!nform test kits distributed with the MJA.
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Biomedical subjects
H M Chandler
Publications and source records attributed to H M Chandler.
Transcendental meditation, mindfulness, and longevity: an experimental study with the elderly.
Can direct change in state of consciousness through specific mental techniques extend human life and reverse age-related declines? To address this question, 73 residents of 8 homes for the elderly (mean age = 81 years) were randomly assigned among no treatment and 3 treatments highly similar in external structure and expectations: the Transcendental Meditation (TM) program, mindfulness training (MF) in active distinction making, or a relaxation (low mindfulness) program. A planned comparison indicated that the "restful alert" TM group improved most, followed by MF, in contrast to relaxation and no-treatment groups, on paired associate learning; 2 measures of cognitive flexibility; mental health; systolic blood pressure; and ratings of behavioral flexibility, aging, and treatment efficacy. The MF group improved most, followed by TM, on perceived control and word fluency. After 3 years, survival rate was 100% for TM and 87.5% for MF in contrast to lower rates for other groups.
Adaptation, Psychological↗
An accelerated enzyme immunoassay for human choriogonadotropin in urine, involving reflow of specimen through capillary tubes.
A very rapid and sensitive assay for human choriogonadotropin (hCG) has been developed involving two beta-subunit-specific monoclonal antibodies. In the assay the test specimen is passed backward and forward (reflow) through a monoclonal-antibody-coated capillary tube for 1 min, then incubated for 1 min with a second monoclonal antibody conjugated to urease (EC 3.5.1.5). After addition of a urease substrate solution, 10 int. units of hCG per liter can be detected visually within 5 min, which compares very favorably with other currently available hCG assay procedures. Advantages of the reflow/capillary tube assay system and optimization of the test procedure are discussed.
Antibodies, Monoclonal↗
A rapid quantitative capillary tube enzyme immunoassay for human chorionic gonadotropin in urine.
A quantitative capillary tube enzyme immunoassay (CTEIA) method for the determination of human urinary chorionic gonadotropin (hCG) has been developed. The method utilizes an antibody-coated capillary tube through which the test fluid is passed and a urease-labelled second antibody in an immunometric format. Any hCG in the test solution is 'captured' by the immobilized antibody which is hybridoma derived and specific for the beta-subunit of hCG. The second hCG-specific antibody, conjugated to the enzyme urease, is used to detect the captured hCG on the internal surface of the capillary tube. The amount of urease bound to the surface is determined by the introduction of a substrate solution containing urea and the pH indicator bromothymol blue. The rate of colour change, from yellow to blue, caused by the release of ammonia from urea by urease, is determined in a spectrophotometer using a cell holder adapted to accommodate capillary tubes. The initial rate of absorbance change is directly proportional to the concentration of hCG in the sample in the range 0-100 mIU/ml. The test can detect concentrations of hCG as low as 10 mIU/ml in a total elapsed time of 5 min.
Animals↗
Screening of pig intestines for K88 non-adhesive phenotype by enzyme immunoassay.
An adhesion test for binding of porcine brush border membranes to Escherichia coli cells that possess the K88 antigen (K88+) has been developed using enzyme immunoassay procedures. K88 pilus protein or K88+ E. coli cells were immobilized in the wells of polystyrene microtitre plates. These plates were incubated in the presence of material obtained by scraping the villous surface of pig small intestines. Adhesion of membrane material to immobilized K88 was detected by adding rabbit anti-brush border IgG followed by urease-labelled sheep anti-rabbit IgG conjugate. Action of bound enzyme on urea/bromo-cresol purple substrate solution (pH 4.8) produced an intense colour change from yellow to purple, enabling the test to be read visually. This test enables simple, rapid testing of large numbers of intestial samples and gives results that agree well with the more cumbersome microscopic adhesion test for adhesion of K88+ E. coli to purified brush border membranes.
Animals↗
Immune response and reactions to various dose regimens for raising hyperimmune antisera in sheep.
To determine the optimum procedure for raising hyperimmune sera to tetanus toxin, three adjuvants, four antigen preparations and two routes of administration in various combinations were investigated in sheep. Oil-in-water adjuvants alone or in combination with aluminum gels were superior to aluminium gels on their own. This disadvantage of aluminium gels was partially but not completely abrogated when the frequency of doses was increased to three per week. Intensity of local reaction was strongly correlated with immune response; the more immunogenic a dose, the more reactive. Reactivity of oily adjuvants could be lessened by use of a more suitable route of administration, thus oily adjuvants appeared suitable for use when administered by the intraperitoneal route even though moderate to severe reactions resulted from subcutaneous injections. Of other variables investigated, toxin did not confer any advantage over toxoid as an immunogen, purified toxoid was a significantly better immunogen than unpurified toxoid and two large bleeds (30% of total blood volume each) every six weeks rather than 20 ml test bleeds did not affect the titre of the hyperimmune serum produced.
Adjuvants, Immunologic↗
A new rapid semi-quantitative enzyme immunoassay suitable for determining immunity to tetanus.
A sensitive enzyme immunoassay for rapidly assessing a patient's state of immunity to tetanus is described. The test, which uses 50 microliter sample of blood, plasma or serum, is done in a capillary tube and, by comparison with two adjacent reference tubes containing standardised sera, places immunity to tetanus in one of three categories--low-negative (less than 0.01 IU/ml), intermediate (0.01-1.28 IU/ml) or high (greater than 1.28 IU/ml). In a study of 90 clinical specimens assayed both by toxin neutralisation bioassay and capillary enzyme immunoassay the enzyme immunoassay accurately assessed the state of immunity to tetanus of the patients concerned.
Antibodies, Bacterial↗
A rapid semi quantitative capillary enzyme immunoassay for digoxin.
A rapid and sensitive enzyme immunoassay (EIA) which does not require highly trained personnel or specialised instrumentation is described for the estimation of digoxin in serum, plasma or whole blood samples. The method is based on the ability of digoxin in a clinical sample to inhibit the binding of urease-conjugated sheep-antidigoxin immunoglobulin to a glass capillary tube coated internally with a human serum albumin-digoxin conjugate. The bound enzyme activity can then be measured using a substrate solution containing urea and a pH indicator, most suitably bromocresol purple. The enzymic hydrolysis of urea produces ammonia which causes a vivid yellow to purple colour change in the pH indicator. Plasma samples from 92 patients receiving digoxin were screened in parallel with reference plasma containing 1.3 or 3.8 nmol/l digoxin. The results were available within a total test time of 30 min, and showed excellent correlation with those obtained by radioimmunoassay.
Cross Reactions↗
Monoclonal hybridoma antibodies against human IgE and their use in a rapid and sensitive enzyme immunoassay for the semiquantitative assessment of total IgE levels in human blood.
A simple semiquantitative enzyme immunoassay (EIA) for the rapid estimation of IgE levels in specimens of human blood, plasma or serum is described. The test requires little labour input and does not require highly trained personnel or instrumentation. By using two monoclonal antibodies of different anti-IgE specificities it is possible, with a single incubation of 20 min at ambient temperature, to detect elevated IgE levels (greater than or equal to 333 IU/ml) within a total test time of 25 min, and low levels of IgE (less than or equal to 10 IU/ml) within 35 min. For diagnosis of elevated/normal IgE levels only, a single incubation of 10 min. at ambient temperature may be used with a total test time of less than 20 min. The EIA system utilizes glass capillary tubes and urease-labelled antibodies, a system that has proven satisfactory in other applications.
Animals↗
Hybridoma antibody-based competitive ELISA in Schistosoma japonicum infection.
A competitive enzyme-linked immunosorbent assay (ELISA) has been developed and compared with the circumoval precipitin test (COPT) for diagnosis of schistosomiasis japonica using Philippine sera. The assay is based on the inhibition, by sera, of the binding of a penicillinase-conjugated hybridoma-derived antibody, I. 134, to a crude Schistosoma japonicum adult worm extract. A change in pH subsequent to addition of the substrate is used as the indicator system. Development of the color change in this assay is relatively slow, a fact which presumably facilitates detection of inhibition by serum. Relative to the COPT, no false positive reactions were obtained and the false negative rate was less than 10%. A wide range of inhibitory titers was obtained using sera in the competitive ELISA similar to that found in a competitive radioimmunoassay using 125I-labeled I. 134. The competitive ELISA will be of more general application for diagnosis of schistosomiasis japonica than the competitive RIA using hybridoma antibodies, and will provide more precise quantitative information than is obtainable in the COPT.
Antibodies↗
Estimation of urinary chorionic gonadotropin levels: a rapid capillary enzyme immunoassay.
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Chorionic Gonadotropin↗
An investigation of the use of urease-antibody conjugates in enzyme immunoassays.
The development of urease (E.C.3.5.1.5) as a label for enzyme immunoassay (EIA) procedures is described and the use of such conjugates illustrated with examples. Urease catalyzes the hydrolysis of urea to carbon dioxide and ammonia. The production of ammonia may be detected readily by a pH shift which we have found best indicated by the vivid colour change (yellow to purple) of bromocresol purple incorporated in the substrate solution. This enzyme-substrate system offers a number of important advantages. The substrate in aqueous solution is stable, titration end points are sharp and readily visible and the enzyme is not inhibited by sodium azide. Thus, test reagents may be prepared with this preservative and stored ready to use. Urease of high specific activity is commercially available and because it does not occur in mammalian tissues, it is suitable for use in EIA tests to detect cell-associated antigens and their antibodies. Finally, the enzyme reaction may be stopped by the addition of organomercurial preservatives, thus allowing storage of developed tests for later examination.
Alkaline Phosphatase↗
A new enzyme immunoassay system suitable for field use and its application in a snake venom detection kit.
A new rapid enzyme immunoassay (EIA) field kit for the detection of antigen in clinical specimens is reported. A kit for the detection and identification of snake venom in specimens collected from envenomated patients is described as an example of such a field kit. Tabulated data were obtained from a pilot study in which kits were issued to hospitals and laboratories reporting regular admission of snake bitten patients or presentation of specimens from such patients. Questionnaires returned from participants in the trial indicated that the test configuration was highly acceptable and an excellent correlation was obtained between clinical diagnosis and the detection and identification of snake venom in specimens of blood, urine or bite-site swab washings.
Animals↗
Some observations on the quality control testing of Clostridium chauvoei vaccines.
Agglutination tests are not suitable for the estimation of the protective antibody level in the sera of vaccinated animals and should not be used for the quality control testing of blackleg vaccines. Some highly virulent strains of Cl. chauvoei are only effectively protected against by vaccines containing cells with a heat labile protective antigen in addition to the heat stable antigen common to all cells of Cl. chauvoei. As these highly virulent strains may be encountered in the field it is important that such strains should be selected for the challenge of vaccinated animals in the quality control testing of blackleg vaccines. It would be useful if an international center could be responsible for the collection and distribution of such strains.
Agglutination Tests↗
The protective antigenicity of protoplasts and sphaeroplasts of a highly protective strain of Clostridium chauvoei.
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Animals↗
The preparation of immunogenic cell walls from a highly protective strain of Clostridium chauvoei.
Conventional methods for the preparation of cell walls of a highly protective strain of Clostridium chauvoei destroy the protective antigen. Bacteria were therefore lysed by the enzyme pronase instead of by the mechanical disintegration methods commonly employed. Final purification and separation of cell walls and membranes was achieved by equilibrium density-gradient centrifugation with sodium iodide in a zonal rotor. The resultant cell walls had a two-layered structure when seen in ultra-thin section and were highly immunogenic when used to immunize mice against challenge with C. chauvoei. Rabbit antisera raised against the cell walls provided passive protection against challenge in mice and the level of protection was not diminished by the absorption of all agglutinins from the sera. These results confirm previous observations that the protective antigen is a heatlabile cell wall antigen which stimulates the production of non-agglutinating protective antibody.
Agglutination Tests↗
Ultrastructure of the basal organelles of flagella of Clostridium chauvoei.
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Cell Membrane↗
Rabbit immunoglobulin responses to the flagella, somatic, and protective antigens of a highly protective strain of Clostridium chauvoei.
The immunoglobulin response of rabbits to the flagells (H), somatic (O), and protective antigens of a highly protective strain of Clostridium chauvoei was studied using antisera that had been fractionated by Sephadex G-200 chromatography. The H antigen elicited the characteristic agglutinin response to a protein antigen--early production of 19S globulin followed by persistent 7S globulin production. The O antigen stimulated a transient agglutinin response which was detected in both the 19S and 7S serum fractions. Protective antibody was assayed by passive protection tests in mice. Using these tests the protective activity of the rabbit sera was found to be confined exclusively to the 7S serum fractions. Purified immunoglobulin G, prepared by DEAE-cellulose chromatography of the above sera, was also tested and found to confer considerable passive protection on mice. It is considered that either the protective antigen fails to stimulate an immunoglobulin M response or that immunoglobulin M is relatively ineffective in conferring protection against infection in the mouse passive protection tests.
Agglutination Tests↗