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This work presents a magnetic purification method of human erythrocyte Acetylcholinesterase (EC 3.1.1.7; AChE) based on affinity binding to procainamide (Proca) as ligand. Acetylcholinesterase is an acetylcholine-regulating enzyme found in different areas of the body and associated with various neurological disorders, such as Parkinson, Alzheymer and Amyotrophic Lateral Sclerosis. AChE from human erythrocyte purification has been attempted in recent years with low degree of purity. Here, magnetic nanoparticles (MNP) were synthesized and coated with polyaniline (PANI) and procainamide (PROCA) was covalently linked to the PANI. The extracted human erythrocyte AChE formed a complex with the MNP@PANI-PROCA and an external magnet separated it from the undesired proteins. Finally, the enzyme was collected by increasing the ionic strength. Experimental Box-Behnken design was developed to optimize this process of human erythrocyte AChE purification protocol. The enzyme was purified in all fifteen experiments. However, the best AChE purification result was achieved, about 2000 times purified, when 100 mg of MNP@PANI-PROCA was incubated for one hour with 4 ml hemolysate extract. The SDS-PAGE of this preparation presented a molecular weight of approximately 70 kDa, corroborating with few previous studies of AChE from erythrocyte purification., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

This work aimed to develop a simple and low-cost method to obtain human serum albumin (HSA) and its consequent application for in vitro drug interaction assays. The HSA was purified by classic principles of plasma precipitation and thermocoagulation, using a multiple-stage fractionation. The quality of the final product was assessed by electrophoresis, protein dosage by the Lowry method and the pharmacopeial thermal stability. At the end, an isotonic solution of HSA with a total protein concentration of 2.7 mg·mL -1 was obtained, which was visualized as a single band corresponding to the molecular weight of 66 kDa. After the thermal stability test, there was no indication of turbidity or color change of the solution. Finally, the HSA was useful for interaction assays with indole-thiazole and indole-thiazolidinone derivatives through UV-vis absorption and fluorescence spectroscopic studies, as well as by docking molecular analysis. Derivatives quenched the intrinsic fluorescence of HSA, disrupted the tryptophan residues microenvironment, and probably bind at Sudlow's site I. Therefore, the simplified methodology developed in this work proved to be effective in obtaining HSA that can be applied to research goals including drug interaction assays., (Copyright © 2021 Elsevier B.V. All rights reserved.)

In this study, we report the synthesis of eight novel indole-thiazole and indole-thiazolidinone derivatives, as well as their ability to interact with DNA, analysed through the UV-vis absorption, fluorescence, circular dichroism (CD), viscosity techniques and molecular docking. The ctDNA interaction analysis demonstrated different spectroscopic effects and the affinity constants (Kb) calculated by the UV-vis absorption method were between 2.08 × 10 5 and 6.99 × 10 6  M -1 , whereas in the fluorescence suppression constants (Ksv) ranged between 0.38 and 0.77 × 10 4  M -1 and 0.60-7.59 × 10 4  M -1 using Ethidium Bromide (EB) and 4',6-Diamidino-2-phenylindole (DAPI) as fluorescent probes, respectively. Most derivatives did not alter significantly the secondary structure of the ctDNA according to the CD results. None of the compounds was able to change the relative viscosity of the ctDNA. These results prove that compounds interact with ctDNA via groove binding, which was confirmed by A-T rich oligonucleotide sequence assay with compound JF-252, suggesting the importance of both the phenyl ring coupled to C-4 thiazole ring and the bromo-unsubstituted indole nucleus., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Heparin was immobilized on magnetic chitosan particles to be used as a tool for human plasma protein identification. Chitosan was magnetized by co-precipitation with Fe 2+ /Fe 3+ (MAG-CH). Heparin was functionalized with carbodiimide and N-hydroxysuccinimide and covalently linked to MAG-CH (MAG-CH-hep). X-ray diffraction confirmed the presence of chitosan and Fe 3 O 4 in MAG-CH. This particle exhibited superparamagnetism and size between 100-300 μm. Human plasma diluted with 10 mM phosphate buffer (pH 5.5) or 50 mM Tris-HCl buffer (pH 8.5) was incubated with MAG-CH-hep, and the proteins fixed were eluted with the same buffers containing increasing concentrations of NaCl. The proteins obtained were investigated by SDS-PAGE, LC/MS, and biological activity tests (PT, aPTT, and enzymatic chromogenic assay). Inhibitors of the serpin family, prothrombin, and human albumin were identified in this study. Therefore, MAG-CH-hep can be used to purify these proteins and presents the following advantages: low-cost synthesis, magnetic separation, ion-exchange purification, and reusability., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

In order to characterize the affinity between specific carbohydrate-binding proteins such as lectins, a model is proposed to study these interactions using a polysaccharide membrane to simulate such adsorption. Here, lectin-carbohydrate interactions were chemiluminescently investigated using lectins conjugated to acridinium ester (AE) and polysaccharides composed of their respective specific carbohydrates. The lectin-AE conjugates were incubated with discs (0.0314-0.6358 cm 2 ) of phytagel, chitosan and carrageenan. The complex formation chemiluminescently detected followed the Langmuir isotherm from which constants were estimated. The association constant (K a ) and maximum binding sites on the membranes were 2.4 × 10 -7  M -1  ± 0.8 × 10 -7  M -1 and 1.3 × 10 -3  mol. mg -1 ± 0.3 × 10 -3  mol. mg -1 (Con A); 0.9 × 10 -6  M -1  ± 0.4 × 10 -6  M -1 and 0.021 × 10 -3  mol. mg -1 ± 0.003 × 10 -3  mol. mg -1 (WGA) and 2.0 × 10 -6  M -1  ± 0.9 × 10 -6  M -1 and 0.069 × 10 -3  mol. mg -1 ± 0.010 × 10 -3  mol. mg -1 (PNA). The proposed model might be useful to study binding affinity and estimate the amount of binding not limited by the sugar content in the membrane., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Molecules bearing indole nucleus present diverse biological properties such as antitumor and anti-inflammatory activities that can be associated both to DNA and protein interactions. This study focused on the synthesis of new indole derivatives with thiazolidines and imidazolidine rings condensed as side chains as well as the evaluation of their ability to interact with the DNA and antitumor and topoisomerase inhibition activities. All derivatives were successfully synthesized and their structures were elucidated by mass spectrometry (MS), infrared (IR), spectroscopy 1 H NMR, 13 C NMR, COSY 1 H- 1 H and HSQC 1 H- 13 C. The antitumor activity was evaluated against different cancer cell lines using the antiproliferative MTT assay. DNA binding ability was analyzed by absorption spectroscopy and fluorescence technique using ethidium bromide (EB) as a fluorescent probe. Changes were observed in spectroscopic properties of the compounds after interacting with ctDNA (calf thymus DNA), with hypochromic and hyperchromic effects, besides blue or red shifts in the maxima of spectra. The indole derivative 5-(1H-Indol-3-ylmethylene)-thiazolidin-2,4-dione (4c) presented the best results in antitumor assay against the breast line tested (T47D), with IC 50 value lower than the positive control, doxorubicin (1.93 and 4.61 μM, respectively). On the other hand, the compound 3-amino-5-(1H-indol-3-ylmethylene)-2-thioxo-thiazolidin-4-one (4a) was active against leukemia cell lines (HL60 and K562) with the high value of the DNA binding constant, Kb of 5.69 × 10 4 . However, this compound (4a) did not inhibit the topoisomerase-I activity evaluated by relaxation assay. These results show that the indole nucleus contribute to the incorporation of molecules into the DNA. Moreover, it was highlighted that basic side chains, such as thiazolidines and imidazolidines, and free amino group, are relevant for design of promising antitumor and DNA binding compounds., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Fluorescent compounds have been widely used for biomolecule labeling in cytochemistry and histochemistry analysis. Here, it is described the optical properties of dimethyl 2-[(acridin-9-yl)methylidene]-malonate (LPSF/IP-81), an acridine derivative. This compound was conjugated to Concanavalin A (Con A) lectin and applied as sugar probe in lectin histochemistry. Evaluation of luminescent properties showed that LPSF/IP-81 is photoluminescent with excitation at 360 nm and emission at 428 nm. Con A hemagglutinating activity and LPSF/IP81 photoluminescence were unaltered after conjugation. Circular dichroism of Con A-LPSF/IP81 conjugate showed the maintenance of the Con A structure. Lectin histochemistry with Con A-LPSF/IP81 conjugate demonstrated different pattern recognition studying normal, fibroadenoma, and invasive ductal carcinoma of human breast. These findings indicate that LPSF/IP-81 can be proposed as an alternative probe for histochemical analysis., (© 2017 Wiley Periodicals, Inc.)

Human antithrombin is a blood derivative widely used in the treatment of coagulation dysfunction. Affinity chromatography using heparin (HEP) derivatives is usually used for antithrombin purification. In this study, an affinity procedure based on a magnetic Dacron-HEP composite is proposed. Dacron was firstly converted to Dacron-hydrazide and magnetised by co-precipitation with of Fe 2+ /Fe 3+ (mDAC). HEP was activated by carbodiimide and N-hydroxysuccinimide and covalently linked to mDAC (mDAC-HEP). EDX and infrared spectra analyses confirmed each synthesis step of mDAC-HEP. This composite exhibited superparamagnetism behaviour. Human plasma was incubated with mDAC-HEP (fresh and stored over a long period) and washed with phosphate buffer containing increasing concentrations of NaCl. Human plasma antithrombin activity was reduced by approximately 20% in the presence of the 1.0M NaCl fraction, and this eluate was able to prolong coagulation time (aPTT) using both preparations. Electrophoresis of the eluates revealed bands corresponding to the expected size of antithrombin (58kDa). The mDAC-HEP particles are reusable. This method presents the following advantages: easy, low-cost synthesis of the composite, magnet-based affinity purification steps, and reusability., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The aim of the present study was to investigate ultrastructural changes induced by (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide (APHCA) treatment on human breast adenocarcinoma cancer cells MCF-7, besides the evaluation of phosphatidylserine externalization and DNA fragmentation in treated cells. Cell viability analysis demonstrated concentration and time-manner cytotoxicity. Treated MCF-7 cells did not expose phosphatidylserine residues to the external plasma membrane surface and DNA fragmentation was not visualized by electrophoresis. Light microscopy showed compromised cell density and presence of vacuolization after APHCA treatment with 60μM. Scanning and transmission electron microscopies revealed hallmarks of autophagy, namely the presence of membrane bebbling and autophagosomes, besides shrunken cells and cell debris in treated MCF-7 cells. However, more specific tests such as the quantification of mammalian autophagy proteins are necessary to determine the kind of death that is trigged by APHCA., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Two new spiro-acridines were synthesized by introducing cyano-N-acylhydrazone between the acridine and phenyl rings followed by spontaneous cyclization. The final compounds (E)-1'-(benzylideneamino)-5'-oxo-1',5'-dihydro-10H-spiro[acridine-9,2'-pyrrole]-4'-carbonitrile (AMTAC-01) and (E)-1'-((4-methoxybenzylidene)amino)-5'-oxo-1',5'-dihydro-10H-spiro[acridine-9,2'-pyrrole]-4'-carbonitrile (AMTAC-02) were evaluated for their interactions with calf thymus DNA, antiproliferative and human topoisomerase I and IIα inhibitory activities. Both compounds presented ability to bind DNA. The binding constant determined by UV-vis spectroscopy was found to be 10 4 M -1 . Antiproliferative assay demonstrated that AMTAC-01 and AMTAC-02 were most active against prostate and melanoma tumor cell lines, respectively. The compound did not present Topo I inhibitory activity. However, both derivatives displayed topoisomerase IIα inhibitory activity comparable to amsacrine, and AMTAC-02 was more potent than AMTAC-01 with methoxy substituent group on phenyl ring. This study demonstrates that the new derivatives are promising molecules with topoisomerase IIα inhibitory and antiproliferative activities., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In this work, the acridine nucleus was used as a lead-compound for structural modification by adding different substituted thiosemicarbazide moieties. Eight new (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide derivatives (3a-h) were synthesized, their antiproliferative activities were evaluated, and DNA binding properties were performed with calf thymus DNA (ctDNA) by electronic absorption and fluorescence spectroscopies. Both hyperchromic and hypochromic effects, as well as red or blue shifts were demonstrated by addition of ctDNA to the derivatives. The calculated binding constants ranged from 1.74 × 10(4) to 1.0 × 10(6) M(-1) and quenching constants from -0.2 × 10(4) to 2.18 × 10(4) M(-1) indicating high affinity to ctDNA base pairs. The most efficient compound in binding to ctDNA in vitro was (Z)-2-(acridin-9-ylmethylene)-N- (4-chlorophenyl) hydrazinecarbothioamide (3f), while the most active compound in antiproliferative assay was (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide (3a). There was no correlation between DNA-binding and in vitro antiproliferative activity, but the results suggest that DNA binding can be involved in the biological activity mechanism. This study may guide the choice of the size and shape of the intercalating part of the ligand and the strategic selection of substituents that increase DNA-binding or antiproliferative properties.

The present work aimed to magnetize Parkia pendula seeds gum and use it as a matrix for Concanavalin A covalent immobilization. This composite was applied in affinity purification of glycoconjugates. Parkia pendula seeds were hydrated and the gum provenient from the supernatant was precipitated and washed with ethanol and dried. The gum was magnetized in co-precipitation using solutions of Fe+2 and Fe+3. Matrix activation was accomplished with NaIO4. Magnetized Parkia pendula seeds gum with covalently immobilized Concanavalin A was used as an affinity matrix for the recognition of bovine serum fetuin glycoprotein. Fetuin elution was carried out with a solution of glucose (300mM) and evaluated through SDS-PAGE. The efficiency of lectin immobilization and fetuin purification were 63% and 14%, respectively. These results indicate that the composite produced is a promising magnetic polysaccharide matrix for lectins immobilization. Thus, such system can be applied for affinity purification allowing an easy recovery by magnetic field.

This work aimed to evaluate the glycophenotype in normal prostate, bening prostatic hyperplasia (BPH) and prostatic adenocarcinoma (PCa) tissues by a chemiluminescent method. Concanavalin A (Con A), Ulex europaeus agglutinin (UEA-I) and Peanut agglutinin (PNA) lectins were conjugated to acridinium ester (lectins-AE). These conjugates remained capable to recognize their specific carbohydrates. Tissue samples were incubated with lectins-AE. The chemiluminescence of the tissue-lectin-AE complex was expressed in relative light units (RLU). Transformed tissues (0.25 cm(2) by 8 µm of thickness) showed statistical significant lower α-D-glucose/mannose (BPH: 226,931 ± 17,436; PCa: 239,520 ± 12,398) and Gal-β(1-3)-GalNAc (BPH: 28,754 ± 2,157; PCa: 16,728 ± 1,204) expression than normal tissues (367,566 ± 48,550 and 409,289 ± 22,336, respectively). However, higher α-L-fucose expression was observed in PCa (251,118 ± 14,193) in relation to normal (200,979 ± 21,318) and BHP (169,758 ± 10,264) tissues. It was observed an expressive decreasing of the values of RLU by inhibition of the interaction between tissues and lectins-AE using their specific carbohydrates. The relationship between RLU and tissue area showed a linear correlation for all lectin-AE in both transformed tissues. These results indicated that the used method is an efficient tool for specific, sensitive and quantitative analyses of prostatic glycophenotype.

Background: Tumor cells show alterations in their glycosylation patterns when compared to normal cells. Lectins can be used to evaluate these glycocode changes. Chemiluminescence assay is an effective technique for quantitative analysis of proteins, nucleic acids, and carbohydrates due to its high sensitivity, specificity, and rapid testing., Objective: To use histochemiluminescence based on lectin conjugated to acridinium ester (AE) for the investigation of glycophenotype changes in cutaneous tumors., Methods: Concanavalin A (Con A), Peanut agglutinin (PNA), Ulex europaeus agglutinin-I (UEA-I), and Maackia amurensis agglutinin (MAA) were conjugated to acridinium ester. Biopsies of cutaneous tumors and normal skin were incubated with the lectins-AE, and chemiluminescence was quantified and expressed as Relative Light Units (RLU). Results. Actinic keratosis (AK), keratoacanthoma (KA), squamous cell carcinoma (SCC), and basal cell carcinoma (BCC) showed lower expression of α -D-glucose/mannose and α -L-fucose residues compared to normal tissue. Cutaneous tumors displayed higher expression of Gal- β (1-3)-GalNAc residues than normal tissue. AK and SCC exhibited higher expression of Neu5Ac- α (2,3)Gal residues than normal epidermis. KA and BCC showed equivalent RLU values compared to normal tissue., Conclusions: Lectin histochemiluminescence allowed quantitative assessment of the carbohydrate expression in cutaneous tissues, contributing to eliminate the subjectivity of conventional techniques used in the histopathological diagnosis.

Patients with acute schistosomiasis were studied before and after oxamniquine treatment. They had been exposed to cercariae 5 to 9 weeks before, and presented compatible clinical manifestations, eosinophilia, and high levels of total IgE. Interferon-gamma (IFN-gamma) and interleukin-4 were measured by ELISA in whole blood samples under soluble egg antigen or soluble adult worm preparation stimulation. After treatment, the reduction of leukocytosis and eosinophilia were not significant, but total IgE levels decreased significantly, in contrast to IFN-gamma levels that were significantly increased. The oxamniquine treatment of acute schistosomiasis patients is followed by an improvement of a Th1 response in vitro. If this response has a protective aspect is unknown, and some investigations need to be realized.

Soluble adult Schistosoma mansoni antigen preparation (SWAP) was covalently fixed onto polyvinyl alcohol-glutaraldehyde discs and an enzyme linked-immunosorbent assay (ELISA) was set up. The best conditions for the assay were established and it was found that small amount of antigen such as 1.5 micrograms was required. A comparison between this procedure and the conventional ELISA was proceeded. A reliable method of antigen immobilization was achieved and the low prices of the employed reagents are economically attractive.

Discs of polyvinyl alcohol cross-linked with glutaraldehyde were synthesized under acid catalysis (H2SO4). Then, the antigen F1 purified from Yersinia pestis was covalently linked to this modified polymer. Afterwards, an enzyme-linked immunosorbent assay (ELISA) was established for the diagnosis of plague in rabbit and human. The best conditions for the method were achieved by using 1.3 micrograms of F1 prepared in 0.067 M phosphate buffer, pH 7.2, containing 1 M NaCl (PBS); anti-IgG peroxidase conjugate diluted 6,000 times and as a blocking agent 3% w/v skim milk in PBS. The titration of positive rabbit serum according to this procedure detected antibody concentrations up to 1:12,800 times. The present method, the conventional ELISA and passive haemagglutination assay are compared.

Xanthine oxidase was covalently immobilized on polyacrylamide gel beads, polyamide-11 and dacron. Hypoxanthine (15 ml of 200 microM), prepared in 0.1 M phosphate buffer, pH 8.0, was circulated through a column containing 1.0 g derivatized enzyme at a flow rate of 1.0 ml/min at 28 degrees C. Specific activities of 0.660, 0.072 and 0.016 Units/mg of protein were demonstrable for the polyacrylamide gel beads, dacron and polyamide-11 derivatives, respectively. The action of these water insoluble enzyme derivatives on 6-mercaptopurine (15 ml of 660 microM) was also investigated, under the same experimental conditions, showing specific activities of 0.063 Units/mg, 0.574 muUnits/mg and 0.118 muUnits/mg, respectively. The 6-mercaptopurine oxidative pathway catalyzed by immobilized xanthine oxidase on dacron stopped at the intermediate compound, 6-mercapto-8-hydroxypurine, so that no 6-thiouric acid was produced, whereas the immobilized preparations using polyacrylamide gel beads and polyamide-11 behaved like the soluble enzyme, namely, 6-thiouric acid was the final product. The behavior of dacron-xanthine oxidase compound was similar to that previously described for the derivatives obtained with carboxymethylcellulose and chitosan. The hypoxanthine oxidative pathway catalyzed by xanthine oxidase immobilized on these three supports was similar to the soluble enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Parasitological examinations were carried out on 663 individuals of three different cities of Pernambuco State, Northeastern Brazil: Recife, Palmares and Bodocó. The population from a drought area of Pernambuco State, Bodocó, was investigated for amoebiasis and compared with Recife, metropolitan city (about 1.3 million of inhabitants) and another inland community, Palmares, located inside of the sugar-cane plantation region of the State. No evidence of invasive strains of E. histolytica were found in these inhabitants, provided that the isolated zymodemes I, III, IV, VIII, IX, X, XVII and XVIII are recognized as nonpathogenic strains of E. histolytica. Furthermore, the prevalence of intestinal helminths and other protozoan infections showed that these individuals are infected by other agents responsible for diarrhoeal diseases.

The yeast Saccharomyces cerevisiae was entrapped within polyacrylamide gel beads by employing a procedure that uses sodium dodecylsulfate as a detergent to improve the spherical configuration of the beads. The resulting preparation showed a rate of fumarate bio-conversion to L-malic acid about 60 times higher than that found for the free cells. Almost all fumarate was converted in 30 min of incubation. The thermal stability of the immobilized cells did not significantly differ from the free cells. An optimal pH of 5.7 was found for the immobilized preparation and no succinic acid was detected as a byproduct in the incubation mixture.

Ferromagnetic dacron is proposed as an alternative solid-phase for magnetic enzyme immunoassays. Human serum albumin (HSA) was covalently immobilized onto ferromagnetic dacron and an enzyme immunoassay was developed using anti-HSA rabbit sera. Peroxidase, o-phenylenediamine (OPD) and hydrogen peroxide were used as the enzymatic label and substrates, respectively. Best results were observed when particles of 63-100 microns (diameter) and 10 micrograms of immobilized antigen were used. Positive reactions were detected until dilutions of 1:51200 of immune sera. Its reproducibility was similar to standard ELISA. Disruption of the immunocomplexes formed and recuperation of the immobilized antigen in other immunoassays also proved to be reliable.

The seroprevalence of human T cell leukemia virus type 1 (HTLV-1) infection was investigated in Brazilians (570): native inhabitants (298) and descendants from Japanese (272) living in Recife and its neighborhoods--North-east of Brazil. Furthermore, polytransfused renal transplanted patients (54) were also examined for the serological status to this virus. The seropositivity to HTLV-1, screened by enzyme-linked immunosorbent assay (ELISA), was low: 1.34% for the local population and 0.73% for the descendants from Japanese. However, the seropositivity for the renal transplanted patients was found to be 11.1%. This higher value suggests that this retrovirus infection seems to be of importance in this clinical condition.

The seroprevalence of antibodies against cytomegalovirus (CMV), human herpesvirus 6 (HHV-6) and human T cell leukemia virus type 1 (HTLV-1), determined by ELISA and by fluorescence in 54 renal transplanted patients, was 96.2%, 88.8% and 11.1%, respectively. These values are relatively high when compared with the results obtained for healthy individuals of the same age groups from Recife, Northeastern Brazil. Active CMV infection was detected by the presence of IgM antibodies and/or virus isolation in 13 (24%) patients. Kidney rejection and renal dysfunction were observed in 11 of these 13 patients, whereas 3 of 6 HTLV-1 antibody-positive individuals presented these complications. All HTLV-1 positive patients were also positive to IgG CMV and HHV-6 antibodies. The importance of the three viruses in this clinical condition is suggested by the high seropositivity rates compared with the healthy population. The group may also represent a potential source of HTLV-1 infection in this non-endemic area.

A dot enzyme linked immunosorbent assay (dot-ELISA) was previously developed to detect specific antibodies in rabbits sera immunized against F1A protein obtained from Yersinia pestis. This antigen was covalently linked onto the surface of dacron (polyethyleneterephthalate). Here, standard conditions are described for the optimization of this procedure: an amount of 20 ng of F1A protein was fixed onto dacron; anti-rabbit IgG peroxidase conjugate diluted 1:8,000 and 30% non-fat instant milk as blocking substance were used throughout the method. This procedure was compared with that employing nitrocellulose as solid-phase which showed to be more sensitive. However, the method based on dacron did not show false positive reactions against non-immunized rabbits sera at low antigen amount and diluted anti-IgG peroxidase conjugate.

Dacron (polyethylenetherephthalate) is proposed as a matrix for dot-ELISA procedures, as an alternative to nitrocellulose. Plates of dacron were partially hydrazinolyzed and hydrazide groups introduced were converted to azide groups. The derivative dacron-antigen was covalently linked on to the plates through these azide groups. The derivative dacron-antigen was exaustively washed according to CROOK and antigen was still fixed onto the plates. Protein F1A purified from Yersinia pestis was used as a model. Titration of sera from immunized and non immunized rabbits against this protein was carried out by employing the dot-ELISA method. No significant difference was observed using dacron-antigen and nitrocellulose-antigen preparations. However, both procedures showed to have a significant better performance in comparison with the passive hemagglutination method. The specificity and reproductibility of the dot-ELISA assay using both preparations showed a similar behaviour. Nitrocellulose preparation was stable at 4 degrees C, 28 degrees C and -20 degrees C for 90 days, whereas the dacron-antigen derivative was stable only when stored at 4 degrees C. Dacron-antigen derivative could be re-used when the spot developing was proceeded using 4-chloro-1-naphtol as substrate.

Ferromagnetic Dacron (polyethyleneterephthalate) is proposed as a matrix to immobilize proteins covalently. Dacron in powder was magnetized by reacting ferrous (Fe+2) and ferric (Fe+3) ions with its hydrazide form at pH 8.3. Ferromagnetic hydrazide Dacron was converted to ferromagnetic azide Dacron and amyloglucosidase (E.C. 3.2.1.3) was covalently bound through the latter group. The catalytic property of the enzyme was preserved (8% of the specific activity estimated for the soluble enzyme) and all the magnetic amyloglucosidase Dacron derivative was recovered by using a magnetic field. No activity was detected in the supernatant.

L-Malate was produced from fumarate by using immobilized Saccharomyces cerevisiae cells entrapped in polyacrylamide. This preparation performed better when pretreated with malonate. Under the experimental conditions described here, succinate was not detected as a by-product of the reaction, as had been reported for other microorganisms.

1. alpha-Amylase immobilized on polyamide 11 showed higher specific activity and retention of activity than the derivatives employing polyacrylamide and polyethyleneterephthalate as supports. 2. Polyamide 11 and polyethyleneterephthalate alpha-amylase derivatives exhibited a higher extent of multiple attacks on starch than the water-soluble enzyme whereas the polyacrylamide derivative presented less. 3. The polyamide 11 alpha-amylase derivative acted on amylose-azure in the same way as the water-soluble alpha-amylase.

The determination of azide groups introduced into Dacron cannot be established by the method based on the Fe3+ - N3- complex because the therephthalic groups present in the polymer also react with Fe3+ to form a product with absorbance at 458 nm.

Inosinic acid (IMP) has been prepared by the deamination of adenosine monophosphate (AMP) with an immobilized adenosine (phosphate) deaminase extracted from the snail Biomphalaria glabrata. The enzyme has been immobilized in polyacrylamide beads. The preparation and characterization of this system are described.