embedding immunoelectron microscopy by heating with several unmasking solutions.

A novel antigen retrieval procedure was carried out in the post-embedding immunogold electron microscopy method to improve the stainability of the samples. This was done by weakly fixing cultured Helicobacter pylori (ATCC43504) and embedding in Lowicryl K4M. Before staining with the anti-H. pylori antibody, the ultrathin sections were mounted on a nickel grid and heated at 121C for 15 min, 99C for 40 min, and 65C for 24 hr in distilled water, 0.1 M phosphate buffer (pH 7.4), 0.01 M EDTA (pH 7.2), 0.05 M Tris buffer (pH 10.0), 0.8 M urea (pH 7.2), 0.01 M citric acid (pH 6.0), or a commercially available target unmasking fluid (S1699; pH 6.0). Antigen retrieval in the Tris buffer solution generally showed better stainability than the classical post-embedding method without any antigen retrieval. At 65C for 24 hr, better stainability of the ultrasections was observed for each of the solutions used except for the phosphate buffer compared to the control. We suggest that the antigen retrieval method should be applied for routine use even by in post-embedding immunogold electron microscopy.     

Evaluation of Antigen Retrieval Buffer Systems

The introduction of antigen retrieval (AR) techniques has dramatically improved the sensitivity of immunohistochemical detection of various antigens in formalin-fixed, paraffin-embedded tissues. The microwave-heating and pressure-cooking procedures are the most effective AR methods reported to date. Although extensive efforts have been made to optimize AR procedures using these two methods, previous studies have not led to a standard protocol applicable to all antibodies derived from different clones. In this study we have investigated the optimal AR buffer conditions for 29 antibodies that are in common use for diagnostic purposes in hospitals worldwide. Borate (pH 8.0) and Tris buffer (pH 9.5) yielded the highest retrieved antigen immunoreactivity against most antibodies as compared to other buffers tested. In addition, the microwave pressure-cooking gave better results than microwave-heating alone. Therefore, borate (pH 8.0) or Tris (pH 9.5) buffer used in conjunction with the pressure-cooking procedure is strongly recommended for standard routine use.