Erythrocyte test preparations for standardizing fluorescent antibodies and the serological diagnosis of influenza

An erythrocytic diagnostic preparation, obtained by the sandwich method and intended for the standardization of antivirus fluorescent antibodies (FA) and the determination of specific IgM in the blood sera of patients with a view to the early serological diagnosis of influenza, has been proposed. The loading of immunosorbents with influenza virus, achieved in this preparation, approaches its theoretical limit, the virions being arranged in a single layer, and constitutes up to 0.4 hemagglutinating units per 1000 red blood cells. The investigation made on 9 commercial and 4 experimental lots of FA has demonstrated that the newly developed test preparations serve as a convenient model for the standardization of FA and the choice of their optimum working dilution in practical diagnostic procedures. In the study of 153 paired blood sera from patients with acute respiratory diseases and clinically healthy persons the proposed variant of the early diagnosis of influenza has proved to be not inferior to the hemagglutination inhibition test and the complement fixation test with respect to the main characteristics of their diagnostic value, while having the advantage over these tests due to the fact that exact laboratory diagnosis can be made by the study of single blood samples as early as on the fourth day of the disease.     

Serological diagnosis and immunological aspects of Proteus infection. V. Design and trial of polyvalent antigenic preparations

The main principles of the development of Proteus polyvalent antigen and erythrocyte diagnosticum, including the selection of the initial strains according to the clinical importance of their H-antigens and to the variety of their partial factors, the combination of monoantigens to form a polyvalent antigenic preparation with due regard to the potency of each component and the use of the preparation in serological tests in accordance with its summary potency, as well as the simultaneous loading of formaldehyde-fixed and tannin- or bisdiazo benzidine-treated sheep red blood cells with sensitins, have been worked out. The diagnostic value of these preparations has been confirmed by the study of serum samples from 579 patients with intestinal and urological infections (among them 153 patients releasing Proteus) and 245 healthy persons.     

Evaluation of an ELISA and immunoblotting for studying the humoral immune response in Anguillicola crassus infected European eel Anguilla anguilla

The applicability of an enzyme-linked immunosorbent assay (ELISA) for the detection of anguillicolosis in feral eels was examined using a crude antigen preparation from the body wall of adult Anguillicola crassus. The screening consisted of samples from 100 feral European eels Anguilla anguilla. As a reference the actual status of infection was determined by dissection of the eels' swim-bladders. The ELISA results were compared with a background value calculated from the results obtained from 43 non-infected farm eels. The screened samples had a high prevalence of A. crassus (83 %); however, the specificity and the negative predictive value of the ELISA were low compared to the high positive predictive value. Nonetheless, the reproducibility (precision) of the test was satisfactory, and for the non-infected reference group specificity was 97.7 %. Although the ELISA, as used in the present study, is not applicable for diagnostic purposes, it represents a useful tool for the investigation of the specific humoral immune response of eels against A. crassus under controlled experimental conditions. Immunoblots using crude antigen preparations from different parts of adult A. crassus as well as a crude somatic third-stage (L3) antigen preparation illustrated that only antigens associated with the body wall of adult A. crassus are potentially suitable for diagnostic purposes. Despite the fact that antibodies against Raphidascaris acus cross-reacted with 3 body wall antigens of A. crassus, the most encouraging results were obtained with the antigen preparation from the outer cuticle of adult A. crassus which yielded a conspicuous, broad band at about 100 kDa.     

Indirect hemagglutination test at the ultrastructural level]

A study on ultra-thin sections was made of the preparations of agglutinate produced during the reaction of the immunoglobulin erythrocytic diagnostic agent with dry corpuscular Rickettsia prowazeki antigen, fluoresceine isothiocyanate labeled, and also SRBC used for the preparation of the diagnostic agent after formalinization, tannin treatment, sensitization with hyperimmune horse serum immunoglobulins and lyophilization, respectively. Formalin and tannin treatment of erythrocytes failed to be reflected on the ultrastructure of their cellular membranes; the treatment with hemosensitin was accompanied by the appearance of spheroid protrusions of the erythrocyte cytoplasmic membrane with the preservation of its three-layer structure. Specific interaction of sensitized erythrocytes with the antigen corpuscles was expressed morphologically in their apposition or connection through a gap of 20--30 nm.     

Effect of sorbed water on the thermal stability of soybean protein

A soybean protein isolate (SPI), and its beta-conglycinin and glycinin componets were obtained from defatted soybean flour by applying dissolution and precipitation based on the difference in their solubility depending on each isoelectric point. The purity evaluated by SDS-PAGE of the beta-conglycinin and glycinin preparations was about 84% and 80%, respectively, resulting in a clear difference in the pH dependence on solubility. A BET plot derived from the water sorption isotherm at 25 degrees C showed that the amount of the monolayer adsorption of these preparations was about 6-9%, the value for the beta-conglycinin preparation being about 1.5 times higher than that for the glycinin preparation. The beta-conglycinin and glycinin preparations were respectively denatured at around 75 degrees C and 86 degrees C in the presence of excess water, whereas the denaturation temperature of both preparations was markedly increased by decreasing sorbed water content below 40%, corresponding well with the unfrozen water content.     

Effect of Sorbed Water on the Thermal Stability of Soybean Protein

A soybean protein isolate (SPI), and its β-conglycinin and glycinin componets were obtained from defatted soybean flour by applying dissolution and precipitation based on the difference in their solubility depending on each isoelectric point. The purity evaluated by SDS–PAGE of the β-conglycinin and glycinin preparations was about 84% and 80%, respectively, resulting in a clear difference in the pH dependence on solubility. A BET plot derived from the water sorption isotherm at 25 °C showed that the amount of the monolayer adsorption of these preparations was about 6–9%, the value for the β-conglycinin preparation being about 1.5 times higher than that for the glycinin preparation. The β-conglycinin and glycinin preparations were respectively denatured at around 75 °C and 86 °C in the presence of excess water, whereas the denaturation temperature of both preparations was markedly increased by decreasing sorbed water content below 40%, corresponding well with the unfrozen water content.     

Infrared spectra of the Gramicidin A transmembrane channel: the single-stranded-beta 6-helix

IR spectra are reported for preparations of Gramicidin A and malonyl Gramicidin A incorporated as the channel state in phospholipid structures. In this preparation Gramicidin A has already been shown to be unequivocally in the single-stranded beta-helical conformation. The result is an amide I frequency of 1633 +/- 1 cm-1. This demonstrates that the single-stranded beta-helix has an amide I frequency that has previously been considered to be diagnostic of antiparallel double-stranded beta-helix and of beta-sheet structures.     

The value of multiple preparations in the diagnosis of malignant pleural effusions. A cost-benefit analysis

A cost-benefit analysis of five techniques employed in processing 108 malignant pleural effusions for cytopathologic examination was performed. Ethanol-fixed, Papanicolaou-stained smears were positive in 68% of the effusions, air-dried Diff-Quik-stained smears in 66%, Cytospins in 83%, cell blocks in 85% and Millipore filters in 85%. Examination of one air-dried smear and one ethanol-fixed smear yielded a diagnostic sensitivity of 82%. Using a combination of two smears and one of three concentrating techniques (Cytospin, cell block or MIllipore filter) would have provided a diagnosis in over 90% of the effusions. The use of four or more preparations provided more sensitivity than did three preparations and decreased the likelihood that a malignant diagnosis would be based on the findings in only one preparation. The costs related to disposable materials and the College of American Pathologists (CAP) work load estimates for specimen preparation, staining and screening were compared. These data may be useful in developing optimal protocols for pleural effusion preparation in laboratories with specific work load requirements and limited resources.     

Fluorescence properties of actin and analysis of the content of native actin in its preparations]

Changes of properties of actin preparations from rabbit skeletal muscles in the course of purification were studied. It is shown that independent on initial properties of actin preparations at successive polimerization, sedimentation and depolimerization cycles: 1) the quantity of protein in supernatant diminishes progressively, 2) intrinsic viscosity of F-actin increases, 3) the value of spectral parameter A = (I320/I365)296., which in characteristic of the fluorescence spectrum position of tryptophan residues, increases and approaches the extremal value, 4) the effect of short wave shift of the spectrum at actin polimerazation becomes more pronounced. The actin preparation with the extremal value of A = 2,6 (native actin) has [eta] = 8,8; deltaAg leads to f approximately 0,25; lambdamax=325 nm. Inactivation of actin results in the long-wave shift of fluorescence spectrum (lambdamax=337 nm, A = 1,30) suggesting the disturbance of exclusively compact globular structure of native protein macromolecular. The ratio is described which enables to use parameter A for the quick estimation of the content of native actin in its preparations. The technical simplicity of the measurement of parameter A enables to use it for the characterization of individual fractions in gelfiltration of actin preparations.     

A simplified method of rapid hemagglutination test for determining the levels of tetanus antibodies]

A new, simplified methodology of preparation of lyophilized chrome-coated turkey blood cells (formalin-treated ) for rapid identification of tetanus antibodies in indirect hemagglutination test is described. Blood cells diagnostic preparations obtained in this way were easier and three times faster to prepare than tanned cell preparations . They maintained unchanged capability for specific agglutination during at least one year of storage when kept at 4 degrees C. proposed methodology enable to start a production of laboratory kits necessary for controlled prophylaxis of tetanus.     

Comparison of ThinPrep and conventional preparations on fine needle aspiration cytology material

OBJECTIVE: To compare the various cytologic features on ThinPrep 2000 (TP) (Cytyc Corporation, Marlborough, Massachusetts, U.S.A.) and conventional preparation (CP) specimens from fine needle aspiration cytology (FNAC) material by a semiquantitative scoring system. STUDY DESIGN: In this prospective study a total of 71 consecutive cases were included. In each case, two passes were performed. The first pass was used for conventional preparations, with direct smears made and fixed immediately in 95% alcohol for Papanicolaou stain. For TP preparation a second pass produced material for processing in the ThinPrep 2000. The TP and CP slides were studied independently by two observers and representative slides of CP and TP compared for cellularity, background blood and necrotic cell debris, cell architecture, informative background, presence of monolayer cells, and nuclear and cytoplasmic details by a semiquantitative scoring system. Statistical analysis was performed by Wilcoxon's signed rank test on an SPSS program (Chicago, Illinois, U.S.A.). RESULTS: TP preparations contained adequate diagnostic cells in all cases and were tangibly superior to CP preparations concerning monolayer cells, absence of blood and necrosis, and preservation of nuclear and cytoplasmic detail (statistically significant, Wilcoxon's signed rank test, P < .000). CONCLUSION: TP preparations are superior to conventional preparations with regard to clear background, monolayer cell preparation and cell preservation. It is easier and less time consuming to screen and interpret TP preparations because the cells are limited to smaller areas on clear backgrounds, with excellent cellular preservation. However, TP preparations are more expensive than CP and require some experience for interpretation.     

Application of neutral-alkaline oectate lyases to cotton fabric boil off

Commercial and pilot pectate lyase preparations (EC 4.2.2.2) have been compared. They differ in their effect on pectins with different esterification degrees (ED). The activity of the pilot preparation with respect to a substrate with ED = 70% is tenfold lower than with respect to unesterified polygalacturonic acid. For commercial preparations, this activity ratio ranged within 1.5–2. At equal pectate lyase activities, the commercial preparations better remove pectin from crude cotton fabric during its boil off. The laboratory preparation is more efficient for improving the capillarity (wettability) of the fabric owing to the cooperative effect of the pectate lyase, cellulase, and hemicellulase present in the preparation.     

Diagnostic value of clot examination for malignant cells in serous effusions

Objective:  To assess the diagnostic value of clot examination for satisfactory processing and confirmation of malignancy in serous effusions in routine cytological evaluation and compare the results with those of conventional smear and cell block preparations.
Methodology:  Body cavity fluids ( n  = 600) received in our laboratory were processed according to a pre-designed protocol for the study as follows: Day1: on receipt of the specimen, smears were made and a cell block was prepared from the sediment. Day2: after overnight sample storage of the remaining specimen at 2–8 °C all fluids were examined for the presence of a clot at the bottom of the container. Fluids in which clot had formed were fixed in formalin. The clot was then placed on a lens paper, wrapped and processed routinely. Diagnostic yields were compared.
Results:  In this study, we included 600 cases of serous fluids from pleural, pericardial and peritoneal effusions. In 73% ( n  = 437) of samples, clot formation was seen, while in 27%, ( n  = 163) no clot had formed. Routine smear and cell block preparations showed malignant cells in 9.6% ( n  = 42). However, with the addition of the clot preparation, the number of cases in which atypical/malignant cells were seen increased from 42 to 85 (19.4%), with a P  < 0.001. Special stains and immunohistochemistry (IHC) were also performed on clot preparations in 10 difficult cases.
Conclusion:  Clot preparation from body cavity fluids on the second day can be used as an adjunct to smear and routine cell block preparation to improve the accuracy and yield of the cytological diagnosis and may also be of great help for special studies such as IHC staining.     

Improved isolation procedures for the purple membrane of Halobacterium halobium.

Techniques for purifying teh purple membrane of Halobacterium halobium are given. This purple membrane contains a chromoprotein with a retinal prosthetic group similar to rhodopsin, the chromprotein found in the visual systems of higher invertebrates and vertebrates. The described purple membrane isolation procedures yield a highly purified preparation as determined by transmitting electron microscopy and gel electrophoresis. Critical analysis of the absorption spectra of the purple membrane was also employed to establish criteria of purity for the preparation. The visible absorption spectra of the purified purple membrane preparation in buffer was found to have a maximum at 559 nm which shifted to 567 nm on light exposure. No indication of any spectral perturbation arising from bacterioruberin-containing membrane, the major contaminant in purple membrane preparations, was found. Furthermore, the ratio of protein aromatic amino acid absorbance at 280 nm to chromophore absorbance at 567 nm was found to be 1.5 in light-exposed preparations compared to the previously reported ratio of 2.3.-3 The decrease in the value of this ratio is also indicative of an increase in the purity of the purple membrane preparation.     

Application of neutral-alkaline pectate lyases to cotton fabric boil off

Commercial and pilot pectate lyase preparations (EC 4.2.2.2) have been compared. They differ in their effect on pectins with different esterification degrees (ED). The activity of the pilot preparation with respect to a substrate with ED = 70% is tenfold lower than with respect to unesterified polygalacturonic acid. For commercial preparations, this activity ratio ranged within 1.5-2. At equal pectate lyase activities, the commercial preparations better remove pectin from crude cotton fabric during its boil off. The laboratory preparation is more efficient for improving the capillarity (wettability) of the fabric owing to the cooperative effect of the pectate lyase, cellulase, and hemicellulase present in the preparation.     

Usefulness of ELISA and radial immunodiffusion tests for evaluation of the degree of purification of influenza diagnostic and vaccine preparations

It is necessary to use new diagnostic tests for careful and rapid evaluation of a degree of purification and immunogenicity of vaccine anti-influenza preparations. In this study in order to obtain this purpose a radial immunodiffusion++ test and immunoenzymatic test (ELISA) were used Recommended by WHO radial immunodiffusion++ test enable to determine a level of haemagglutinin of particular types and subtypes of influenza virus in polyvalent preparations. However, this test is time consuming therefore for hemagglutinin level determination ELISA test was adapted. This test is hundred times more sensitive and can be applied with success for determination of hemagglutinin level of influenza virus A or B. For evaluation of a degree of purification of vaccine preparations ELISA was elaborated, in which as an index of purification of preparation a level of ovalbumin is determined. This test is specific and extremely sensitive, and it is possible to determine ovalbumin level with accuracy of 1ng in 1 ml of preparation.     

Fluorescence anisotropy from diphenylhexatriene in rat liver plasma membranes

Rat livers were fractionated to obtain intracellular membrane preparations and a highly purified preparation of bile canaliculi. The fraction containing bile canaliculi was homogenized and subfractionated to give fractions representing fragments of contiguous membrane and of canalicular microvilli. The relative purity and extent of contamination of each preparation was determined. When the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene was incorporated into aliquots of each fraction at the same probe: lipid ratio and the steady-state anisotropy of its fluorescence measured, it was found that the plasma membrane preparations were much more ordered than the intracellular membrane preparations. Of the plasma membrane preparations, that containing the canalicular microvilli was the most ordered, even allowing for any contribution of contaminants. Thus the microvillus membrane of the bile canaliculus appears to be the most ordered domain of the plasma membrane of the hepatocyte. The high order in this domain may be a factor in reducing the susceptibility to bile salt damage during bile secretion, since it is this region which is exposed to high concentrations of bile salts in vivo.     

Increasing diagnostic accuracy with a cell block preparation from thin-layer endometrial cytology: a feasibility study

OBJECTIVE: To investigate (1) the feasibility of preparing cell blocks by inverted filter sedimentation (IFS-CB) from endometrial samplings processed by the ThinPrep (TP) technique (Cytyc Corp., Boxborough, Massachusetts, U.S.A.), and (2) the possibility of increasing the diagnostic accuracy of TP endometrial cytology by examining the tissue architecture as an adjunctive method of detecting endometrial lesions. STUDY DESIGN: Three hundred one endometrial samplings were obtained, using the Endogyn endometrial device (Biogyn S. n.c., Italy), from perimenopausal and postmenopausal women. The endometrial samplings were collected in a vial with liquid fixative for the TP processing. One TP slide was prepared from each case. If adequate material remained in the vial after the TP slide preparation, it was processed for IFS-CB preparation. RESULTS: IFS-CB preparation was processed in 263 cases (87%) with adequate material. Diagnoses on IFS-CB preparations obtained by endometrial sampling matched those of the hysterectomy specimens. The addition of IFS-CB histology to the cytologic diagnosis by TP increased the diagnostic accuracy of endometrial cytology to 96.3% and 100% for benign/atrophic endometrium and adenocarcinoma, respectively (p = 0.39 and 0.46). In hyperplasia without atypia and hyperplasia with atypia, the diagnostic accuracy increased significantly, to 96% and 95.3%, respectively (p = 0.037 and < 0.001). CONCLUSION: This study illustrates the merit of linking TP cytology with direct endometrial sampling, including small tissue fragments and material adequate for IFS-CB preparation. TP cytology provides an accurate cytologic diagnosis and the possibility of IFS-CB preparation, which could be a valuable diagnostic adjunct to TP cytology.     

Preparation of a stable fresh frozen primary lipoprotein[a] (Lp[a]) standard

LP[a] is one of the most atherogenic lipoproteins consisting of an LDL-like core particle and a covalently linked glycoprotein of variable size. Due to its structural features, its heterogeneity and instability, there are great difficulties in standardizing quantitative immunochemical Lp[a] assays. One particular problem is the preparation of a pure primary standard, which is sufficiently stable to be used for value assignment of secondary reference material. Here we describe a method to purify Lp[a] to virtual homogeneity. When mixed with glycerol at a ratio of 1:1, the preparation is stable in the deep frozen state for more than 12 months. This latter material gave dose;-response curves in several immunochemical assays that were parallel to fresh or frozen sera, freshly prepared Lp[a], and other proposed reference materials. After determination of the protein content by amino acid analysis, it was possible to assign concentrations in molar and mass units to these preparations considering the theoretical molecular weights of the particular apo[a] isoform. Thus we propose to use this procedure for preparation of a "gold standard" for Lp[a] assays.     

Serologic diagnosis of intestinal yersiniosis. The design of a stable erythrocyte preparation for the indirect hemagglutination reaction

Nine types of erythrocyte diagnostica of serovars O3 and O9, differing in the methods of obtaining sensitins and the physical state of erythrocytes, were put on trial. The preparations were used for the titration of hyperimmune sera and blood sera obtained from about 500 healthy persons, 300 patients with Yersinia enteric infection and 300 patients with other diseases. Freeze-dried diagnostica, when compared with liquid ones, were found to be less sensitive, but more stable and specific. Sensitins isolated by the methods of Westphal ad Boivin showed the highest degree of specificity. The authors believe freeze-dried sheep red blood with activated Boivin's antigen adsorbed onto them to be the optimal preparation for use in the passive hemagglutination (PHA) test. The preparation was found to retain its serological activity for as long as 2-3 years. The titer 1:160 (1:200) in the PHA test is recommended as the minimal diagnostic indicator. Erythrocyte diagnosticum is more sensitive, specific and stable than bacterial one. Since 1984 dried Yersinia erythrocyte diagnostica (serovars O3 and O9) have gone into quantity production at the Leningrad Research Institute for Vaccines and Sera.