Search

The integration of several controlled parameters within a single test system is experiencing increased demand. However, multiplexed test systems typically require complex manufacturing. Here, we describe a multiplexed immunochromatographic assay that incorporates a conventional nitrocellulose membrane, which is used together with microspot printing, to construct adjacent microfluidic "tracks" for multiplexed detection. The 1 mm distance between tracks allows for the detection of up to four different analytes. The following reagents are applied in separate zones: (a) gold nanoparticle conjugates with antibodies against each analyte, (b) other antibodies against each analyte, and (c) antispecies antibodies. The immersion of the test strip in the sample initiates the lateral flow, during which reagents of different specificities move along their tracks without track erosion or reagent mixing. An essential advantage of the proposed assay is its extreme rapidity (1-1.5 min compared with 10 min for common test strips). This assay format was applied to the detection of cardiac and inflammatory markers (myoglobin, D-dimer, and C-reactive protein) in human blood, and was characterized by high reproducibility (8%-15% coefficient of variation) with stored working ranges of conventional tests. The universal character of the proposed approach will facilitate its use for various analytes., Competing Interests: The authors declare that they have no known competition for financial interests or personal relationships that could have appeared to influence the work reported in this paper.

A multiplex analytical system has been developed to carry out microformat latex agglutination tests with video digital registration. The system makes it possible to apply less than 1 microl drops of latex and samples in the matrix format to a carrier and to make the test in 30 points at once, as well as to record and to interpret the results of the test, by keeping analytical information for each sample. Comparison of the results of determination of the serum levels of C-reactive protein, rheumatoid factor, and antistreptolysine in the system developed by the authors with the results obtained in the macroagglutination test by the immunoturbidimetric technique leads to the conclusion that the methods are comparable. The proposed type of a latex agglutination test based on a matrix approach and miniaturization assures efficient production and the quality of laboratory studies.

A microarray analytical system for performing tests of latex agglutination reaction in microformat with digital image registration was developed. The system allows the application of latex microdrops to the surface of the carrier in the form of a regular microarray and mixing of the latex droplets with the individual samples in each droplet of the microarray. The reaction is performed in a total mixture volume of about 1 microl for each of 30 samples simultaneously with video registration and interpretation of the results using a scanning device and specially developed software. The results of the semi-quantitative determination of C-Reactive Protein, Rheumatoid Factor and Anti-Streptolysin O concentrations by traditional macro- and proposed micro-arrayagglutination method were compared with the immunoturbidimetric measurements used as reference method. It was concluded that the suggested method for performing latex agglutination reactions on the basis of a microarray approach with digital image evaluation of results can provide a high throughput and reliable results and also offers significant advantages to the traditional latex agglutination tests with visual interpretation. Comprehensive documentation and objectification of readouts show a siginificant improvement to the present methodology.

The present paper describes the use of the scanner "Expert-Lab Agglutination" system to record the results of latex-agglutination tests in making a reaction on the lids of 96-well immunoassay plates. The described system provides a possibility of having a primary image of all tests carried out on a carrier. Software offers the prospect of contrasting the images of individual and control samples, increasing the size of images, and comparing the latter. The use of special image treating techniques may also yield objective figures characterizing the rate of a reaction. The possibility of archiving primary information and retrospectively appraising the correctness of the result of an analysis at any required moment is of fundamental importance to the latex-agglutination test.

Whether a commercially available scanner might be used as a vertical photometer of 96-well microplates for enzyme immunoassay (EIA) was studied. The analytical characteristics of the scanner in this regard were assessed, by comparing the findings with the respective photometric data obtained by a "VICTOR" EIA analyzer. It is concluded that the scanner registration system in combination with "Expert-Lab EIA" may be used in all types of EIA. The scanner system provides additional possibilities as compared with the conventional photometric analyzers.

The respiration parameters of mitochondria from rat heart muscle and from fast-twitch and slow-twitch skeletal muscle skinned fibers were comparatively analyzed. Electrophoretic patterns of fiber protein composition were also compared. It was found that fibers with low affinity of mitochondria for ADP (i.e., heart and slow-twitch skeletal muscle soleus) contain a 27.5-kD protein that is absent from the fibers that exert high affinity for ADP (i.e., fast-twitch skeletal muscle gastrocnemius). Partial proteolysis, which increases the affinity of mitochondria of the heart and slow-twitch skeletal muscles for ADP, results in the disappearance of this protein. The results suggest that this protein may be an intracellular factor that controls the permeability of the outer mitochondrial membrane for ADP.

A new technique for the creation of compounds with targeted action is described. In such compounds, termed respecrins (receptor-specific, screened toxin), a physiologically active component carries an epitope-containing fragment of the target antigen and is screened or masked by antibody specific to this antigen. Interaction of the respecrin with the target cell results in dissociation of this immunocomplex, and thereby activation of the physiologically active component. The kinetics of dissociation of a respecrin during interaction with a cell were investigated by flow cytometry. Possible applications of the principle are illustrated by investigation on the antigen-dependent activation of the mitogenic activity of staphylococcal enterotoxin A.

By immunodiffusion analysis the presence of embryonal prealbumin (EPA) in extracts of different tumors has been studied. EPA is most frequently detected in extracts of bone tumors (75.0%) with the titre up to 1 : 256 and in tumors of soft tissues (74.5%) also with the titre up to 1 : 256. The EPA immunochemical test is believed to be of value for monitoring tumors of the connective-tissue origin.

A quantitative immunoenzymatic method (ELISA) has been developed for detection of embryonic prealbumin (EPA-1). This method allows EPA-1 to be detected in human serum in an amount of 4 to 100 ng/ml. The high levels of EPA-1 (over 4 ng/ml) were observed in all cases (27 patients) of tumors of connective tissues, in 50 of 51 in brain tumors, in 39 of 341 in gastrointestinal tract tumors, in 4 of 13 in breast tumors, and in 21 of 51 tumors of the respiratory tract. The levels of EPA-1 in donors and pregnant woman sera normally did not exceed 4 ng/ml. However, about 3% of the samples of these sera had the levels of EPA-1 from 4 to 12 ng/ml.

Embryonal prealbumin-2 /EPA-2/ was shown to be a protein, which was actively synthesized in fetus during embryogenesis. The highest content of EPA-2 was found in soft fetal tissues; this suggesting that the protein synthesis occurred in organs of mesodermal origin. To the moment of delivery the content of EPA-2 in fetus was distinctly decreased and the protein was not detected using the immunodiffusion technique in blood serum of adult persons, i e. the EPA-2 synthesis was markedly reduced. Low content of EPA-2 was observed in human gastrointestinal tract, locomotive system, spleen and skin tissues. The EPA-2 synthesis was increased distinctly in malignant tumors, especially, of connective tissue, whereas in tumors of epithelial tissue the content of EPA-2 often depended on presence of connective tissue elements.

Four specific antigens (trophoblastic beta 1-globulin, placental lactogen, alpha 1- and alpha 2-globulins of human placenta) were identified using antisera to the native amniotic fluid. Five antigens with the mobility of prealbumins, alpha 1-globulins, alpha 2-globulins and beta 2-globulins which bear no resemblance with the previously studied antigens were identified using antisera to the acid fraction of the amniotic fluid. Both the prealbumins and alpha 2-globulin were found in the blood serum of foetuses of different age and of newborn infants; these proteins were absent from the blood serum of pregnant women and donors. They received the names of embryonic prealbumine 1, embryonic prealbumine 2 and embryonic alpha 2-globulin. The protein with the mobility of alpha 1-globulins was found in the amniotic fluid of foetuses and in the blood serum of pregnant women only and received the name of amniotic alpha 1-globulin. The concentration of the antigens in question was studied in the developing foetuses and in the blood serum of pregnant women at different stages of pregnancy.

IEA of brain specific antigen 10-40-4 in human and animal organs and tissues was elaborated. The method permits measuring the concentration of the antigen within the range from 1 to 120 ng/ml. The use of the method made it possible to confirm brain specificity of protein 10-40-4, since its brain content is 1500 to 2000 times higher than in other organs, and to estimate the percentage of cross-reaction between antigenic determinants of brain specific antigen 10-40-4 from different species of animals.