Dual-enzyme cascade--an amplified method for the detection of alkaline phosphatase

A method in which a two-enzyme cascade is used for rapid and sensitive detection of alkaline phosphatase is described. A masked inhibitor, 4-(3-oxo-4,4,4-trifluorobutyl)phenyl phosphate, is dephosphorylated by the action of alkaline phosphatase. The resulting compound, 1,1,1-trifluoro-4-(4-hydroxyphenyl)-butan-2-one, acts as a potent inhibitor of the second enzyme, a liver carboxylesterase. A determination of the residual esterase activity provides a highly sensitive indication of the original phosphatase concentration. The sensitivity of this dual-enzyme cascade is approximately 125-fold greater than that observed for the direct detection of phosphatase activity with p-nitrophenyl phosphate.     

Chemiluminescent detection of alkaline phosphatase in PhastGel.

A chemiluminescent assay has been applied to the detection of alkaline phosphatase on PhastGel containing lysates of preimplantation mouse embryos. The very sensitive detection capabilities reported for the chemiluminescent system led to the investigation of its applicability to the characterization of the alkaline phosphatases in one embryo or less and to compare the sensitivity of two different commercial alkaline phosphatase chemiluminescent assays to a colorimetric assay.     

An improved fluorogenic substrate for the detection of alkaline phosphatase activity

We designed a new alkaline phosphatase (ALP)-sensitive fluorogenic probe in which a self-immolative spacer group, p-hydroxybenzyl alcohol, is linked to a profluorogenic compound to improve substrate specificity. Enzymatic hydrolysis converts the fluorogenic substrate 1 to a highly fluorescent reporter 3, thus allowing for the fast and quantitative analysis of ALP activity with greatly increased affinity for the enzyme.     

Qualitative and quantitative detection of alkaline phosphatase coupled to an oligonucleotide probe for somatostatin mRNA after in situ hybridization using unfixed rat brain tissue

In situ hybridization (ISH) of somatostatin (SOM) mRNA was carried out on sections of rat brain using an alkaline phosphatase (AP) coupled oligonucleotide probe. Different hybridization and AP development conditions were tested for qualitative and quantitative detection of target mRNA on sections of unfixed tissue. Hybridization signal intensities after 24 h of hybridization were high. Comparison with adjacent formaldehydefixed tissue sections and hybridization for various lengths of time (2–42 h) indicated that in unfixed tissue retention of SOM mRNA was at least as high as after fixation, and that the mRNA was not degraded during hybridization. The use of tetranitroblue instead of nitroblue tetrazolium chloride in the AP detection medium provided a superior signal-to-noise ratio, and medium stability was improved for quantitative studies on unfixed sections by adding 10% polyvinyl alcohol at pH 8.5. Microphotometric measurements of mean optical densities (MOD) of the formazan reaction product in a defined area within individual neurons of the lateral central amygdaloid nucleus showed a linear increase over the first 23 h of AP reaction time. The mean MOD values per neuron were comparably high in various equally thick sections of the nucleus and increased with section thickness in a linear manner. The findings indicate that the ISH and detection reagents penetrate the entire section and that there is a linear relationship between the amount of AP reaction product measured and the amount of mRNA present in the measured area. Thus, ISH using an AP-coupled oligonucleotide on sections of unfixed tissue appears suitable for quantitative mRNA detection.     

Indoxyl-tetranitro blue tetrazolium method for detection of alkaline phosphatase in immunohistochemistry

A sensitive method for detection of alkaline phosphatase in immunohistochemistry, using lymphoid cells, has been optimized. The conditions for staining are 0.23 mM 5-bromo-4-chloro-indoxyl phosphate, 0.55 mM tetranitro blue tetrazolium, 2.0 mM levamisole, 5.0 mM sodium azide, 10.0 mM magnesium chloride, and 0.15 mM 1-methoxyphenazine methosulfate dissolved in 100 mM Tris-HCl buffer, pH 9.5.     

Conjugate formation in urea: coupling of insoluble peptides to alkaline phosphatase for ELISA and in situ detection of antibody-forming cells

We report here a new method to produce synthetic peptide/alkaline phosphatase (AP) conjugates in the presence of urea. The method allows the use of peptides that are not soluble to a sufficient degree in aqueous buffers. The presence of 8 M urea during the construction of the synthetic peptide/AP conjugates does not influence enzyme activity nor the affinity of the anti-peptide antibodies for the conjugated peptide. We demonstrate that these synthetic peptide/AP conjugates can be used for detection of specific antipeptide antibody-forming cells (AFC) in vivo. This method for constructing enzyme conjugates with insoluble proteins or peptides suggest not only new possibilities for detection of specific AFC in vivo but also for applications in receptor-ligand studies, ELISA (enzyme-linked immunosorbent assay), and spot ELISA for detection of antibody-secreting cells in vitro.     

Immunohistochemical detection of antibody in tissue sections of non-perfused and ex vivo-perfused organs using a tetrazolium alkaline phosphatase substrate

Summary We have used nitroblue tetrazolium (NBT) as a color reagent to localize antibody-bound alkaline phosphatase in frozen tissue sections. In the method described, NBT is reduced to a stable black diformazan reaction product that contrasts well with nuclear counterstains such as hematoxylin and stands out strongly in black and white photographs. We have found NBT to be a suitable color reagent for the alkaline phosphatase: anti-alkaline immunohistochemical technique. The reaction product also contrasts well with fast red and can therefore be used as second reagent for two color immunoenzyme studies. In this report, we describe a novel two color immunoenzyme method to assess the ex vivo binding of antibodies against Class II histocompatibility antigens in whole organs connected to a perfusion circuit.     

Immunohistochemical detection of antibody in tissue sections of non-perfused and ex vivo-perfused organs using a tetrazolium alkaline phosphatase substrate

We have used nitroblue tetrazolium (NBT) as a color reagent to localize antibody-bound alkaline phosphatase in frozen tissue sections. In the method described, NBT is reduced to a stable black diformazan reaction product that contrasts well with nuclear counterstains such as hematoxylin and stands out strongly in black and white photographs. We have found NBT to be a suitable color reagent for the alkaline phosphatase: anti-alkaline immunohistochemical technique. The reaction product also contrasts well with fast red and can therefore be used as second reagent for two color immunoenzyme studies. In this report, we describe a novel two color immunoenzyme method to assess the ex vivo binding of antibodies against Class II histocompatibility antigens in whole organs connected to a perfusion circuit.     

Alkaline phosphatase in human lymphocytes. II. A method for ultracytochemical detection of alkaline phosphatase in lymphocytes

For the ultracytochemical identification of alkaline phosphatase in lymphocytes gained from the peripheral blood of healthy individuals a sensitive method is described which allows the low enzyme activity of these cells to be determined. This was possible because the authors succeeded in stabilizing lead ions in the alkaline medium by forming a complex directly between tris-(hydroxymethyl) aminomethan and lead (II) citrate. AP localized ultrachemically in lymphocytes in particular formations similar to phosphasomes of neutrophilic granulocytes. In those lymphocytes stimulated by lipopolysaccharides a high enzyme activity could be observed and, in addition to phosphasomes, the product of response can also be found in canal-like structures of the endoplasmatic reticulum. These findings contribute to clarify the ultrastructural localization of alkaline phosphatase in lymphocytes and may be regarded as an aid in discovering the importance of the enzyme in the biology of lymphocytes or in its activation, respectively.     

DNA hybridization assay using ATTOPHOS, a fluorescent substrate for alkaline phosphatase.

A fluorometric procedure for the detection of DNA-DNA hybrids is described. The procedure involved the detection of probe-bound alkaline phosphatase with the fluorescent substrate ATTOPHOS. This substrate is converted to ATTOFLUOR by alkaline phosphatase and fluoresces strongly at 550 nm when excited with a wavelength of 440 nm. DNA hybridization assays were performed both with dilutions of purified target plasmid DNA (pSE9 or PBR322) and whole bacterial cells. Streptavidin-alkaline phosphatase conjugates were added to react with bound probe. Fluorometric assays, as well as colorimetric assays, using 5-bromo-4-chloro-3-indolylphosphate + nitroblue tetrazolium for alkaline phosphatase activity were performed. The fluorescence of the substrate was measured at time intervals, and the slope of the regression line calculated. A slope four times greater than that of background was considered positive. One hundred femtograms or 2.2 x 10(4) molecules of homologous DNA were detected with the fluorescent assay as compared with 10,000 femtograms or 2.2 x 10(6) molecules of homologous DNA with the colorimetric assay. Similar results were obtained with whole cells. Approximately 1 x 10(3) homologous cells were detected fluorometrically and 1 x 10(5) cells were detected colorimetrically. Based on these results, we conclude that, in our hands, the DNA hybridization assay described here using ATTOPHOS as the substrate for alkaline phosphatase is a very sensitive assay for the detection of DNA-DNA hybrids.     

An optimized method for the chemiluminescent detection of alkaline phosphatase levels during osteodifferentiation by bone morphogenetic protein 2

Differentiation of osteoprogenitor cells into osteoblasts is a pivotal step during the normal development and repair of bone. Upregulation of endogenous cellular alkaline phosphatase activity (AP) is a commonly used intracellular marker for the assessment of osteoprogenitor cell differentiation into the osteoblastic phenotype. Current methods for assaying AP involve colorimetric detection of the enzyme's activity using the synthetic substrate p-nitrophenol phosphate. In this paper, we explored an alternative method of detecting AP using the chemiluminescent substrate disodium 3-(4-methoxyspiro[1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.1(3,7)]decan]-4-yl) phenyl phosphate (CSPD) for enhanced AP sensitivity and a more simplified assay. Using calf intestinal alkaline phosphatase as a standardizing enzyme, we determined that the chemiluminescent detection system was four orders of magnitude more sensitive than the standard colorimetric method of detection. Moreover, the chemiluminescent assay was faster and markedly simpler to perform. To maximize the utility of this assay system, two osteoprogenitor cell lines were compared for their ability to generate alkaline phosphatases in vitro when exposed to recombinant human bone morphogenetic protein (rhBMP-2). The W20-17 cell line was substantially more sensitive to rhBMP-2 than the C3H10T1/2 cell line, where each cell line produced detectable increases in AP after exposure to rhBMP-2 levels of 5 and 25 ng/ml, respectively. The experimental design for AP responsiveness to rhBMP-2 was further optimized for chemiluminescent detection with the W20-17 cell line by comparing the effects of reporter cell seeding density and the day of assay. In summary, the data presented in this paper demonstrate a faster, simpler, and more sensitive chemiluminescent method to monitor changes in AP levels during osteodifferentiation.     

The biological function of the R2a regulatory gene for alkaline phosphatase in Escherichia coli

Previous attempts to purify lysyl oxidase have been frustrated by the failure to recover activity during ion exchange or affinity chromatography. We have found that lysyl oxidase from chick cartilage shows marked stability in buffers containing urea and in these solutions can be recovered in high yield from DKAE-cellulose and collagen-derivatized Sepharose. The purified enzyme was active against both collagen and elastin substrates but devoid of monoamine oxidase activity. An absolute requirement for oxygen for activity was found.     

A method for determining alkaline phosphatase activity in marine phytoplankton using spectrofluorometry

A method for determining relative percent intensity alkaline phosphatase activity (APA) using enzyme labeled fluorescence coupled with spectrofluorometry is presented. Compared to traditional microscopy and flow cytometry, we increase statistical power and reduce sample-handling issues. Combined with a biological standard, our method can quantify APA of natural plankton assemblages.     

Comparison of methods for following alkaline phosphatase catalysis: spectrophotometric versus amperometric detection

An amperometric method for alkaline phosphatase is described and compared to the most widely used spectrophotometric method. Catalytic hydrogenation of 4-nitrophenylphosphate (the substrate in the spectrophotometric method) gives 4-aminophenylphosphate (the substrate in the amperometric method). The latter substrate has the formula C6H6NO4PNa2.5H2O and a Mr of 323. The Michaelis constant for 4-aminophenylphosphate in 0.10 M, pH 9.0. Tris buffer is 56 microM, while it is 82 microM for 4-nitrophenyl phosphate. The amperometric method has a detection limit of 7 nM for the product of the enzyme reaction, which is almost 20 times better than the spectrophotometric method. Similarly, with a 15-min reaction at room temperature and in a reaction volume of 1.1 ml, 0.05 microgram/l alkaline phosphatase can be detected by electrochemistry, almost an order of magnitude better than by absorption spectrophotometry. Amperometric detection is ideally suited for small-volume and trace immunoassay.     

Decoding signals for membrane protein assembly using alkaline phosphatase fusions.

We have used genetic methods to investigate the role of the different domains of a bacterial cytoplasmic membrane protein, MalF, in determining its topology. This was done by analyzing the effects of MalF topology of deleting various domains of the protein using MalF-alkaline phosphatase fusion proteins. Our results show that the cytoplasmic domains of the protein are the pre-eminent topogenic signals. These domains contain information that determines their cytoplasmic location and, thus, the orientation of the membrane spanning segments surrounding them. Periplasmic domains do not appear to have equivalent information specifying their location and membrane spanning segments do not contain information defining their orientation in the membrane. The strength of cytoplasmic domains as topogenic signals varies, correlated with the density of positively charged amino acids within them.     

Quantification of the histochemical reaction for alkaline phosphatase activity using the indoxyl-tetranitro BT method

Summary The indoxyl—tetranitro BT method for the demonstration of alkaline phosphatase activity has been optimized and its validity for quantitative histochemistry tested. The study has been performed with model films of polyacrylamide gel incorporating homogenate of rat liver and with cryostat sections from the same livers. Addition of polyvinyl alcohol to the incubation medium greatly improved the localization of the final reaction product in cryostat sections. In polyacrylamide films, the formazan production specifically due to alkaline phosphatase was highest when using a medium containing 100mm Tris-HCl buffer, pH 9.0, 0.2–1.0mm substrate, 0.32mm 1-methoxyphenazine methosulphate, 10mm MgCl₂, 5mm sodium azide and 1mm tetranitro BT. For the incubation of cryostat sections in the presence of polyvinyl alcohol, the same medium could be used but the optimum concentrations of substrate and tetranitro BT appeared to be 1–2mm and 5mm respectively. The test minus control reaction was specific for alkaline phosphatase activity and could be inhibited completely with tetramisole. The test minus control reaction was linear with time up to 30 min with model films and up to 15 min with cryostat sections. The formazan production was also linear with the amount of homogenate incorporated in model films and with section thickness up to 18 µm and therefore, the reaction obeyed the Beer—Lambert law. Variation of the substrate concentration yielded aK_M of 0.05mm for aqueous media and aK_M of 0.55mm for polyvinyl alcohol-containing media. The inhibition with tetramisole appeared to be competitive withK_i = 0.07mm for aqueous media andK_i = 0.7mm for polyvinyl alcohol-containing media. These values indicate that the indoxyl—tetranitro BT method is considerably more sensitive than any metal salt or diazonium salt method developed so far. It is concluded that the optimized method described here is a specific, sensitive and valid quantitative histochemical method for the demonstration of alkaline phosphatase activity.     

Amplified luminometric assays of alkaline phosphatase using riboflavin phosphates

An asasy for alkaline phosphatase is described which is based on the hydrolysis of riboflavin phosphates (5′FMN or 4′FMN) to produce riboflavin. This is converted to 5′FMN using riboflavin kinase, and then asayed using the bacterial bioluminescent system from Vibrio harveyi or V. Fischeri. The most sensitive assay is obtained using 4′FMN, which can measure less than 20 amol after a 1-hour incubation.