An improved double antibody radioimmunoassay method for the measurement of ragweed antigen E

Antigen E, the principal antigen of short ragweed pollen, usually has been measured by immunodiffusion or, more recently, by radioimmunoassay. Several technical improvements were made in the radioimmunoassay method which allow the test to be completed in less than a day and which increase sensitivity to 30 picograms per ml of antigen E.     

An improved procedure for accurate assays of factor VIII.

An improved one-stage method for accurate assays of factor VIII combines highly reproducible end points with elimination of temporal drift and of subjective factors involved in graphic analysis. Activated deficient plasma substrate (ADPS) is used as a single reagent for parallel tests. The assays can be performed manually or, with much greater precision, on an 8-channel coagulation meter of new design in which end points are recorded automatically and depend on an abrupt clearing of agitated cloudy suspensions. The coagulation time readings are reproducible to 0.5%. Factor VIII levels are read off a standard table or computed from a general equation which is readily programmable on a pocket calculator and is valid over a wide range of concentrations, including very low plasma levels.     

An oil-well method for time-resolved microfluorescence assays

A simple fluorescence assay method has been devised for direct time-resolved recordings of NADH-coupled enzyme reactions in a microliter sample volume in an oilwell. The apparatus allows practically instantaneous mixing and stirring of two droplets containing the enzyme and substrate, respectively, and which are viewed with a microscope spectrophotometer. This procedure can be used for instance for enzyme kinetic studies of ATPases on a microscale. Thus, by monitoring NADH-fluorescence changes we determined the time course of ATP splitting in nanogram quantities of myosin subfragment 1. In principle, the method can also be used to determine the time course of any other reaction causing fluorescence changes or luminescence changes.     

An improved method for the detection of IgE antibody of defined specificity by ELISA using rat monoclonal anti-IgE antibody

An improved method for the detection of IgE antibody of defined specificity by ELISA using rat monoclonal anti-IgE antibodies is described. The innovation consists of coating the plates first by a monoclonal rat anti-murine IgE antibody, adding the sera to this antibody-coated plates and then adding the biotin-conjugated antigen after the sera. The plates are then reacted with streptavidin-peroxidase and developed. This procedure eliminates possible competitions with other isotypes of the same specificity. The method is useful especially to quantitate IgE with defined specificity in the presence of high amounts of isotypes of the same specificity.     

An improved method for purification of lymphocytes

A method that combines glass-bead column filtration, Ficoll-Hypaque gradient separation, discontinuous sucrose gradient, and drastic reduction of cell transfers is described. The procedure gives a high yield of pure human lymphocytes from small amounts of blood, good preservation of B cell/T cell ratio, and sufficient material for subsequent biochemical studies.     

Multicenter clinical trial and laboratory utilization of an enzymatic detection method for gonococcal antigens

Seven institutions participated in a comparative study evaluating standard culture method and a new enzyme immunoassay (EIA, Gonozyme, Abbott Diagnostics) for the detection of Neisseria gonorrhoeae. Five hundred twenty-three patients were entered from hospitals of various sizes representing different population densities, ethnic and economic sectors, and gonococcal prevalence. Statistical analysis showed sensitivity, specificity, and positive and negative predictive values of 80.3%, 94.6%, 66.2%, and 97.3% for the total population tested. For the high-prevalence (greater than 15%) population the respective values were as follows: 78.6%, 91.5%, 68.8%, and 94.7%. Specimens from females had a lower sensitivity than those from males. In the low-prevalence population (less than 10%) results were as follows: 100%, 97.8%, 50%, and 100%. A cost comparison emphasized the benefit of the Gram's stain and culture. It also indicated that, unless batched or assayed at high volume, Gonozyme is not cost competitive for laboratories using standard culture methods. The impact of the EIA method, in general, and Gonozyme, specifically, on the microbiology section also was investigated. Integration would require altering of established work patterns and loss of flexibility and freedom of standard plating technics. The fact that Gonozyme is a "presumptive" test limits it to being a complementary assay, not an alternative. The authors conclude that Gonozyme is optimally suited to a high-volume laboratory, screening a low-prevalence female outpatient population, where specimen transport is a problem and gonococcal resistance to penicillin has not been demonstrated. This would include sexually transmitted disease clinics, reference laboratories, and state health departments.     

An improved and rapid method for the isolation of rat lymph node or spleen T lymphocytes for T cell proliferation assays.

To detect and compare the capacity of antigen presenting cells to present antigen in a T cell proliferation assay, it is necessary to obtain a pure population of antigen-primed T cells that gives low background proliferative responses. Therefore in this paper we present a newly developed isolation method of antigen-primed T lymphocytes from rat spleen or lymph nodes. This method uses a nylon wool column to deplete most of the adherent cells and B cells, followed by an indirect elimination method with magnetic beads to remove contaminating Ia-positive cells. We compared this method with two commonly used isolation methods, namely a 1.5 h adherence step, followed by a nylon wool column and a Sephadex G-10 column and a 1.5 h adherence step followed by a passage through two consecutive columns of Sephadex G-10. The best T cell enrichment (98% OX-19/52-positive cells) was achieved with the newly developed method, in which the contamination of Ia-positive cells, predominantly B cells and dendritic cells (DC), was diminished to less than 2%. The background response of this population was low and differed significantly with the common methods. Antigen-specific T cell responses induced by splenic DC, expressed as stimulation index, gave very specific responses and showed a steep rise with increasing DC concentrations compared to the common methods. Therefore we conclude that we developed an improved, rapid and reproducible method for the isolation of rat spleen or lymph node T lymphocytes suitable for T cell proliferation assays.     

An improved method for water vapor detection

We describe improvements in and details for the construction, calibration and use of a device using a thermal conductivity cell for the measurement of low-level rates of water evaporation (E) from a small surface area. E is measured from 0.0 to 1.0 mg·min^?1 with a correlation coefficient of 0.999 between measured and independently verified rates and amounts of water evaporation. Data are available as a recordable analog d.c. voltage as well as in digital display for E and for the amount of water evaporated during an operator defined time period. The device we describe is noninvasive and it is designed to be constructed of conventional components. It is useful not only for measuring transcutaneous water diffusion in normal and diseased skin, but also it is adequately sensitive and rapidly responding to follow thermoregulatory and psychogenic sweating in small (nom. 1.0 cm²) skin areas. It can also be used to measure accurately and precisely the rates at which water is adsorbed by and removed from inanimate materials, as well as to determine how much water they store.     

Polymer microarrays: identification of substrates for phagocytosis assays

A polymer microarray of 120 polyurethanes was used to identify polymers that promoted the adhesion of bone marrow dendritic cells (BMDC). Identified polymers were coated onto glass cover slips and shown to be efficient substrates for the immobilisation of these primary cells, which underwent efficient phagocytosis while still presumably maintaining their immature state.     

An improved method for development of toxoid vaccines and antitoxins

Botulinum neurotoxins are the most potent toxins known and causative agents of human botulism. Treatment comprises of administering purified polyclonal antitoxin or the prophylactic use of a vaccine containing formaldehyde inactivated toxoid. Whilst formaldehyde inhibits toxin activity, it induces so many structural changes in the molecule that immunisation often results in low levels of neutralising antibodies. We describe here for the first time a simple, less time consuming, novel method for producing a non-toxic toxoid that is structurally and antigenically more similar to the native toxin. Toxin is chemically inactivated by alkylation with iodoacetamide in the presence of reversibly denaturing conditions. This reduces neurotoxic activity by at least 7-orders of magnitude to undetectable levels. Following immunisation, in vivo neutralising antibody levels were 600-times higher than those produced with formaldehyde toxoid, despite generating equivalent ELISA antitoxin binding titres. These studies demonstrate that the new toxoid retains more of the native toxins structure and critical epitopes responsible for inducing life-saving neutralising antibody. Toxoid produced by the new method should substantially improve both antitoxin and vaccine production and be applicable to other toxins and immunogens.     

Development of an improved method for measuring neutralizing antibody to rotavirus.

An improved assay was developed for measuring neutralizing antibody titers against rotaviruses. This procedure used the same initial steps as performed to determine antibody titers by the focus reduction neutralization (FRN) assay. However, instead of counting infected cells after staining with fluorescein, reductions in virus infectivity by neutralizing antibody were determined by quantitation of viral antigen production using an ELISA. A linear relationship was found between ELISA absorbance values and focus forming units for each of four prototype rotaviruses, representative of serotypes 1-4. Thus, the serum dilution that resulted in neutralization of 60% of infectious virus (i.e. the neutralizing antibody titer) could be readily determined from absorbance values. Titers found by this method were similar to and as reproducible as those found by the FRN assay. Because this method is less laborious and the results are obtained by objective rather than subjective methods, it represents an improvement over the FRN assay.     

An improved perfusion fixation method for the testis

A reliable and uniform vascular perfusion fixation method for the testis has been developed by using an initial washout solution containing a vasodilator and an anticoagulant. This is followed by a brief fixation with a sodium phosphate buffered formaldehyde-glutaraldehyde solution of conventional strength, and then a second more concentrated aldehyde fixative solution containing picric acid. The method takes into account some of the unique features of the vascular supply of the male genital tract for its favorable perfusion and fixation. The advantages of this method are: (1) consistently favorable preservation of the testis; (2) simple and inexpensive apparatus; and (3) stable and relatively innocuous stock solutions.     

一种改进的大引物PCR定点诱变方法

目的 建立一种改进的、简便易行的大引物PCR定点诱变技术。方法 在两轮PCR中设计了两种不同的质粒DNA模板，两者分别缺少正向或反向外侧引物可结合的互补序列；当两种模板和两条外侧引物同时存在于一个反应管时，可以完全避免对野生型目的基因全长的扩增。第1轮PCR由正向外侧引物和突变引物合成大引物，这一产和无需凝胶纯化而直接用于第2轮PCR。第2轮PCR的第1阶段由大引物延伸合成全长突变终产物，两条外侧引物则在第2阶段指数扩增，所有终产物均含所需致突变位点。结果 用此方法对中国人β地中海贫血15种稀少突变类型进行定点诱变，并将诱变得到的全长人β珠蛋白基因克隆到pGEM－T载体后全长测序，所有克隆均鉴定为所需突变型。结论 这种改进的大引物PCR定点诱变技术省时、省工，诱变的成功率可达100％，适于实验室常规应用。     

An improved method for Daugman's iris localization algorithm

Computer-based automatic recognition of persons for security reasons is highly desirable. Iris patterns provide an opportunity for separation of individuals to an extent that would avoid false positives and negatives. The current standard for this science is Daugman's iris localization algorithm. Part of the time required for analysis and comparison with other images relates to eyelid and eyelash positioning and length. We sought to remove the upper and lower eyelids and eyelashes to determine if separation of individuals could still be attained. Our experiments suggest separation can be achieved as effectively and more quickly by removing distracting and variable features while retaining enough stable factors in the iris to enable accurate identification.     

An evaluation of two simple synthetic peptide based anti-HIV assays.

Two simple synthetic peptide based assays for anti-HIV (Agen SimpliRED and Genetic Systems GENIE) were evaluated for their ability to distinguish samples that contained anti-HIV-1 from samples that did not. The anti-HIV negative samples were from uninfected subjects that had either given false positive reactions on existing screening assays or indeterminate reactivity on Western blot. The anti-HIV-1 positive samples had been shown to contain antibody by a number of assays, and clinical details of the patients were known. The SimpliRED and GENIE assays demonstrated similar performances, with sensitivities of 99.77% and 100%, and specificities of 97.78% and 99.16%, respectively.