New separation-free assay technique for SNPs using two-photon excitation fluorometry

A new separation-free method for detection of single nucleotide polymorphisms (SNPs) is described. The method is based on the single base extension principle, fluorescently labeled dideoxy nucleotides and two-photon fluorescence excitation technology, known as ArcDia™ TPX technology. In this assay technique, template-directed single base extension is carried out for primers which have been immobilized on polymer microparticles. Depending on the sequence of the template DNA, the primers are extended either with a labeled or with a non-labeled nucleotide. The genotype of the sample is determined on the basis of two-photon excited fluorescence of individual microparticles. The effect of various assay condition parameters on the performance of the assay method is studied. The performance of the new assay method is demonstrated by genotyping the SNPs of human individuals using double-stranded PCR amplicons as samples. The results show that the new SNP assay method provides sensitivity and reliability comparable to the state-of-the-art SNaPshot™ assay method. Applicability of the new method in routine laboratory use is discussed with respect to alternative assay techniques.     

A simple method for hyaluronic acid quantification in culture broth

The carbazole assay has been used for determination of the percentage of hyaluronic acid in biological fluids. However, it is difficult to measure the concentration of hyaluronic acid in culture broth because glucose and polysaccharides remaining after cultures can react with sulfuric acid and carbazole. The glucose and polysaccharide remnants must be completely removed in order to get the correct value for hyaluronic acid. The turbidity assay, another method for estimating the concentration of hyaluronic acid, is based on the formation of insoluble complexes between hyaluronic acid and cetyltrimethylammonium bromide. This method is very easy and fast compared with the carbazole assay. Because concentrations of hyaluronic acid measured by the turbidity assay were ranged around 100% of those measured by the carbazole assay, the content of hyaluronic acid in culture broth can be determined by the turbidity assay. The turbidity method also has the advantage of being safer than the carbazole assay.     

Stability‐indicating spectrofluorimetric method for the assay of riboflavin and photoproducts: Kinetic applications

A stability‐indicating spectrofluorimetric method has been developed for the simultaneous assay of riboflavin (RF) and photoproducts, formylmethylflavin (FMF), lumichrome (LC) and lumiflavin (LF) in aqueous solution. The method is based on the extraction of LC formed in acid solution and LC and LF formed in alkaline solution with chloroform at pH 2.0 and their assay by fluorescence measurements at 478 and 530 nm, respectively. The aqueous phase, on readjustment of the pH to 6.5, is used to extract FMF with chloroform and its assay is carried out at 530 nm. The aqueous phase is then used for the assay of RF at 530 nm. The proposed method gives more accurate results for the assay of RF compared to those of the United States Pharmacopeia (USP) spectrofluorimetric method which does not take into account the presence of RF photoproducts having similar fluorescence characteristics. The proposed method along with the USP method has been applied to the study of the kinetics of photolysis of RF, assay of stored commercial vitamin preparations and their radiated samples. The results show that the USP method does not distinguish between the fluorescence of RF and its photoproducts, and, therefore, gives erroneous results with about 11% excess in the quantity of the vitamin compared to that of the proposed method. This is due to the interference of the fluorescence of photoproducts in the assay of RF. The method has been validated for various analytical parameters according to the guideline of the International Council for Harmonization (ICH).     

A new fluorometric assay method for quinolinic acid

A new fluorometric assay method for quinolinic acid is introduced in this study. Quinolinic acid-hydrazine complex, a stable fluorescent compound, is formed after heating quinolinic acid with hydrazine at 215–220°C for 2 min. Fluorescence excitation and emission maxima of the complex are at 285 and 380 nm, respectively. This assay method is rapid and rather sensitive. It takes about 30 min to ascertain the amount of quinolinic acid as low as 50 ng. Specificity of this method is high among biological compounds. An ultrasensitive assay method for uinolinic acid (as low as 20 pg) with diphenylhydrazine instead of hydrazine is also found. After separating the quinolinic acid-diphenylhydrazine complex from residual diphenylhydrazine, this ultrasensitive assay method may be practically applicable.     

Procedures for the Assay of Carbenicillin in Body Fluids

The assay of carbenicillin in clinical specimens is complicated by the fact that carbenicillin also contains a small amount of benzylpenicillin, thereby precluding the use of conventional penicillin assay organisms. This report gives details of a microbiological assay method involving the use of a strain of Pseudomonas aeruginosa which is very sensitive to carbenicillin but insensitive to benzylpenicillin. The outline of a microassay method with this organism is presented, and a method for the assay of specimens containing mixtures of carbenicillin and other antibiotics is described.     

Spectrophotometric determination of serum nitrite and nitrate by copper-cadmium alloy

A macro and micro assay for the spectrophotometric determination of serum nitrite and nitrate was developed. Nitrite/nitrate in biological samples can be estimated in a single step by this method. The principle of the assay is the reduction of nitrate by copper-cadmium alloy, followed by color development with Griess reagent (sulfanilamide and N-naphthylethylenediamine) in acidic medium. This assay is sensitive to 1 microM nitrate and is suitable for different biological fluids, including sera with a high lipid concentration. The copper-cadmium alloy used in the present method is easy to prepare and can completely reduce nitrate to nitrite in an hour. The present method provides a simple, cost-effective assay for the estimation of stable oxidation products of nitric oxide in biological samples.     

Assay for cellobiose dehydrogenase in the presence of laccase

The currently used assay for cellobiose dehydrogenase (CDH), an enzyme produced by many wood degrading fungi, lacks specificity and can give false results. The presence of laccase interferes with the standard assay. We have developed an assay for CDH that is insensitive to the presence of both laccase and other phenoloxidases. The method is based on the decrease of reducing end groups in lactose determined by the DNS method. Ferricyanide is present as electron acceptor. Advantages and drawbacks of CDH assay methods are discussed     

New assay for enzymatic phosphate release: application to aspartate transcarbamylase and other enzymes

The colorimetric method for phosphate determination described in the preceding paper is adapted for the assay of orthophosphate liberated in the aspartate transcarbamylase reaction. The method provides for simple, accurate, and sensitive measurement of enzyme activity. The assay uses ammonium molybdate and zinc acetate to form a colored complex with the enzymatically released phosphate; mild conditions which minimize the nonenzymatic background degradation of the substrate, carbamoyl phosphate, are used. Since the assay procedure is relatively rapid, it is especially attractive in situations where results are desired immediately. The method can be used for the assay of any enzyme which releases inorganic phosphate, even in the presence of labile organophosphate compounds.     

Application of improved coupling assay method for peroxidase of diseased thyroids: report of three cases

Recently we have developed an assay method for peroxidase-catalyzed coupling of iodotyronine residues of thyroglobulin, which is applicable to human diseased thyroid tissues. In the present study, the assay method as well as usual peroxidase assay methods were applied to thyroids of three patients (No. 1: familial goiter with impaired thyroglobulin synthesis, No. 2: mild chronic thyroiditis, No. 3: dyshormonogenetic goiter) who showed organification of iodine with high TSH levels and low thyroid hormone levels in sera. In general, these patients showed relatively high activities measured by guaiacol oxidation assay, iodide oxidation and coupling assay compared with those of control thyroids. Iodothyronine content in thyroglobulin was very low except thyroxine in No. 2. These results indicate that factors other than peroxidase may be responsible for the cause of the hypothyroid state. The coupling assay method used here is therefore useful for the detection of the 'coupling defect' in patients in a hypothyroid state.     

Rapid Microbiological Assay of Amino Acids

The rapid microbiological method for determination of amino acids was established. It is composed of 3 steps of culture; inoculum culture, intermediate culture, and assay culture. The inoculum culture is the same as that of ordinary method using Leuc. mesenteroides P–60. For the intermediate culture, which is carried out between the inoculum and assay cultures, the basal medium supplemented with appropriate amount of the amino acid to be determined is employed. The large amount of cells at logarithm phase grown in the intermediate culture are dispersed and used as inoculum for the assay culture. By this technique the assay can be performed by 2.5 to 3.5 hr of assay culture after 2 to 3 hr-intermediate culture.

The technique can be applied to the determination of amino acids in the mixture and the results agree with those obtained by ordinary method.     

High-throughput quantification of hydroxyproline for determination of collagen

An accurate and high-throughput assay for collagen is essential for collagen research and development of collagen products. Hydroxyproline is routinely assayed to provide a measurement for collagen quantification. The time required for sample preparation using acid hydrolysis and neutralization prior to assay is what limits the current method for determining hydroxyproline. This work describes the conditions of alkali hydrolysis that, when combined with the colorimetric assay defined by Woessner, provide a high-throughput, accurate method for the measurement of hydroxyproline.     

PCR-ELISA检测HBV DNA的研究

目的:建立一种敏感、特异的乙型肝炎病毒(HBV)DNA检测方法。方法:应用PCR扩增技术和核酸杂交技术结合酶促显色技术(即PCRELISA技术)来检测血清中的HBVDNA。结果:应用PCRELISA技术能够检出许多PCR琼脂糖凝胶电泳所检测不到的HBVDNA,大大地提高了检出率,而且,特异性强。结论:PCRELISA方法灵敏度高,特异性强,检测结果数据化,不受主观因素的影响 。     

A fluorimetric method using fluorescein di-beta-D-galactopyranoside for quantifying the senescence-associated beta-galactosidase activity in human foreskin fibroblast Hs68 cells

The senescence-associated beta-galactosidase (SA-betaG) assay is one of the few accepted markers of cell aging. However, the cytochemical method using 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-Gal) as substrate is limited in sensitivity and is only semiquantitative. Here, we modified the X-Gal method by replacing X-Gal with fluorescein di-beta-D-galactopyranoside (FDG) as substrate for SA-betaG, and the activity was measured fluorimetrically. We showed in Hs68 cells that the FDG fluorescein fluorescence increased with increasing passages of the cells in parallel with the X-Gal method. A major advantage of the FDG method is that it is a quantitative method for the SA-betaG activity. For example, we showed that the FDG fluorescein in p30(+1) of Hs68 cells was generally stronger than that in p26(+1) cells, whereas the X-Gal method gave similar results (95 and 100%) for p26(+1) and p30(+1) cells. The FDG method was precise with a relative standard deviation lower than 10%. We further demonstrated that FDG and X-Gal could be added simultaneously for SA-betaG assay because the FDG fluorescein diffused readily through formaldehyde-fixed cell membrane and could be detected in the suspension buffer. Thus, a double-substrate method, i.e., X-Gal for rapid qualitative assay and FDG for quantitative assay, can be conducted simultaneously to provide a simple and reliable assay of SA-betaG activity as a marker of cell aging.     

A simple and sensitive fluorometric assay for tyrosine hydroxylase.

A simple, rapid, and sensitive fluorometric assay for measuring the activity of tyrosine hydroxylase is described. The enzyme activity is detected by converting tyrosine to 3,4-dihydroxyphenylalanine (dopa), which is then subjected to conversion to the highly fluorescent product by the trihydroxyindole method. The assay method is very reproducible, more sensitive than a radiochemical method for the determination of tyrosine hydroxylase activity using the isolation of [³H]water commonly used, and linear from 0.2 to 12 nmol of dopa. The method should be applicable for the assay of the enzyme with a wide range of activity.     

Gold nanoparticles-based colorimetric assay for rapid detection of Salmonella species in food samples

A rapid, sensitive and cost-effective method was developed for detection of foodborne pathogens, particularly Salmonella species. The method utilizes single stranded DNA (ssDNA) probes and non-functionalized gold nanoparticles to provide a colorimetric assay for the detection of PCR amplified DNA. Different food samples were tested with the PCR-based colorimetric assay parallel with the conventional culture method. The sensitivity and specificity of colorimetric assay was 89.15 and 99.04% respectively with reference to conventional culture method. The total time required to detect the Salmonella spp. present in food samples by the developed method is less than 8 h, including 6 h incubation. It was observed that the colorimetric assay was 10 times more sensitive than gel-based detection with the same concentration of DNA used for analysis.     

两种甲型肝炎病毒抗原快速检测方法的建立

目的建立两种甲型肝炎病毒抗原(HAV-Ag)检测试剂盒,并对其检测效果进行评价。方法生物素标记甲型肝炎病毒抗体(HAV-Ab)与辣根过氧化物酶标记亲和素联合应用建立甲型肝炎病毒抗原BA-ELISA检测法;同时使用辣根过氧化物酶标记HAV-Ab作放大系统建立双抗体夹心甲型肝炎病毒抗原ELISA检测试剂,对比两种检测方法的特异性、灵敏度及实际应用效果。结果用生物素标记甲型肝炎病毒抗体-辣根过氧化物酶标记亲和素作放大系统建立的甲型肝炎病毒抗原BA-ELISA检测法,较双抗体夹心ELISA检测方法灵敏度高1～2个稀释度;两种检测法均对10余种病毒无交叉,P/N值BA-ELISA检测法较高。结论甲型肝炎病毒抗原BA-ELISA检测法是一种灵敏度高,特异性好,方便快捷的检测方法,可广泛应用于甲型肝炎病毒研究及临床检测中。而甲型肝炎病毒抗原双抗体夹心ELISA检测法,检测灵敏度适中,操作简单,更适用于甲肝疫苗生产检定。     

Simple high-performance liquid chromatographic method to verify the direct barbituric acid assay for urinary cotinine

An isocratic HPLC method is described to determine urinary concentrations of nicotine and cotinine after derivatization with cyanogen chloride and barbituric acid. This method has been used to assess the reliability of the direct barbituric acid assay to determine smoking status. It is concluded that the direct barbituric acid assay is a very reliable indicator of smoking status, provided that urine blank samples are prepared to correct for background absorbance. If the direct barbituric acid assay is in disagreement with self-reported smoking status, this HPLC procedure is a useful method to resolve the discrepancy.     

Analysis of radiation-induced apoptosis in human lymphocytes: flow cytometry using Annexin V and propidium iodide versus the neutral comet assay

BACKGROUND: The neutral comet assay was devised to measure double-stranded DNA breaks, but it has also been used to measure apoptosis based on its characteristic DNA fragmentation patterns. There is still uncertainty about the reliability of this method. By comparing the comet assay with a flow cytometry method that uses Annexin V binding to apoptotic cells, we have provided further evidence for evaluating the usefulness of the comet assay for detecting apoptosis. METHODS: Apoptosis was induced in human peripheral blood mononuclear cells (PBMC) by ionizing radiation and measured using the comet assay and a flow cytometry method that measures Annexin V and propidium iodide (PI) staining. RESULTS: The Annexin V flow cytometry assay distinguished among early apoptosis, late apoptosis, and an apoptotic or necrotic phase in which the cells were labeled with both Annexin V and PI. The comet assay detected only the latter two phases of apoptosis. CONCLUSIONS: The comet assay is a useful tool for measuring the late stages of apoptosis whereas the Annexin V assay measures higher amounts of apoptosis because it can detect cells in an earlier stage of the apoptotic pathway.     

A novel sensitive immunoassay method based on the Invader technique

A novel and sensitive immunoassay method has been developed in which the conventional sandwich immunoassay and the highly sensitive DNA detection method, the Invader method, are combined. The signal amplification function of the latter method has been successfully used to enhance the sensitivity of the sandwich immunoassay. The new assay method may be called the Immuno-Invader assay. The assay format involves three important steps: (1) a target antigen is captured and flagged with a biotin-conjugated detection antibody by the sandwich method, (2) streptavidin and a biotin-conjugated oligonucleotide are added to form a complex with the detection antibody, and (3) the oligonucleotide in the complex is detected using the Invader method. The method was applied to the assay of human tumor necrosis factor-α (hTNF-α). Detection limits obtained were 0.1 pg/ml hTNF-α when a luminescent europium chelate was used with a time-resolved measurement mode, and 0.8 pg/ml when fluorescein was used with a normal prompt fluorescence measurement mode. On the other hand, the detection limit of a commercially available hTNF-α enzyme-linked immunosorbent assay that uses horseradish peroxidase was 3.5 pg/ml. These results demonstrate the feasibility and potential of the new assay method for highly sensitive immunoassay.     

Enhanced fluorescence resonance energy transfer immunoassay with improved sensitivity based on the Fab'-based immunoconjugates

Fluorescence resonance energy transfer (FRET) is a powerful technique to monitor protein-protein interaction. Recently, we developed homogeneous and noncompetitive immunoassay based on the enhanced FRET by leucine zipper interaction. Here we improved the assay by establishing a general method for preparation of the Fab'-based immunoconjugate. Anti-human serum albumin Fab' numbers 11 and 13 were chemically conjugated with recombinant proteins consisting of thioredoxin, flexible linker, and green fluorescent protein color variant tethered with a leucine zipper motif. Compared with single chain antibody variable region-based fusion proteins prepared by the gene fusion method in our previous study, the resultant Fab'-based immunoconjugates accomplished an assay with nearly 10 times greater sensitivity. Furthermore, the conjugation method enabled us to apply the assay generally to measurement of another high-molecular weight antigen for which antibodies prepared for sandwich immunoassay are commercially available. Because of the facility and generality of the preparation method for the immunoconjugate, the assay is expected to be applied to many antigens that require rapid diagnosis and moderate measurement range.