Automatic spot preparation and image processing of paper microzone-based assays for analysis of bioactive compounds in plant extracts.

The colorimetric determination of the concentration of phytochemicals in plant extract samples using a spotting automatic system, mobile phone camera and a computer with developed software for quantification is described. Method automation was achieved by using a robotic system for spotting. The instrument was set to disperse the appropriate aliquots of the reagents and sample on a Whatman paper sheet. Spots were photographed and analysed by ImageJ software or by applying the developed MatLab based algorithm. The developed assay was found to be effective, with a linear response at the concentration range of 0.03-0.25g/L for polyphenols. The detection limit of the proposed method is sub 0.03g/L. The paper microzone-based assays for flavonoids and amino acids/peptides were also developed and evaluated as applicable. Comparing the results with conventional PµZP methods demonstrates that both methods yield similar results. At the same time, the proposed method has an attractive advantage in analysis time and repeatability/reproducibility.

Evaluation of a sensitive colorimetric FXIII incorporation assay. Effects of FXIII Val34Leu, plasma fibrinogen concentration and congenital FXIII deficiency.

There is an increasing interest in the role of coagulation factor XIII (FXIII) in cardio- and cerebrovascular diseases. It has recently been reported that a common G-->T point mutation in the A-subunit gene of FXIII, which codes for a valine (val) to leucine (leu) change (FXIIIVal34Leu), is protective against thrombotic diseases but seems to increase the risk of intracerebral bleeding. We developed a colorimetric incorporation assay for detection of FXIII activity based on incorporation of 5-(biotinamido) pentylamine (BAPA) into fibrin or fibrinogen. With this new assay, we studied the effects of FXIIIVal34Leu mutation, plasma fibrinogen concentration and congenital FXIII deficiency on FXIII activity. There are no data available about the ability of different FXIII assays to detect altered activity in FXIIIVal34Leu genotypes. We therefore compared our results determined by the incorporation method with a commonly used photometric method based on ammonia release after cross-linking of glycine-ethylester to a specific glutamine containing peptide substrate. We also determined FXIII A-subunit antigen (Ag) levels using enzyme-linked immunosorbent assay (ELISA) technique. The FXIIIVal34Leu genotype could not be detected either by the photometric method nor by the FXIII A-subunit ELISA. The incorporation assay showed an increased specific FXIII activity in subjects possessing the leu allele. The photometric assay and ELISA gave similar results independent from genotype. In patients with congenital FXIII deficiency before and after substitution, however, ELISA and the incorporation assay gave similar results, whereas the photometric assay showed consistently higher values. Our results show that the incorporation assay, not the photometric assay based on ammonia release, can be used for detection of elevated activity in subjects with FXIIIVal34Leu. Because of specificity and over a wide range sensitivity, the assay can also be used for determination of FXIII deficiency and monitoring of FXIII substitution therapy.

Development of a rapid and sensitive chromogenic heparin assay for clinical use.

A new sensitive chromogenic heparin assay is developed, which is well suited for clinical use. For the assay two reaction mixtures are required which can be lyophilized and reconstituted on the day of use. These reagents are stable during at least 6 h. Only two time-dependent pipetting steps are necessary. Any compound that inactivates thrombin, or can potentiate thrombin inactivation by an inhibitor, can be measured with this assay, including standard heparin, low molecular weight heparins, hirudin, alpha-NAPAP, pentosan polysulphate and dermatan sulphate. It is shown that heparin can be measured accurately in whole blood and in plasma. By addition of dextran sulphate to one of the reagents a platelet factor 4-insensitive assay is developed, so heparin can be measured even in blood that is partially activated and thus contains platelet factor 4 which neutralizes heparin.

Multicenter evaluation of a chromogenic substrate method for photometric determination of prothrombin time.

A multicenter study of a chromogenic substrate method for photometric determination of prothrombin time was conducted in order to evaluate its clinical application. Seven laboratories participated in the study using a total of 742 plasma samples from 417 patients on oral anticoagulant therapy, 261 healthy subjects and 64 patients with different diseases especially of the liver as well as 30 patients with hereditary deficiency of coagulation factors II, V, VII, X. The chromogenic PT method was compared to a standardized coagulometric PT assay which uses the same sensitive human placenta thromboplastin calibrated against international reference preparations. A high correlation of the prothrombin ratio values of the chromogenic and the coagulometric assay was obtained in 402 plasma samples (r = 0.940; y = 1.02x - 0.1). The study showed that the chromogenic PT reagent is sensitive to deficiency of the coagulation factors of the extrinsic pathway but not affected by heparin up to 1 IU/ml because of the heparin antagonist added. The precision (coefficient of variation) of the photometric method ranged between 0.6 and 3% (intraassay CV) and between 1.4 and 5.8 (interassay CV). The International Sensitivity Index (ISI) obtained for the used lot was 1.09. The therapeutical range in percentage activity for patients in a stable phase of an anticoagulant therapy was found to be from 15 to 27 percent of normal. The results of the clinical evaluation proved the good comparability of the new chromogenic PT test with coagulometric methods, its high factor sensitivity, good reproducibility and easy performance.

Development of a rapid kinetic assay for the function of the classical pathway of the complement system and for C2 and C4.

A simple functional complement assay (FCA) is described which is based on kinetic turbidimetry. 25 microliter of sample are combined with 250 microliter of a suspension of antibody coated ovine erythrocytes in a microprocessor controlled photometer. After a lag phase the activation of complement leads to a rapid lysis of the cells which is measured photometrically at 578 nm. The time for a decrease of absorbance of 0.1 is defined as the endpoint of the reaction. Normal citrated plasma which is the preferred specimen for the assay shows a reaction time of about 42 sec whereas pathological or diluted samples show a prolongation. With specific antibodies it was demonstrated that the assay was sensitive for a deficiency of factors of the classical pathway. Factors of the alternative pathway do not influence the assay. In combination with deficient sera a modified form of the assay allows also the determination of C2 and C4. Because of its simplicity, speed and accuracy these assays may be well suited to perform functional complement assays in a routine laboratory.

[Methodological aspects of the photometric determination of prothrombin time using chromogenic substrates]. / Methodische Aspekte zur photometrischen Bestimmung der Prothrombinzeit mittels chromogener Substrate.

A method for photometric determination of prothrombin time (PT) with a chromogenic peptide as substrate is described. The reagent contains human placental thromboplastin, a chromogenic substrate, calcium, a heparin antagonist and buffer. The new prothrombin time method has been calibrated against international reference preparations for thromboplastin. The reagent is sensitive to deficiency of all coagulation factors of the extrinsic pathway. However, it is not sensitive for heparin up to 1 IU/ml. The precision of this fast and simple method is comparable to that of mechanised assays for clinical chemistry.

New chromogenic substrates for thrombin with increased specificity.

Chromogenic substrates for thrombin with high specificity are necessary for several functional assays, especially for the performance of photometric PT and APTT. A new approach to improve the specificity of chromogenic peptide substrates is made coupling tripeptide sequences selective for thrombin to derivatives of 5-amino-2-nitro benzoic acid (ANBA). Especially when the chromophore's side chain is substituted by amines or amino acids hydrolysis rates by other enzymes like kallikrein, plasmin or factor Xa are decreased significantly compared to corresponding para-nitroanilides of the same amino acid sequence. On the other hand, most of these compounds are still sensitive thrombin substrates. KM-values for thrombin and other enzymes are in the same order of magnitude as corresponding pNA-peptides. ANBA peptide substrates may be useful to measure thrombin selectively in a mixture of other proteases like plasmin, factor Xa or kallikrein and for the colorimetric determination of PT and APTT.

Polyethylene glycol can be validly omitted from chromogenic peptide substrate assay for antithrombin III.

To investigate whether the characteristics of a commercial test kit for antithrombin III (Berichrom Antithrombin III) could be influenced by surfactants such as Tween-80 or polyethylene glycol (PEG), we performed some experiments with the original kit reagents and with the reagents dissolved in surfactants. Neither the reliability of the calibration curve nor the data for precision and assay kinetics were amended by the addition of either PEG to the (human) thrombin reagent or Tween-80 to the chromogenic substrate. In the same test system, assays with some other chromogenic substrates and with bovine thrombin showed comparable behavior. Evidently, if one follows the working scheme proposed for this kit, the use of surfactants is not warranted.

Determination of protein C in plasma.

Three new methods to characterize the function of protein C in plasma are described and compared to established procedures. Two methods make use of a snake venom derived specific protein C activator which allows rapid, sensitive and standardizable assays in plasma without sample pretreatment. The other technique is a two dimensional immunoelectrophoresis in the presence of calcium or EDTA, respectively.

ChromoTimeSystem--a new generation of coagulation analyses.

A new system of reagents and a dedicated instrument for coagulation and fibrinolysis assays, the ChromoTimeSystem is described. All assays are performed in the Chromotimer, a microprocessor controlled 4 channel photometer which is connected to micro computer. Most of the reagents use chromogenic substrates. PT, APTT, fibrinogen, reptilase time and thrombin time are all performed with 25 microliters of sample and 250 microliters of reagent. Neither predilution nor preincubation steps are necessary improving these photometric assays in respect to practicability and speed compared to coagulometric techniques. The use of photometry in contrast to the varying physical principles of classical coagulation analysis will be a step forward into standardization.

The primary structure of the beta-lactoglobulin of the waterbuffalo (Bubalus arnee).

The complete amino acid sequence of the beta-lactoglobuline of the waterbuffalo (Bubalus arnee) was established. The sequence of peptides obtained by cleavage with BNPS-Skatole, CNBr and trypsin were determined automatically by the help of the sequenator. Only two differences were found in the beta lactoglobulin of the waterbuffalo compared with the bovine beta-lactoglobulin B.