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Newly developed active pharmaceutical ingredients (API) often experience low solubility in aqueous media and thus possess poor oral bioavailability. The SmartFilm®-technology is a novel approach to overcome poor solubility. The technique uses commercial paper in which API can be loaded in amorphous state, thus increasing dissolution rate dc/dt and solubility c s when compared to bulk material. However, the preservation of the amorphous state is a prerequisite for an efficient use of the smartFilm-technology and thus the crystalline state needs to be inspected during storage. Preferably, this should be done non-destructively. Traditional techniques, such as x-ray diffraction (XRD) or differential scanning calorimetry (DSC), do not allow for non-destructive crystallinity investigations, whereas Terahertz (THz) spectroscopy is a non-destructive technique, that is sensitive to the crystalline state of many molecular crystals. Therefore, the potential of THz-spectroscopy for crystallinity state inspection of API in smartFilms and tablets made from smartFilms was investigated in this study. The THz results obtained were compared to results obtained from XRD and DSC measurements. Whereas DSC measurements failed to reliably detect crystalline API in the smartFilms, XRD and THz-spectroscopy showed similar results and revealed that it was possible to prepare smartFilms loaded with >23% (w/w) amorphous API. Results indicate the great potential of THz spectroscopy for the non-destructive determination of the crystalline state of APIs in smartFilms and/or tablets made from paper., 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 © 2020 Elsevier B.V. All rights reserved.)

Previously (Glushakova et al. 2017), a cellulose-based cationic (Q) paper derivatized with quaternary ammonium groups was shown to be a convenient platform to collect, preserve, and store nucleic acids (NAs) derived from mosquito vectors infected with pathogens for surveillance. NAs bind electrostatically to Q-paper, but the quantity of NA bound depends on the paper's binding capacity. To optimize the original technology for mosquito surveillance, factors that affected NA absorbance on Q-paper were evaluated. Sixteen variations of Q-paper were prepared with modifications of the derivatizing reagents and derivatization temperature. The binding capacities of these variations were determined first with 1,3,5-benzenetricarboxylic (BTCA), then viral RNA (purified or in infected mosquito samples) was used for validation. For this, samples with Zika (ZIKV) and chikungunya (CHIKV) RNA or virus-infected Aedes aegypti mosquito bodies were applied to sixteen Q-paper variants. Washing the paper samples with water versus elution with aqueous salt (1 M) gave samples that were analyzed for viral RNA by a PCR-based direct Luminex hybridization assay. The comparison ranked the Q-paper binding capacities from the lowest to the highest. The Q-paper with the highest RNA binding capability was further validated with ZIKV- and CHIKV-infected mosquito saliva., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The possibility to compress ordinary paper into tablets was systematically investigated in this study. Results proved that tablets can be made from paper, independent of the type of paper used. The tablets appear shiny and with a smooth surface. The pharmaceutical quality was acceptable, i.e. all tablets fulfilled the requirements for tablets according to the European Pharmacopeia. Drug-loaded tablets were produced by compression of drug-loaded paper. Drug loading did not alter the pharmaceutical quality. However, the uncoated tablets possessed an extremely fast disintegration, i.e. intense swelling upon contact with water, which might hamper the swallowing after oral administration. To avoid swelling tablets were successfully coated with a polymer film, leading to a prevention of swelling but immediate disintegration in simulated gastric fluid. In fact, tablets made from paper are a novel and promising strategy for improved oral drug delivery. They can be easily produced without any further excipients and possess pharmaceutical quality according to the European Pharmacopeia., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The extended frozen semen (EFS) batches produced from infectious bovine rhinotracheitis (IBR) sero-positive cattle and buffalo bulls housed in various semen stations in India are transported to the testing laboratory in liquid nitrogen (LN 2 ) for screening bovine herpesvirus-1 (BoHV-1). This procedure is laborious and poses LN 2 related hazards. An alternative logistics for transportation of samples was investigated. Use of Flinders Technology Associates (FTA ® ) elute card was evaluated for transportation of extended bovine semen to screen BoHV-1 DNA by real-time PCR targeting gB gene and the method was compared with the OIE approved Chelex resin based method. A protocol for extraction of BoHV-1 DNA from FTA ® card spotted with extended semen was optimized. The viral DNA was found to be stable on FTA ® card for at least 28 days when the cards are stored at 4°-37 °C. The analytical sensitivity for the assay was determined using variable dilutions of BoHV-1 spiked semen and positive plasmid harbouring gB gene (97bp) spotted onto FTA ® card and it was found to be 10 0.8 TCID 50 /ml or 100 copies respectively in real-time PCR. The test could detect as low as 10 0.008 TCID 50 /ml or 1 copy of positive plasmid when more number of replicates (n = 6) of the same sample were tested. This sensitivity was found to be comparable to Chelex method and both the methods demonstrated a very strong correlation (r = 0.9774; 95% CI: 0.9620-0.9860) in terms of Ct value (p < 0.0001). The diagnostic sensitivity and specificity of the FTA method in comparison to the Chelex method was 83.08% (95% CI: 71.73%-91.24%) and 93.23% (95% CI: 89.38%-96.01%) respectively when 316 samples were screened by both the methods. The degree of agreement between these two tests was good (Kappa value: 0.738; 95% CI: 0.646-0.829). The method was found to be robust and highly repeatable in inter-assay and intra-assay precision testing. The result suggests that the FTA ® card holds promise as an alternative system for transportation of EFS for downstream screening of BoHV-1 DNA., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Chikungunya virus (CHIKV) represents a growing and global concern for public health that needs inexpensive and convenient methods to collect mosquitoes as potential carriers so that they can be preserved, stored and transported for later and/or remote analysis. Reported here is a cellulose-based paper, derivatized with quaternary ammonium groups ("Q-paper") that meets these needs. In a series of tests, infected mosquito bodies were squashed directly on Q-paper. Aqueous ammonia was then added on the mosquito bodies to release viral RNA that adsorbed on the cationic surface via electrostatic interactions. The samples were then stored (frozen) or transported. For analysis, the CHIKV nucleic acids were eluted from the Q-paper and PCR amplified in a workflow, previously developed, that also exploited two nucleic acid innovations, ("artificially expanded genetic information systems", AEGIS, and "self-avoiding molecular recognition systems", SAMRS). The amplicons were then analyzed by a Luminex hybridization assay. This procedure detected CHIKV RNA, if present, in each infected mosquito sample, but not in non-infected counterparts or ddH 2 O samples washes, with testing one week or ten months after sample collection., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Male specific RNA (F-RNA) coliphages are used as indicators of fecal contamination and for source tracking. However, collecting fecal samples for analysis from remote sites is problematic due to the need for an uninterrupted cold chain to guarantee sample suitability for downstream molecular detection of these coliphages. Here, we investigated the feasibility of using filter paper as a collection and storage vehicle for F-RNA coliphages. Various concentrations (10(1) to 10(4)pfu) of two F-RNA coliphages, MS2 and Qβ, were prepared in lambda buffer or a 10% bovine manure slurry, spotted onto filter paper disks, dried, and maintained at 37 °C for up to 37 days. Nucleic acids were extracted from the spotted filter paper disks at 0, 6, 13, and 37 days post inoculation and analyzed by real time RT-PCR. F-RNA coliphages at concentrations of 10(2)pfu/filter paper unit were readily detected, and only a slight decrease in nucleic acid detection was observed over time. Furthermore, the sensitivity of real time RT-PCR detection of the F-RNA coliphage RNA was similar between the developed filter paper sampling methodology and traditional cold storage. These results indicate that filter paper is a suitable storage and transport medium for F-RNA coliphages when refrigeration is not possible., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Haemorrhagic fever viruses cause emerging infections worldwide, and blood or serum is the main sample used for diagnosis. However, storage and transportation of such samples from remote areas to regional laboratories may be complicated and expensive. In this study, a novel approach was evaluated for the detection of Puumala hantavirus (PUUV) RNA and Rift Valley fever virus (RVFV) RNA. Whole-blood samples spiked with viable virus particles were tested in parallel with clinical samples from patients with acute haemorrhagic fever with renal syndrome (nephropathia epidemica). Individual blood samples were spotted on filter paper, dried, and used for RNA extraction at later time points. PUUV RNA was detected by RT-PCR after storage at room temperature for up to six weeks. In contrast, only low copy numbers of RVFV RNA were detected after 1-2 days even though viable RVFV was eluted from the dried filter papers after the same time. The use of filter paper to collect and store blood samples for PUUV RNA detection is therefore a simple and reliable procedure. This approach might facilitate sampling and analysis of other RNA viruses from human or animal sources and could be used for field studies in remote areas or in developing countries., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Avian influenza viruses (AIVs) infect a wide range of host species including domestic poultry and wild birds; also, AIVs may infect humans in whom some highly pathogenic viruses (HPAIV) may cause acute fatal disease. Accurate laboratory diagnosis of AIV infections requires time-consuming and logistically complex precautionary measures for shipment of specimens or viruses to avoid biohazard exposure. The feasibility was investigated of the Flinders Technology Associates filter paper (FTA® card) for infectivity of AIVs and to preserve viral RNA for detection by RT-qPCR, sequencing and by DNA microarray assay. The infectivity of AIV subtype H6N2 and HPAIV subtype H5N1 was inactivated completely within one hour after adsorption to the FTA card at room temperature. FTA-adsorbed viral RNA remained stable for five months. Swab samples obtained from chickens infected experimentally with H5N1 virus and spotted directly onto the FTA® cards allowed a sensitive and straightforward diagnosis by RT-qPCR. FTA® cards were also suitable for examination of field samples, although AIV RNA was detected with reduced sensitivity in comparison to direct examination of swab fluids. The use of FTA® cards will facilitate safe transport of samples for molecular diagnosis of AIV avoiding the need for an uninterrupted cold storage., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Experiments were conducted to illustrate some of the challenges associated with measuring dynamic MDI aerosols. Experimental HFA-134a solution MDIs containing 8 or 20% ethanol were measured using the Andersen cascade impactor using three different inlets. It was demonstrated that the size distribution of MDI aerosols changes substantially during the measurement process. The measured size distribution was shown to be dependent on the degree of evaporation that has occurred prior to size measurement. Additionally, the degree of evaporation prior to measurement also influences the number of modes present in the measured size distribution. While MDI aerosols appeared to have a separate large particle mode when measured using the U.S. Pharmacopeial induction port ("USP inlet"; [U.S. Pharmacopeia, 1996. Physical tests and determinations <601> aerosols, metered dose inhalers, and dry powders. Pharmacopeial Forum 22, pp. 3065-3095]), the aerosols were shown to be monomodal when measured using a large volume inlet. The apparent large particle mode observed with the USP inlet seem rather to be droplets from the same monomodal distribution that have not fully evaporated. The complex interaction of the MDI plume and inlet configuration was described. Inlet design was shown to influence inlet deposition, measured particle size, and even deposition in the actuator mouthpiece. Inlet deposition was shown to be highly size-dependent with large droplets being collected more efficiently than smaller droplets.

This study was conducted to evaluate the pretreatment effects of different in vivo moxibustion on the permeation of a model high molecular compound, FITC-dextran, with a mean molecular weight of 4 kDa (FD-4), through excised hairless rat skin. Direct or indirect moxibustion (0.10 g moxa) was pretreated consecutively 4 times every 5 min on the abdomen of hairless rats, and the permeation of FD-4 was determined through the excised skin over 8h from 30 min after starting the first moxibustion. This consecutive moxibustion pretreatment showed a significant increase in the skin temperature as well as skin permeation of FD-4 compared with the control group (no moxibustion pretreatment). Quantitative parameters showed an increase in skin temperature and skin permeation: the area under the skin temperature over control temperature-time curve during one burning cycle (5.0 min) (AUCtemp) or the maximum skin temperature during moxibustion (Tmax) and the cumulative amount of FD-4 permeated through skin over 8h (Q8) or steady-state flux were increased by moxibustion pretreatment. Then, the effect of pedestal thickness (distance from the moxa cylinder and skin surface), shape of the moxa cylinder (5mm diameter, 13 mm height or 9 mm diameter, 7 mm height), burning materials (moxa or aromatic incense), pedestal component (paper, potato or ginger) and moxibustion pretreatment method (direct or indirect moxibustion) was evaluated on the AUCtemp or Tmax and Q8 or flux. The amount of protein leached from the skin surface was also determined as an inflammatory index by this moxibustion pretreatment. When the skin temperature was increased to 60 degrees C, the Q8 or flux as well as the amount of protein leached were markedly increased. When the skin temperature was controlled to 42 to 45 degrees C by an adequate selection of pedestal thickness, shape of the moxa cylinder, burning materials, pedestal component and moxibustion pretreatment method, on the other hand, protein leaching remained unaltered, but the Q8 or flux significantly increased with the Tmax. This study thus provides credible evidence that moxibustion pretreatment increases the skin permeation of high molecular compounds.

Recent reports from Japan implicated wild Sika deer (Cervus nippon) in the zoonotic transmission of hepatitis E to humans. Seroprevalence studies were performed to determine if imported feral populations of Sika deer in Maryland and Virginia posed a similar risk of transmitting hepatitis E virus (HEV). Hunters collected blood on filter paper discs from freshly killed deer. The discs were desiccated and delivered to a collection point. The dried filters were weighed to estimate the amount of blood absorbed and were eluted and collected in one tube via a novel extraction system. The procedure was quantified and validated with negative and positive serum and blood samples obtained from domestic Sika deer before and after immunization with HEV recombinant capsid protein, respectively. None of the 155 tested samples contained antibody to HEV, suggesting that Sika deer in these populations, unlike those in Japan, do not pose a significant zoonotic threat for hepatitis E. However, the new method developed for collecting and eluting the samples should prove useful for field studies of many other pathogens.

The combination of Flinders Technology Associates filter papers (FTA cards) and real-time PCR was examined to establish a simple and rapid technique for the detection of porcine reproductive and respiratory syndrome virus (PRRSV) from whole pig blood. A modified live PRRS vaccine was diluted with either sterilised saline or pig whole blood, and the suspensions were applied onto the FTA cards. The real-time RT-PCR detection of PRRSV was performed directly with the samples applied to the FTA card without the RNA extraction step. Six whole blood samples from at random selected piglets in the PRRSV infected farm were also assayed in this study. The expected PCR product was successfully amplified from either saline diluted or pig whole blood diluted vaccine. The same PCR ampliocon was detected from all blood samples assayed in this study. This study suggested that the combination of an FTA card and real-time PCR is a rapid and easy technique for the detection of PRRSV. This technique can remarkably shorten the time required for PRRSV detection from whole blood and makes the procedure much easier.

This study evaluates the feasibility of the use of the FTA Gene Guard System (a commercial product consisting of filter paper impregnated with patented chemicals supplied by the Whatman company) for the shipment, storage and detection of RNA rabies viruses by a simplified hemi-nested reverse transcriptase polymerase chain reaction. HnRT-PCR of the rabies virus nucleoprotein gene with specific primers showed that viral RNA extracted from crude infected tissues remained stable after fixation on the filter paper under diverse environmental conditions for at least 35 days. The sequence analysis of the products amplified from five out of the seven known genotypes of Lyssaviruses showed the stability of viral RNA viruses after fixation on the filter paper. Furthermore, the sensitivity of the hnRT-PCR following RNA fixation on the filter paper was equivalent to that of standard hnRT-PCR. In conclusion, the stability of viral RNA and the inactivation of infectivity make the FTA technology useful for the storage, transport, collection and subsequent molecular analysis of viral rabies RNA, facilitating epidemiological investigations in the field.

Leaf samples from 155 maize streak virus (MSV)-infected maize plants were collected from 155 farmers' fields in 23 districts in Uganda in May/June 2005 by leaf-pressing infected samples onto FTA Classic Cards. Viral DNA was successfully extracted from cards stored at room temperature for 9 months. The diversity of 127 MSV isolates was analysed by PCR-generated RFLPs. Six representative isolates having different RFLP patterns and causing either severe, moderate or mild disease symptoms, were chosen for amplification from FTA cards by bacteriophage phi29 DNA polymerase using the TempliPhi system. Full-length genomes were inserted into a cloning vector using a unique restriction enzyme site, and sequenced. The 1.3-kb PCR product amplified directly from FTA-eluted DNA and used for RFLP analysis was also cloned and sequenced. Comparison of cloned whole genome sequences with those of the original PCR products indicated that the correct virus genome had been cloned and that no errors were introduced by the phi29 polymerase. This is the first successful large-scale application of FTA card technology to the field, and illustrates the ease with which large numbers of infected samples can be collected and stored for downstream molecular applications such as diversity analysis and cloning of potentially new virus genomes.

Whole blood dried onto filter paper constitutes a potentially useful material for molecular testing of viruses, including dengue. In order to assess the stability of viral RNA, we carried out dengue-RNA detection in whole blood infected with dengue virus that had been previously spotted onto filter paper. Filter papers were stored at room temperature, 4 and -70 degrees C and processed for PCR assay at intervals of 2, 4, 6 and 9 weeks. Our results demonstrated that dengue-RNA was stable in filter paper for 9 weeks at all tested temperatures. Furthermore, we evaluated these conditions using frozen sera and dried blood samples onto filter paper from 52 patients with confirmed clinical diagnosis of dengue infection. PCR results showed a 100% specificity and 93% sensitivity for dried blood samples. This storage method facilitates the transportation and analysis by nucleic acid amplification techniques even when freezing conditions are not available.

Noroviruses are the most common cause of nonbacterial gastroenteritis outbreaks. A novel method was tested that utilizes sodium dodecyl sulfate (SDS)/EDTA-pretreated chromatography paper strips to collect and transport norovirus-containing fresh stool samples. As confirmation, the strips were infected with different dilutions of human norovirus-containing faeces and stored at different temperatures (-80, -20, 4 degrees C, room temperature and 37 degrees C). Reverse transcription (RT)-PCR analysis was carried out after storage of the strips for 1, 7, 14, 60, and 120 days. The presence of amplifiable norovirus RNA on the strips was inversely correlated with the duration of storage and the storage temperature. Norovirus RNA remained detectable for a sufficient amount of time at room temperature or even in warmer climatic conditions to allow transportation of the samples from a field site to a reference laboratory using regular postal mail. To prove that viral infectivity was lost upon contact with the SDS/EDTA-treated strips, feline calicivirus F9 (10(7) TCID(50)/ml) was loaded onto the strips. After elution and dialysis, no cytopatic effect could be observed upon incubation on Crandell-Reese feline kidney cells. The SDS/EDTA-pretreated chromatography filter paper strips are an inexpensive, biosafe and adequate method to collect, transport and store norovirus samples.

This paper describes a suite of routines using IgorPro, a powerful analysis and graphing software package for the Macintosh computer, to enhance the ability to analyze, manipulate, and display data recorded with the Discovery acquisition software marketed by DataWave Technologies. The routines are able to time-align fast and slow data channels, and are especially useful for analyses that involve both neural and behavioral data. The software was designed for eyeblink conditioning and vocalization experiments, but it can easily be used for analyzing other types of neurobehavioral data. The data are first prepared on the PC with routines that inspect the header of the data file and translate the data file into a compact binary format that can be read by IgorPro. An option is also available to splice out data from unnecessary portions of an intertrial interval. The new file is then put on the Macintosh computer for display and analysis by IgorPro. These routines enable both neural and behavioral data to be quickly and easily reduced, manipulated, and statistically and graphically summarized.

Immunity to measles virus, rubella virus, and mumps virus was determined by EIA in serum samples and in dried whole blood specimens spotted on Whatman filter paper (5 mm in diameter). Both specimens were obtained from each patient by venepuncture and finger prick. Ten microliters of whole blood is enough to detect antibodies to these three different viruses. The comparison of the results obtained by EIA from 227 serum and whole blood samples have demonstrated close agreement: 98.6% for measles virus, 99.1% for rubella virus, and 96.0% for mumps virus. Moreover, 96 whole blood samples can be tested in a microtiter plate and can be stored at room temperature for 15 days or at +4 degrees C for several months. Therefore, whole blood dried on filter paper is a convenient alternative method for collecting and transporting specimens, it is easier and safer than venepuncture, and could be used for large-scale epidemiological studies, especially in newborns. This method could solve the problem of sampling, especially in young children, and could simplify studies of vaccine efficacy.

A simple and inexpensive technique is described for the assessment of regional cerebral blood volume (CBV) using a color reaction based on the pseudoperoxidase activity of hemoglobin. Cryostat sections are placed on a commercially available reagent strip originally designed for quantitative estimation of blood content in urine. After melting of the brain section, a blue color reaction appears which is darker in areas with higher hemoglobin content. The image is analyzed by a computerized image-processing system and evaluated by standards prepared from brain homogenate with different hemoglobin content. From the optical density data, regional CBV is calculated with correction for the difference between microvascular and large vessel hematocrit. The method was validated by a radiolabel procedure. Values of regional CBV are reported in gray matter areas of control rabbits and in brains embolized by autologous clot. This simple inexpensive technique is a useful supplementary tool in multiparametric metabolic studies.

To detect human cytomegalovirus (HCMV) DNA, filter paper spotted with peripheral blood from newborns 4 to 7 days after birth was dried and subjected to the polymerase chain reaction (PCR) followed by Southern blot hybridization with a nonradioactive oligonucleotide probe. The detection rates were 25.1% in healthy individuals and 33.0% in low body weight neonates weighing not more than 2500 g at birth, most of whom appeared to have been infected transplacentally or by other means within the uterus. HCMV was detected after only heating the dried blood on the filter paper, and may be applied as a screening method for the early diagnosis of HCMV.

Synthetic peptides have been used to define specificity determinants and to distinguish reactivities of numerous protein kinases and phosphoprotein phosphatases. Direct analysis of peptide phosphorylation is most often determined using P81 phosphocellulose paper to separate modified peptide and unreacted [gamma-32P]ATP; however phosphopeptide dephosphorylation is usually determined by extraction and quantitation of phosphomolybdate complexes or ion exchange chromatography. We describe here the adaptation of the rapid, direct P81 paper protein kinase assay for the determination of phosphopeptide dephosphorylation. The S6-21 peptide (AKRRRLSSLRASTSKSESSQK), which is derived from the multiphosphorylated carboxyl terminal domain of the S6 ribosomal protein, was phosphorylated by a human placenta S6 kinase and dephosphorylation by purified phosphoprotein phosphatase type 1 in the presence of a variety of buffers, and inhibitors/activators was determined using the new assay. Results comparable to those obtained with the ion-exchange chromatography were obtained, and the assay was significantly less expensive, more rapid, and more accurate than methods previously used to quantitate phosphopeptide dephosphorylation.

Walking track analysis is an investigative technique that allows a researcher to assess objectively the functional capacity of the limb in the rat sciatic nerve model. This study has shown that paint and paper is a better method for imaging the rat foot plantar surface than other methods described in the literature. The block printing paint has the distinct advantage of keeping radial diffusion error to a minimum. Paint more correctly images the important anatomical landmarks of the rat plantar surface. There is better traction (less slippage) rendering the prints easier to interpret. The authors conclude that the paint and paper is a superior method to the other methods currently available.

Kinetoplast DNA (kDNA) has been isolated from the human cutaneous Leishmania isolates, L. tropica major, L. aethiopica and an unknown Kenyan isolate, Leishmania SP48. DNA sequence relationships among these isolates have been studied by restriction enzyme digestion and two phase hybridisation to Southern blots of kDNA covalently coupled to diazobenzyloxymethyl (DBM) paper. The results of this analysis confirm that rapid kDNA sequence evolution is occurring in the Old World leishmanias although some sequence conservation in defined regions of the mini-circle sequence is present. These results emphasise the danger of constructing a rigid Leishmania classification on buoyant density data alone. The covalent binding of kDNA electrophoretic separations to DBM paper permits the construction of a DNA sequence "library' which can be used in the classification and diagnosis of unknown Leishmania isolates.

A method for elution of HIV antibodies from whole blood or serum impregnated filter paper discs was developed. The results from testing of 73 eluates in an enzyme linked immunosorbent assay and the immunoblotting test agreed with the results obtained by ordinary serum testing. Significant loss of antibody activity was not observed, neither in the eluates after storage for 1 mth at -20 degrees C nor in the filter paper discs after storage for 3 mths at +4 degrees C. This technique may be useful in facilitating sample collection and transportation, particularly in remote areas of the world.

A blot-immunobinding test was used to detect anti-sperm antibodies in human sera and to identify the corresponding auto- or iso-antigens on human sperm. A high proportion of sera at a 1:100 dilution from fertile persons, as well as infertile patients, contains antibodies reactive with sperm. This phenomenon might be physiological. At 1:2,000 dilution, a higher binding capacity was detected in the sera from infertile groups, but a few fertile persons were also positive. Antibodies to a single antigenic determinant with Mr of approximately 14,000 were found in a significantly higher proportion among males with unexplained infertility.

Considerable controversy exists concerning the origin and composition of Alzheimer neurofibrillary tangles (ANT) and of paired helical filaments (PHF), the abnormal cytoplasmic fibers which ultrastructurally are the major components of ANT. Thus far, the unusual solubility properties of PHF have hindered the analysis of ANT. A new procedure is presented for isolating purified PHF which are soluble in the presence of sodium dodecyl sulfate. The purification protocol involves differential and rate zonal centrifugation, treatment with the detergents sarcosyl and sulfobetain 3-14, and sonication. The isolated PHF from Alzheimer disease/senile dementia of the Alzheimer type (7 cases) and Down's syndrome (one case) have been characterized structurally by negative-stain electron microscopy, biochemically by PAGE, and immunologically by both the ELISA technique and Western blot analysis using a monoclonal antibody prepared against ANT. Distinct polypeptides were shown to be associated with this structure and not seen in preparations from young and age-matched normal brains.

The ability to collect whole blood directly onto filter paper pre-cut to the size used in the dot-immunobinding assay (DIA) is a practical adjunct to this procedure. Its applicability for field studies is suggested.