Enzyme-linked immunosorbent assay determination of specific rubella antibody levels in micrograms of immunoglobulin G per milliliter of serum in clinical samples.

A "microgram assay" is described in which solid-phase enzyme-linked immunosorbent assay is used for the determination of specific rubella immunoglobulin G (IgG) antibody levels in micrograms per milliliter of serum. The quantitation was based on a standard curve obtained by using a reference serum, for which the specific IgG content was assayed by immunochemical purification. IgG was first purified and specific rubella antibodies were separated by an immunoadsorbent prepared by linking rubella virus antigens to Sepharose 4B. By using IgG-specific conjugate, the levels of specific rubella IgG antibodies could then be determined from clinical samples. Seronegative samples showed antibody levels less than 1 microgram/ml, whereas levels up to several hundred micrograms per milliliter were detected in some postinfection sera. The correlation between microgram antibody levels and hemagglutination inhibition titers was linear. The method offers a simple and sensitive antibody assay which could be used both for the laboratory diagnosis of acute rubella and for the evaluation of immunity.     

The outcome of patients presenting with crush syndrome after the Marmara earthquake

In this study, we evaluated the clinical and laboratory data of the patients presenting after the Marmara earthquake. Crush syndrome was diagnosed in 60 patients (30 M, 30 F, mean age: 31.3+/-13.8 years). They were buried under the rubble for a mean period of 12.3+/-15.1 hours. On admission, 27 patients were oligoanuric and the mean serum creatinine, creatinine phosphokinase and potassium levels were 4.4+/-3.2 mg/dl, 18453.1+/-24527.2 IU/L, and 4.9+/-1.7 mEq/L, respectively. The most frequent site of trauma was the lower extremity. Dialysis treatment was initiated in 40 patients (19 M, 21 F; mean age: 32.7+/-13.0 years). Mean number of hemodialysis sessions/patient was 8.9+/-6.8. Nine (23%) patients among the dialyzed and 4 (20%) among the non-dialyzed died leading to an overall mortality of 21.6%. This low mortality rate suggests that the death rate from acute renal failure due to crush syndrome could be decreased by extensive follow-up.     

Comparison of enzyme-linked immunosorbent assay with enzyme-linked fluorescence assay with automated readers for detection of rubella virus antibody and herpes simplex virus.

The enzyme-linked immunosorbent assay (ELISA) was compared with the enzyme-linked fluorescence assay (ELFA) for the detection of rubella antibody and herpes simplex virus antigen. Test parameters, specimens, antigen or antibody, and conjugates for the two types of assays were identical except that p-nitrophenyl phosphate was used as the substrate for the ELISA and 4-methylumbelliferyl phosphate was used as the substrate for ELFA. Automated readers were used for both assays. Antibody titers and sensitivity of antigen detection were quite similar for ELISA and ELFA. ELFA for rubella antibody, however, could be conducted with less antigen or shorter substrate incubation time (5 min for ELFA versus 30 min for ELISA). For herpes simplex virus antigen detection, ELFA could also be read after a shorter substrate incubation time (15 min for ELFA versus 30 min for ELISA). Clear polystyrene microtiter plates routinely used for ELISA could be used for ELFA, but clear polyvinyl chloride plates had high background fluorescence. Black polystyrene and polyvinyl chloride plates gave lower background fluorescence than did clear plates. ELFA is of particular value as a substitute for ELISAs in which long substrate incubations are required or antigens of only low titer are available.     

Genetic heterogeneity in familial hyperinsulinism [published erratum appears in Hum Mol Genet 1998 Sep;7(9):1527]

Familial hyperinsulinism (HI) is a disorder characterized by dysregulation of insulin secretion and profound hypoglycemia. Mutations in both the Kir6.2 and sulfonylurea receptor (SUR1) genes have been associated with the autosomal recessive form of this disorder. In this study, the spectrum and frequency of SUR1 mutations in HI and their significance to clinical manifestations of the disease were investigated by screening 45 HI probands of various ethnic origins for mutations in the SUR1 gene. Single-strand conformation polymorphism (SSCP) and nucleotide sequence analyses of genomic DNA revealed a total of 17 novel and three previously described mutations in SUR1 . The novel mutations comprised one nonsense and 10 missense mutations, two deletions, three mutations in consensus splice-site sequences and an in- frame insertion of six nucleotides. One mutation occurred in the first nucleotide binding domain (NBF-1) of the SUR1 molecule and another eight mutations were located in the second nucleotide binding domain (NBF-2), including two at highly conserved amino acid residues within the Walker A sequence motif. The majority of the remaining mutations was distributed throughout the three putative transmembrane domains of the SUR1 protein. With the exception of the 3993-9G-->A mutation, which was detected on 4.5% (4/88) disease chromosomes, allelic frequencies for the identified mutations varied between 1.1 and 2.3% for HI chromosomes, indicating that each mutation was rare within the patient cohort. The clinical manifestations of HI in those patients homozygous for mutations in the SUR1 gene are described. In contrast with the allelic homogeneity of HI previously described in Ashkenazi Jewish patients, these findings suggest that a large degree of allelic heterogeneity at the SUR1 locus exists in non-Ashkenazi HI patients. These data have important implications for genetic counseling and prenatal diagnosis of HI, and also provide a basis to further elucidate the molecular mechanisms underlying the pathophysiology of this disease.      

Huntingtin interacts with cystathionine beta-synthase

We have screened a rat brain library to identify proteins which interact with the 5'-end of huntingtin (amino acids 1-171), including the polyglutamine tract, in the yeast two-hybrid system. We detected an interaction with cystathionine beta-synthase (CBS) [L-serine hydrolyase (adding homocysteine), EC 4.2.1.22], which was confirmed in vitro using His-tagged CBS expressed in Escherichia coli , which was able to specifically bind both rat and human full-length huntingtin. Neither normal nor expanded polyglutamine repeat alone interacted with CBS in the yeast two-hybrid system and nor did constructs containing SBMA or DRPLA with normal or expanded polyglutamine tracts. CBS therefore appears to bind specifically to huntingtin. CBS deficiency is associated with homocystinuria, which is known to affect various physiological systems, including the central nervous system. Homocysteine, one of the substrates of CBS, is known to accumulate in homocystinuria and is metabolized to homocysteate and homocysteine sulphinate, both known to be powerful excitotoxic amino acids. It has been suggested that Huntington's disease involves the action of excitotoxic amino acids and this interaction with CBS may suggest a mechanism for such excitotoxic damage.      

LONG TERM EFFECTS OF PROSTHESES ON STUMP IN LOWER LIMB AMPUTEES: A CRITICAL ANALYSIS OF 100 CASES

One hundred lower limb amputees wearing prostheses for more than 5 years were studied and long terms effects on stumps were noted. Common changes were tissue destruction, tissue proliferation, contact dermatitis, circulatory changes, recurrent folliculitis, bursa formation, and eczematous changes. Out of 100 stumps examined long terms changes were observed in 25 stumps.KEY WORDS: Amputation stumps, Amputees, Tissue destruction, Dermatitis