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Biomedical subjects

J D Conradie

Publications and source records attributed to J D Conradie.

At least 37 records · Page 2Linked to original sources
PubMed1985

Safer blood--supplementation of blood products with anti-HBs.

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection and quantitation of antibodies (anti-HBs) to the surface antigen of the hepatitis B virus (HBsAg). The ELISA uses HBsAg as the solid phase, and, after conjugation to horseradish peroxidase, also as the conjugate. Conditions for this assay were optimized and a rapid (1.5 hours) ELISA has evolved which works very satisfactorily for the large-scale screening of donated blood. We have used this ELISA to examine donated blood in Natal and concluded that we cannot initiate a programme of anti-HBs supplementation of parenteral blood products without hyperimmunization and plasmapheresis of selected, voluntary donors.

Blood Donors↗
PubMed1985

Rapid purification of HBsAg from human plasma.

A method is described for the rapid isolation of purified HBsAg from infected donor plasma. This method uses polyethylene glycol precipitation and a single isopycnic ultracentrifugation step in which the gradient is formed in situ. It works equally well using a small swingout rotor or a zonal rotor.

Centrifugation, Isopycnic↗
PubMed1984

Mouse monoclonal anti-HBs and its use in the screening of donated blood by Elisa.

A method is described for the production of mouse monoclonal antibodies to HBsAg. 4 clones produced antibodies directed at the a and one at the d determinant of HBsAg. These antibodies were conjugated to horse radish peroxidase and used in an Elisa for the detection of HBsAg in donated blood. Antibody C10 produced conjugate which rendered the Elisa as sensitive as a commercially obtainable immunoradiometric assay with which it was compared. Conditions for this assay were optimised and it may be used as a rapid (1 h) test for detecting HBsAg-positive blood. It is practicable to read this Elisa by eye instead of photometrically and it can be thus used under field conditions or in emergencies.

Animals↗
PubMed1983

ELISA solid phase: partial denaturation of coating antibody yields a more efficient solid phase.

We have examined conditions which will improve the solid phase in ELISAs for HBsAg and ferritin in human serum. Sheep and rabbit antibodies were used. It was found that pre-exposure of antibody to low pH, 3 M urea and temperatures as high as 82 degrees C will enhance ELISA colour in these assays. Further experiments indicate that these conditions perturb the structure of the antibody molecules and results in the exposure of new hydrophobic regions. It is speculated that these more hydrophobic molecules can bind to regions on the plastic surface normally not coated by non-perturbed molecules.

Alkylation↗
PubMed1983

Hepatitis B surface antigen in donated blood--screening and confirmation by enzyme-linked immunosorbent assay.

Principles of and test procedures enzyme-linked immunosorbent assay (ELISA) screening and confirmatory tests for hepatitis B surface antigen (HBsAg) in donated blood are described. The assay is of "third-generation' sensitivity and is practical and economical for large-scale screening of blood donors. Results from 119,000 tests show ELISA to be superior to the passive haemagglutination inhibition (PHAI) test for HBsAg. The ELISA described here is considerably cheaper than any comparable commercially available isotopic kit for HBsAg screening but is slightly less sensitive.

Animals↗
PubMed1982

An ELISA procedure for detecting human anti-endotoxin antibodies in serum.

We report an ELISA method suitable for the large-scale screening of blood bank stores to identify those blood units containing high concentrations of antiendotoxin antibodies. In Natal, 8.3% of total units collected had antiendotoxin antibodies at concentrations greater than 40 micrograms/ml, values that may be therapeutically useful. We found that one technician could screen enough samples per year to produce 800 litres of such high-titre plasma.

Antibodies, Bacterial↗
PubMed1981

Prenatal detection of neural tube defects by maternal serum alphafetoprotein assay.

Mass screening for fetal neural tube defects using maternal serum alpha-fetoprotein assay as the initial test is financially justified in South Africa, despite the relatively low incidence of these defects in this country, using enzyme-linked immunosorbent assay. This technique is efficient and inexpensive and lends itself to mass screening. We report a pilot study in which 3153 maternal serum samples were received during a 4-month period. Of these 3102 were assayed and the outcome of pregnancy was recorded in 2069 cases. On the first assay 147 (4.7%) of the women had serum alpha-fetoprotein levels which were high for the gestational dates given, but in retrospect about half of these had been misinterpreted because of incorrect dates. Among the remainder, 6 women proved to be carrying twins, 4 aborted spontaneously, 10 delivered prematurely or had infants with intra-uterine growth retardation, and 4 had a fetus with a neural tube defect. Two affected fetuses were missed by screening, in both cases because serum was taken after 20 weeks gestation. In this sample the incidence in Indians (1/1000)and Coloureds (1/600) was as expected, but in Whites it was much higher (1/130), which probably reflects a sampling error and indicates that the epidemiology of neural tube defects in South Africa requires investigation.

Adolescent↗
PubMed1981

A simple and rapid method of washing and drying micro-titre plates used in ELISA.

A simple device constructed out of perspex sheeting and capable of washing 96-well micro-titre plates used in ELISA is described. It consists of three parts: (a) the 'shower' - a tank with 96 stainless steel syringe needles through which washing fluid runs into the wells of the micro-titre plate, (b) a sump into which washing fluid is collected and (c) a sliding base plate allowing the accurate positioning of the micro-titre plate below the shower. The efficiency of this washing device was established by comparing the results from several specific ELISAs with tha obtained after washing duplicate plates by hand. The use of a specially constructed centrifuge to spin-dry plates is also described.

Enzyme-Linked Immunosorbent Assay↗
PubMed1980

Serum alphafetoprotein determination by enzyme-linked immunosorbent assay.

Enzyme-linked immunosorbent assay (ELISA) in microtitre trays has been adapted to the determination of alpha-fetoprotein (AFP); the method is described here. This assay has a lower limit of sensitivity of AFP 1 microgram/l and results compare well with those obtained with a commercial radio-immunoassay (RIA) kit (r = 0,994; y = 949x-3,00). ELISA has several advantages over RIA; it is considerably less expensive, the reagents are stable for longer periods and no elaborate equipment is required.

Costs and Cost Analysis↗
PubMed1980

Quantitation of serum ferritin by enzyme-linked immunosorbent assay (ELISA).

We have developed an enzyme-linked immunosorbent assay (ELISA) for determining ferritin in serum. In this assay the solid phase consists of rabbit antiferritin IgG adsorbed onto PVC microtitre plates. Ferritin from standards or samples binds to the solid phase and is then detected by a conjugate consisting of rabbit antiferritin-IgG and horseradish peroxidase. The entire assay can be performed in 5 hours, with good precision. The assay has a useful range of 1 to 64 microgram/l and serum samples therefore have to be diluted 10-fold. The coefficient of variation for 6 samples (1,5-40,1 microgram/l) assayed 12 times on the same plate varied from 4% to 9% (intra-assay). When these 6 samples were assayed on different plates and on different days by two technicians the coefficients of variation varied from 4% to 11% (inter-assay). The lowest detectable level of ferritin was 1 microgram/l. Forty-six serum samples from blood donors were assayed by the method described here (gamma) and compared with results obtained by RIA (alpha). The resulting regression equation was gamma = 1,073 alpha -- 4,33; r = 0,984.

Animals↗
PubMed1977

Isolation and identification of the Cmicron4-domain of IgM.

Intact Cmicron4-domain was isolated by molecular exclusion chromatography of reduced and alkylated Fc5 micron prepared by tryptic digestion at 60 degrees C of a monoclonal IgM. Two fragments were obtained of which one contained carbohydrate and the other none. These fragments were successfully separated by chromatography on insolubilised Concanavalin A. Cmicron4-domain was identified by means of amino acid sequence, amino acid composition, molecular weight and immunological analyses. It has a molecular weight of 14,700 daltons and results from tryptic cleavage at Lys-445 of the micron-chain.

Amino Acid Sequence↗
PubMed1976

The importance of quaternary structure in the expression of the C1-binding site of IgM.

The ability of C1 to bind to the Fc5mu-fragment of a monoclonal IgM was determined by means of a haemolytic C1-inhibition assay. Fc5mu fragments were produced by trypsin digestion at five different temperatures ranging from 54-62 degrees (2 degrees increments) and were purified by immunoadsorption through a column of monospecific anti-Fabmu and by molecular exclusion chromatography. Analytical ultracentrifugation showed the final preparations to be free of aggregates. A plot of mug Fc5mu required to inhibit 50% of available C1 versus temperatures of production of the fragment yielded a curve with a minimum at 58-60 degrees. Upon mild reduction and alkylation of these Fc5mu fragments their C1-fixing capacity became approximately the same irrespective of temperature of production. Fc5mu was also prepared at 25 degrees in the presence of 5 M urea, purified by immunoadsorption as before and aliquots then exposed to temperatures ranging from 40-70 degrees (5 degrees increments) for 15 min. After aggregates had been removed by chromatography a similar minimum in C1-fixation was again observed at 60 degrees. Reduction and alkylation once more abolished these differences. Fc5mu and its reduced and alkylated subunits, produced at 60 degrees and then exposed to various concentrations of urea (0-7 M) for 24 hd did not yield a minimum in C1 fixation. Reduced and alkylated Fcmu incubated at various temperatures (40-70 degrees) also did not fix C1 differentially. Examination in the near and far u.v. region of the circular dichroism spectra of different Fc5mu preparations showed a gradual loss of structure associated with restricted aromatic chromophores and secondary (beta) structure with increased temperature. Urea denaturation had a more pronounced and irreversible effect on Fc5 mu conformation. These changes could not be correlated with the CU-fixation patterns observed. It would therefore appear that elevated temperatures induce a static change in the pentameric FC-part of IgM which in turn directly influences or modulates the availability of the C1-binding site. The importance of disulphide bonds in maintaining these temperature-induced changes in Fc5mu was also indicated.

Binding Sites↗