Indirect enzyme‐linked Immunosorbent assay for detection of cow's milk in goat's milk

An indirect enzyme‐linked immunosorbent assay (ELISA) has been developed for the specific detection of cow's milk (1–25%) in goat's milk. The test uses polyclonal antibodies raised in rabbits against bovine whey proteins (BWP). The anti‐BWP antibodies were recovered from the crude antiserum by immunoadsorption and elution from a column containing immobilized BWP. The anti‐BWP antibodies were biotinylated and rendered cow's milk specific by mixing them with lyophilized ovine and caprine whey proteins. Streptavidin‐peroxidase was used to detect the biotinylated anti‐BWP antibodies bound to bovine milk proteins immobilized on 96‐well plates. The colour developed by the subsequent enzymic conversion of the substrate gave clear absorbance differences when assaying mixtures of goat's milk containing variable amounts of cow's milk.     

Comparison of radioimmunoassay and ELISA methods for detection of antibodies to chromatin components

A solid phase radioimmunoassay has been compared with an enzyme-linked immunosorbent assay (ELISA) for efficacy in measuring anti-chromatin antibodies. The low backgrounds achieved with the radioimmunoassay method produced a high signal-to-noise ratio and enabled detection of the human test antiserum at a dilution of 1:102,400. By contrast, the ELISA could detect the same antiserum only at a dilution of 1:3200 and above. The radioimmunoassay was consistently more sensitive than the ELISA for detection of anti-chromatin antibodies in a number of human and mouse sera and ascites fluid containing a monoclonal antibody. Factors affecting sensitivity in both assays are discussed.     

Systemic and mucosal antibody responses specific to SARS-CoV-2 during mild versus severe COVID-19

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Detection of Mycoplasma hominis antigen in clinical specimens by using a four-layer modification of enzyme immunoassay (EIA)

A 4-layer modification of enzyme immunoassay (EIA) was developed for the detection of Mycoplasma hominis antigen in clinical specimens. Microtiter plates were sensitized with rabbit anti-mycoplasma immunoglobulin, guinea pig anti-mycoplasma immunoglobulin was used as the secondary antibody, and horseradish peroxidase-conjugated anti-guinea pig immunoglobulin was used as the indicator antibody. The specificity of the assay was confirmed by using guinea pig immunoglobulins from preimmunization sera. The sensitivity of the assay is down to 10 ng/ml of antigen protein. Marked cross-reactivity was demonstrated for different strains within the species M. hominis, whereas the other genital mycoplasma species tested showed no reactivity in the assay. A comparison was made of EIA and conventional culture of vaginal specimens from 24 women. All 6 specimens positive by culture were also positive for M. hominis antigen by EIA. Antigen detection by EIA is a sensitive, rapid and simple method for the detection of M. hominis in clinical specimens.     

A computer-interfaced photometer and systematic spacing of duplicates to control within-plate enzyme-immunoassay variation

A Multiskan photometer for reading microtiter plate enzyme immunoassays was linked with a time sharing computer to facilitate control of assay variation and analysis of results. The interface that converted photometer output to RS-232-C format required changes to divide the output into segments short enough for input to the computer. To measure within-plate variation and investigate how the method of allocating sample duplicates to plate wells may affect the estimation of sample variance, uniformity tests were conducted with 47 plates. Coefficients of variation (CV) among wells within-plates ranged from 4.6 to 20.7% and in two-thirds of the plates exceeded 10%. Duplicates allocated to adjacent wells (method 1) gave consistently higher CV for sample means than duplicates allocated to opposite plate quadrants (method 2). In general, the CV by method 2 was about 30% smaller than that by method 1. Analysis of variance confirmed the effectiveness of the quadrant pattern of duplicate allocation as a method of controlling variation that arises from well position effects.     

Determination of glucokinase and hexokinase activity in liver extracts previously purified on Molselect G-50 dextran gel

Hexokinase and glucokinase activity in the supernatant of a rabbit liver homogenate obtained at 18,000g was determined by a spectrophotometric method. Preliminary purification to remove low-molecular-weight components by gel filtration on Molselect G-50 dextran was shown to prevent reduction of NADP unconnected with the hexokinase reaction.Presented by Academician of the Academy of Medical Sciences of the USSR V. S. Il'in.Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 82, No. 7, pp. 891–892, July, 1976.     

Hemodynamic factors in the pathogenesis of diabetic nephropathy

Summary The pathogenesis of the diabetic glomerular lesion is unknown. However, cumulative indirect evidence favors hemodynamic factors associated with the abnormal endocrine environment as the cause of diabetic angiopathy. Experimental evidence suggests that the increased hydrostatic pressures in capillary beds, a hallmark of the early stages of insulin-dependent diabetes, are associated with macromolecular leakage leading to the typical thickening of glomerular capillary basement membrane and increased glomerular mesangial matrix even prior to the occurrence of systemic hypertension. Patients with renal or carotid artery stenosis seem to be protected against diabetic nephropathy and retinopathy on the stenosed side. The first signal of diabetic nephropathy even before deterioration of the renal function is microalbuminuria detected by sensitive methods such as radioimmunoassay. Not only in hypertensive, but even in normotensive diabetic patients with microalbuminuria antihypertensive therapy has been shown to reduce albumin excretion rate and to slow the progression of diabetic nephropathy. Once overt diabetic nephropathy has been established, hypertension is a constant accompaniment of the disease. Thus, hypertension may be a cause as well as a result of diabetic nephropathy. Tight control of blood sugar in close association with antihypertensive treatment reducing blood pressure to a lower normal limit, possibly with agents that specifically decrease glomerular capillary hydraulic pressure are the corner stone in protection against progression of the diabetic angiopathy.Abbreviations ECF extracellular fluid - ESRD end-stage renal disease     

A micro cell culture method utilizing modified microscope slides for use in fluorescent antibody and enzyme immunoassays

Summary A technique for culturing small quantities of mammalian cells on modified microscope slides is described. The modified microscope slides were Bellco Glass, Inc., toxoplasmosis slides and the cell cultures used were early passage bovine embryonic lung cells and continuous cell lines of porcine and canine origins. The slide cell cultures were either uninfected or infected with selected viruses or the obligate intracellular protozoanEncephalitozoon caniculi for utilization in direct and indirect fluorescent antibody testing or in peroxidase antiperoxidase immunosorbant assays.     

Renal function in relation to three candidate genes in a Chinese population

We recently found in a white population that the genes encoding angiotensin-converting enzyme (ACE, I/D polymorphism), -adducin (Gly460Trp), and aldosterone synthase (–344C/T) jointly influence renal function. We therefore investigated in a Chinese population the associations between the serum concentrations of creatinine and uric acid and these three genetic polymorphisms. We genotyped 471 ethnic Han Chinese subjects from 125 nuclear families recruited in northern China via random population sampling (75%) and at specialized hypertension clinics (25%). We performed population-based and family-based association analyses using generalized estimating equations (GEE) and quantitative transmission disequilibrium test (QTDT), respectively, while controlling for covariables. The participants were 39.7 years old and included 235 women (49.9%). The blood pressure measured at the subjects homes averaged 126/80 mmHg. Mean values were 71 µmol/l for serum creatinine, 111 ml min^–1 1.73 m^–2 for calculated creatinine clearance, and 236 µmol/l for serum uric acid. With adjustment for covariables, GEE analyses of single genes demonstrated that serum uric acid, but not serum creatinine, was positively associated with the ACE D allele. Serum uric acid concentrations were 15.8 µmol/l (95% confidence interval 3.3–28.2) and 25.7 µmol/l (11.1–40.2) higher in DD homozygotes than in ID and II subjects, respectively. Further GEE analyses of the three genes combined showed that the association between serum uric acid and the ACE polymorphism was confined to carriers of the -adducin Gly and/or aldosterone synthase C alleles. Sensitivity analyses in parents and offspring separately as well as QTDT analyses were confirmatory. Among 114 informative offspring carrying the -adducin Gly allele serum uric acid was significantly and positively associated with the transmission of the ACE D allele (=20.7 µmol/l). In conclusion, the present study extends our previous findings on the combined effects of the three candidate genes and supports the concept that these genetic polymorphisms jointly influence renal function.     

High rate of false positives in blood donor screening for antibodies to hepatitis C virus

Summary Anti-hepatitis C virus antibody screening of blood donors in different countries revealed prevalences ranging from 0,4–1,4%. These results were obtained with an enzyme immunoassay based on a recombinant hepatitis C virus antigen. We applied a specific inhibition assay (neutralization assay) and a recombinant immunoblot assay to determine the specificity of positive reactions in the enzyme immunoassay.Of 2836 blood donor sera tested, 10 (0,35%) were reactive in the enzyme immunoassay, however, only 3 sera (0,1%) proved to be specifically anti-HCV positive in the inhibition assay. The recombinant immunoblot assay gave similar results. The prevalence of anti-hepatitis C virus antibodies among blood donors has been overestimated in recent publications. Furthermore the high rate of false positives in the enzyme immunoassay may explain reports claiming that only a minor part of EIA positive blood units transmitted the hepatitis C virus to recipients.The inhibition assay was also applied to sera of haemophiliacs and of patients with hepatopathy which had reacted positively in the anti-hepatitis C virus antibody enzyme immunoassay. The antihepatitis C virus specificity was confirmed for all sera from the haemophiliacs group (100%) and for 77% of the hepatopathy patients group. Thus, the anti-hepatitis C virus enzyme immunoassay has a high predictive value when it is used to screen groups with high risks of parenteral hepatitis C virus infection, however, its predictive value is very low when it is used for blood donor screening.Abbreviations EIA enzyme immunoassay - HCV hepatitis C virus - RIBA recombinant immunoblot assay - SOD superoxide dismutase