Visualization of antigenic proteins on Western blots

A new technique for the detection of antibodies bound to proteins blotted onto nitrocellulose paper was developed. The method is rapid, sensitive, and does not require radioactive probes. Proteins transferred to nitrocellulose paper are first reacted with primary antibody followed by reaction with an alkaline phosphatase conjugated second antibody. The phosphatase activity is then visualized using an agar gel impregnated with the histochemical phosphatase stain 5-bromo-4-chloro-3-indolyl phosphate (BCIP) (J. P. Horwitz, J. Chua, M. Noel, J. T. Donatti, and J. Freisler (1966) J. Med. Chem. 9, 447; Sigma Chemical Co., Technical bulletin No. 710-EP (1978]. Antigen-antibody complexes give rise to sharp, permanent blue stained bands both on the nitrocellulose paper and in the agar overlay gel. This procedure allows detection of bands containing less than 20 ng of protein.     

Detection of antigens on nitrocellulose paper immunoblots with monoclonal antibodies

A very sensitive method for the detection of antigen-antibody complexes on nitrocellulose paper immunoblots is described. The protein antigens are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by their electrophoretic transfer onto a nitrocellulose sheet (“Western blot”). The protein antigens bound to the nitrocellulose paper are exposed to the monoclonal antibody and the antibody-antigen complexes are detected on the paper by an immunoenzymatic reaction. The improved sensitivity of this method is the result of (i) the use of the detergent Tween 20 in blocking the nonspecific binding of the antibodies to the nitrocellulose paper, (ii) the use of a peroxidase-antiperoxidase (PAP) reaction, and (iii) the intensification of the diaminobenzidine reaction product with nickel and cobalt ions in phosphate buffer.     

FerriDye: colloidal iron binding followed by Perls' reaction for the staining of proteins transferred from sodium dodecyl sulfate gels to nitrocellulose and positively charged nylon membranes

A staining method for proteins on (positively charged) nylon and nitrocellulose membranes is described. The two-step method uses cationic cacodylate iron colloid which is substituted with Tween 20 at an OD460 nm = 0.5, followed by Perls' reaction with acid potassium ferrocyanide. It stains transferred proteins deep blue with low background. The sensitivity is intermediate between that of conventional stains and AuroDye, the colloidal gold stain. This is the first sensitive staining method for proteins transferred on (positively charged) nylon membranes. These membranes have documented advantages in immunoblotting. It will therefore be a useful tool for correlating the position of bands or spots of proteins detected with overlay assays with the complete electropherogram in a duplicate protein blot.     

应用斑点免疫结合法检测植物病毒

用改进的斑点免疫结合法,检测了烟草花叶病毒、黄瓜花叶病毒、豇豆花叶病毒、芋花叶病毒、柑桔速衰病毒和柑桔顽固病螺原体。无论用单克隆抗体还是多克隆抗体,全血清还是提纯的IgG,均获得满意的结果。直接法与间接法结果相同。在测定豇豆花叶病毒和芋花叶病毒时,与酶联免疫吸附试验和免疫电镜进行比较。表明无论是对纯化病毒还是感病植物汁液,其测定的敏感性均优于后二法,豇豆花叶病毒的可测感度是0.35ng,芋花叶病毒为0.83ng。以含0.5％吐温20的Tris缓冲液封闭硝基纤维素薄膜固相载体的未结合部位,其效果与牛血清白蛋白相同。应用碱性磷酸酶标记抗体以及羟基吲哚磷酸盐和氮蓝四唑为底物较合适,这种底物可在室温下长期保存,且反应产物为不褪色的紫色。易于观察和保存。     

Detection of sub-picogram quantities of specific DNA sequences on blot hybridization with biotinylated probes.

A sensitive method for detecting biotinylated DNA probes on dot and Southern blots is described which is based on the principle outlined by Leary et al (1). This system has two main components: detection of biotinylated DNA by a two-step procedure with streptavidin and poly(alkaline phosphatase); and blocking background with Tween 20. 32fg and 80fg of lambda phage DNA was detected on dot and Southern blot hybridizations respectively. 150fg of beta-globin was detected on Southern blots of genomic DNA. This method is fast, reproducible and can detect single copy genes in 0.25 micrograms genomic DNA on Southern blots.     

Assessment of a method for immunochemical detection of antigen on nitrocellulose membranes

Immunoblotting techniques are widely used for detection of antigen immobilized on nitrocellulose membranes. There are many immunolabeling methods and staining methods available to disclose the presence of antigen in such techniques. Five common staining methods each for alkaline phosphatase and horseradish peroxidase were examined. The staining methods with the highest sensitivity and the lowest background were selected for studies comparing five immunological labeling methods using human IgG as a model antigen. Results were evaluated on the basis of the least amount of detectable antigen and background staining. The most sensitive dot-blot method was then tested for its applicability to Western blots. For both dot-blots and Western blots, the immunoalkaline phosphatase methods are more sensitive than the corresponding immunoperoxidase methods. The use of biotinylated secondary antibodies and an avidin-enzyme conjugate is recommended. Disclosure of alkaline phosphate is best achieved with naphthol AS phosphate as substrate and fast blue BB as chromogen. Peroxidase is best stained using H2O2 and diaminobenzidine (DAB). Potential endogenous enzyme activities are demonstrable by blotting methods but can be inhibited by including levamisole in the disclosure reaction medium for calf intestinal alkaline phosphatase indicators, or by incubation of blots with sodium azide and hydrogen peroxide before immunolabeling when using horseradish peroxidase indicators.     

A high-affinity reversible protein stain for Western blots

We describe a reversible staining technique, using MemCode, a reversible protein stain by which proteins can be visualized on nitrocellulose and polyvinylidine fluoride (PVDF) membranes without being permanently fixed to the membrane itself. This allows subsequent immunoblot analysis of the proteins to be performed. The procedure is applicable only to protein blots on nitrocellulose and PVDF membranes. MemCode is a reversible protein stain composed of copper as a part of an organic complex that interacts noncovalently with proteins. MemCode shows rapid protein staining, taking 30s to 1 min for completion. The method is simple and utilizes convenient application conditions that are compatible with the matrix materials and the protein. The stain is more sensitive than any previously described dye-based universal protein staining system. The turquoise-blue-stained protein bands do not fade with time and are easy to photograph compared to those stained with Ponceau S. Absorbance in the blue region of the spectrum offers good properties for photo documentation and avoids interference from common biological chromophores. The stain on the protein is easily reversible in 2 min for nitrocellulose membrane and in 10 min for PVDF membrane with MemCode stain eraser. The stain is compatible with general Western blot detection systems, and membrane treatment with MemCode stain does not interfere with conventional chemiluminescent or chromogenic detection using horseradish peroxide and alkaline phosphatase substrates. The stain is also compatible with N-terminal sequence analysis of proteins.     

Enhancement of the sensitivity for in situ detection of alkaline phosphatase using a homopolymer of 2-acrylamide 2-methylpropanesulfonate

In order to enhance the sensitivity of detection of alkaline phosphatase immobilized on nitrocellulose, a homopolymer of 2-acrylamide 2-methylpropanesulfonate (poly-AMPS) and agarose were added to the reaction mixture using 5-bromochloroindoxyl phosphate and nitroblue tetrazolium. By addition of the above ingredients, the amount of the reaction product deposited on the nitrocellulose filters on which alkaline phosphatase had been dot-blotted was increased, and darkening of the background (un-dot-blotted area) was prevented. Reflective densitometry revealed that the addition of poly-AMPS and agarose increased the sensitivity of the detection approximately three times higher than without the use of these compounds.     

Sensitive colloidal metal (gold or silver) staining of protein blots on nitrocellulose membranes

A sensitive staining method for protein blots on nitrocellulose membranes is described and compared with commonly used dye staining methods. It uses colloidal metal sols (gold or silver) stabilized with Tween 20 and adjusted to pH 3. It is based on the selective high-affinity binding of colloidal metal particles to the proteins and produces a red-purplish color (gold) or dark grey (silver). The sensitivity of this new staining method is in the same range as silver staining of polyacrylamide gels and matches the sensitivity of overlay assays. It will therefore be a useful tool for correlating the position of bands or spots of proteins detected with overlay assays with the complete electropherogram in a duplicate protein blot.     

A simplified lysis method allowing the use of biotinylated probes in colony hybridization

A method is described for the lysis of bacterial cells grown on nitrocellulose filters which allows the use of nonradioactive (biotin labeled) probes in colony hybridization. Used in conjunction with a colorimetric assay involving streptavidin and alkaline phosphatase this lysis method allows the detection of clones containing a target nucleic acid sequence. Sites of positive hybridization produce dark blue-purple signals, while nonreacting clones give very light blue signals. The occurrence of false-positive and background signals is minimal. With pBR322 as the target sequence it was possible to detect approximately 20 clones in the presence of 10(5) plasmid-free cells. It was also possible to detect low-frequency plasmid-free cells in a population of clones containing the target sequence.     

Selective extraction of marker enzymes of bovine milk fat globule membrane by nonionic detergents.

Large amounts (66-97%) of marker enzymes such as alkaline phosphatase, 5'-nucleotidase, phosphodiesterase I, and gamma-glutamyl transpeptidase of bovine milk fat globule membrane (MFGM) were selectively solubilized by nonionic detergents such as Triton X-100, Tween 20, Nonidet P-40, Liponox NCK, and Emulgen 109-P. On the other hand, the extractability of MFGM protein with these detergents was less than 50%. Judging from the recovery of total activity, it is likely that alkaline phosphatase, phosphodiesterase I, and gamma-glutamyl transpeptidase are activated by nonionic detergents, whereas 5'-nucleotidase is somewhat inhibited by the detergents, except for Tween 20, and acid phosphatase is strongly inhibited by all detergents. In addition, solubilization of the protein with the nonionic detergents was found to be somewhat selective by SDS-polyacrylamide gel electrophoresis. There was no appreciable difference between the five brands of nonionic detergents used as regards the extractability of protein and the enzymatic activity of the extracted marker enzymes of MFGM, except that the solubilizing ability of Tween 20 was relatively low.     

Commercial detection methods for biotinylated gene probes: Comparison with32P-labeled DNA probes

This study had two objectives: (a) to determine whether biotinylated DNA probes could be substituted for³²P-labeled DNA probes to detect the presence of the TEM-1 -lactamase gene in crude bacterial preparations, and (b) to evaluate two commercial detection systems for biotinylated probes—an alkaline phosphatase kit produced by Bethesda Research Laboratories (BRL) and an acid phosphatase kit produced by Enzo Biochem. Both the kits produced nonspecific reactions with TEM-1-negative organisms. Treatment with chloroformphenol and proteinase K did not remove these nonspecific reactions. When plasmid DNA was purified by electrophoresis and transferred to nitrocellulose filters by the Southern blot method, there was no qualitative difference between the biotinylated and radioactive probes. However, the³²P-labeled probes were quantitatively 100 times more sensitive than the biotinylated probes. In addition, the Enzo Biochem kit and the³²P-labeled probes could be used with charged nylon membranes, whereas the BRL kit could be used only with nitrocellulose filters.     

Binding of Streptavidin to Bacteria or Fungi and Its Applications in Detecting These Microbes

We have investigated the characteristics and utilities of streptavidin-binding to gram-negative and gram-positive bacteria and Candida spp. The pre-treatment of these microbes with chemical reagents such as CHCl₃, NaOH, and Tween 20 have allowed colorimetric visualization under light microscopy or quantitation on nitrocellulose membranes, using streptavidin/biotinylated alkaline phosphatase conjugates. Analysis of this binding was confirmed by western blot. These binding reactions were due to the specific interaction of streptavidin with biotinylated proteins present in the microbes. Competition assays with free biotin or inhibition by an antibiotin antibody confirmed binding to these proteins. With knowledge of these strongly specific interactions, we attempted to reveal the biotinylated proteins within these microbes using clinical specimens. Using phagocyte-smears from blood, urine, and ascites, these intracellular microbes were easily detected by light microscopy. One of the septic blood samples stained by our technique revealed semi-digested microbial signals despite the absence of a signal with routine staining. This detection system, which combines streptavidin as a probe and biotinylated proteins as a microbial marker, is useful in staining for intracellular bacteria or fungi (e.g., microbial infections in phagocyte-smears).     

A new fluorescence method for alkaline phosphatase histochemistry.

We have developed a new fluorescence method for the histochemical localization of alkaline phosphatase activity. Calcium phosphate deposited at the sites of alkaline phosphatase activity in a Gomori-type reaction are identified by calcium binding fluorochromes. The calcium binding fluorochromes calcein, calcein blue, and xylenol orange were investigated, with each fluorochrome being included in the alkaline phosphatase incubating medium and used in a single-step procedure. Alkaline phosphatase activity was studied in freeze-substituted, resin-embedded human liver and jejunal biopsies, and each fluorochrome produced intense fluorescence of different colors at sites of alkaline phosphatase activity. Calcein, calcein blue, and xylenol orange produced green, blue, and red fluorescence, respectively. Sites of enzyme activity were accurately localized without evidence of diffusion, and there was an absence of non-enzyme-catalyzed binding of any of the fluorochromes to tissue. This fluorescence method, which is particularly suited to investigating the localization and distribution of the activity of different enzymes in the same section, was used to investigate the distribution and co-localization of alkaline phosphatase and aminopeptidase M in human liver and jejunum.     

Double in situ hybridization for detection of Herpes simplex virus and cytomegalovirus DNA using non-radioactive probes.

We describe a double in situ hybridization assay for the simultaneous detection of Herpes simplex virus (HSV) and cytomegalovirus (CMV) DNA in infected cell cultures using non-radioactive-labeled probes. This work used a biotinylated HSV DNA probe, which can be revealed by an avidin-biotin-peroxidase complex and a digoxigenin-labeled CMV DNA probe, visualized by anti-digoxigenin F(ab) fragments conjugated with alkaline phosphatase. Light microscopy visualization was achieved by the contrasting colors of appropriate peroxidase and alkaline phosphatase reaction products (red and dark blue, respectively). The time required to perform the double hybridization assay was about 3 hr. This double hybridization assay proved to be sensitive, specific, and provided good resolving power.     

Quantitation of the blocking effect of tween 20 and bovine serum albumin in ELISA microwells

ELISA provides a highly sensitive procedure for quantitating antigens and antibodies. In that assay, microwells are coated initially with a specific ligand and then saturated with inert molecules to minimize nonspecific background. Coating can be improved by pretreating the microwells with poly-l-lysine (PLL). Proteins and Tween 20 are most often used to block vacant binding sites in enzyme-linked immunosorbent assay (ELISA). In the present study the blocking effects of Tween 20 and bovine serum albumin (BSA) were estimated using an original novel approach. In the assay the magnitude of saturation of the microwells was quantitated by measuring the enzymatic activity of alkaline phosphatase adsorbed to residual vacant sites in the microwell. Tween 20 completely saturated ELISA microwells at concentrations higher than 2 microg/ml. If the microwells were pretreated with PLL, even high concentrations of the detergent did not completely saturate the wells. In contrast, BSA completely saturated both PLL-treated and nontreated microwells at 5 microg/ml. Complementation of Tween 20-induced saturation of PLL-treated microwells was achieved only by addition of BSA at concentration required for BSA alone to reach complete saturation. This approach is applicable for assessing binding to ELISA microwells of any reagent of choice either as a ligand or as a blocking reagent.     

The use of Tween 20 in a sensitive turbidimetric assay of lipolytic enzymes

A turbidimetric esterase assay was developed using a Tween 20 solution in the presence of CaCl2 and Lysobacter enzymogenes esterase (EC 3.1.1.1) as the enzyme source. The reaction was followed by measuring the increase in the optical density at 500 nm (OD500) due to the hydrolytic release of the fatty acids from Tween 20 and their precipitation as the calcium salts. Concentrations of 1.8% Tween and 3 mM CaCl2 were found to be optimal for the assay of 0.036 to 0.15 esterase units in a 4-mL reaction mixture over a 30-min period. The esterase reactions were linear with time at least up to 1.2 OD500 and the rate of increase in the OD500 was proportional to the enzyme concentration. Low initial reaction rates were seen with low esterase activity, presumably because of the limited solubility of the fatty acid - calcium salt in a 1.8% Tween solution. This turbidimetric method is much simpler and at least 36 times more sensitive than the titrimetric assay with Tween 20, and at least four times more sensitive than a spectrophotometric assay with p-nitrophenyl palmitate. This assay has been used to determine the activities of cell-associated and excreted esterases produced by Lysobacter enzymogenes and Pseudomonas aeruginosa, and of lipolytic enzymes from porcine liver, Chromobacterium viscosum, Candida cylindracea, and wheat germ.     

An alternative nonradioactive method for labeling DNA using biotin

An alternative nonradioactive labeling method and a highly sensitive technique for detecting specific DNA sequences are described. The labeling method requires the "Klenow" fragment of DNA polymerase I and random hexanucleotides (synthesized or naturally extracted) as a primer for the production of highly sensitive DNA probes. The system has three main steps: (i) labeling of DNA with biotinylated 11-dUTP; (ii) detection of biotinylated DNA by a one-step procedure with streptavidin-alkaline phosphatase complex; (iii) blocking of background with Tween 20. Twenty attograms (2 X 10(-17) g) of pBR322 plasmid DNA was detected by dot-blot hybridization. Upon Southern blot hybridization, 7.4 fg (7.4 X 10(-15) g) of pBR322 HindIII DNA was detected using the biotinylated pBR322 plasmid DNA probe; 40.8 ag and 7.4 fg of lambda HindIII DNA were detected with the biotinylated whole lambda DNA probe by dot and Southern blot hybridization, respectively. Specific bands were also detected with the biotinylated argininosuccinolyase probe upon Northern blotting of mouse poly(A+) RNA. Further applications for in situ hybridization are also described.     

The determination of lipopolysaccharide admixtures in protein preparations

A new method for the determination of lipopolysaccharide (LPS) admixtures in protein solutions has been developed. The method includes the periodate oxidation of LPS, biotinylation with biotin hydraside, immobilization on a nitrocellulose membrane and the development of biotinylated LPS in the streptavidin--alkaline phosphatase system. Proteins are previously removed from the solution by treatment with hot phenol. Development with the use of 5-bromoinodyl phosphate and nitrotetrazolium blue makes it possible to detect about 30 pg of LPS immobilized on the nitrocellulose membrane.     

A novel fluorometric assay for ADP-ribose pyrophosphatase activity

ADP-ribose pyrophosphatase (ADPRase) hydrolyzes ADP-ribose to ribose-5-phosphate and AMP. The ADPRase activity have been assessed by coupling the reaction to alkaline phosphatase and colorimetrically measuring the amount of inorganic phosphate released from AMP that is one of the products of ADPRase. Another but less sensitive colorimetric method has been employed: the reaction mixture was treated with charcoal to adsorb the adenine-containing compounds such as AMP and ADPR and subsequently remaining ribose-5-phosphate was measured colorimetrically. However, the measurement of inorganic phosphate cannot be feasible to assay ADPRase in phosphate-containing samples and the determination of ribose-5-phosphate also is less sensitive. Here we develop a fluorescent assay for ADPRase that utilizes 1, N(6)-etheno ADP-ribose, a fluorescent analogue of ADP-ribose. This method measures fluorescent 1, N(6)-etheno adenosine that is produced by coupling the hydrolysis of 1, N(6)-etheno ADP-ribose to dephosphorylation with alkaline phosphatase. The fluorometric assay is comparable in sensitivity and useful for ADPRase assay in phosphate-containing samples.