Quantitative and qualitative detection of cytomegalovirus-specific antibodies using two types of enzyme-linked immunosorbent assay

An indirect ELISA and an inhibition ELISA were developed for the detection of cytomegalovirus (CMV)-specific immunoglobulin G (IgG) and CMV-specific total immunoglobulin, respectively. Both assays were more specific than the complement fixation (CF) test, and titres of positive sera were 660 times higher by IgG ELISA and 6 times higher by inhibition ELISA than titres by the CF test. Titres by IgG ELISA were reliably determined using the absorbance obtained at a single serum dilution of 1/1,000 in conjunction with a standard graph. Both ELISAs compared favourably with each other in sensitivity and specificity in determining CMV immune status. The inhibition ELISA, in particular, provides a simple and reliable method of screening sera, which requires no control antigen or predilution of sera. It should prove useful for large-scale screening procedures, such as blood donor testing.     

A practice guideline and decision aid for blood transfusion

An attempt was made to reduce exposure of patients to blood products by using a point-of-ordering decision support system and strict adherence to a practice guideline, by observing physician behavior in the multidisciplinary intensive-care unit (ICU) of a tertiary-care medical center. Hemoglobin (Hg) level at the time of transfusion, total units of red blood cells (RBCs) per admission, units per patient per ICU day, fraction of patients receiving no transfusions, and incidence of single-unit transfusions covering 628 patients were measured. In Phase 1, RBC transfusion behavior was observed without intervention. In Phase 2, a special order form for RBCs that suggested a transfusion threshold of 8.6 g/dL of Hg was introduced. In Phase 3, the suggested threshold was lowered to 7.0 g/dL and required all transfusions that did not meet the new guideline to be prospectively reviewed by a transfusion medicine physician. The Hg level at transfusion fell from 8.5 g/dL to 8.2 g/dL (p = 0.008). The use of single-unit transfusions fell from 32 percent to 17 percent (p = 0.001), but there was no change in the number of patients receiving any blood, the total units per admission, or units per patient per day. In this setting, a practice guideline with a point-of-decision support system did not influence blood usage. Intermediate outcomes (such as the level of anemia at transfusion or compliance to a guideline) should not substitute for     

Detection of human cytomegalovirus-specific IgG antibodies by a sensitive solid-phase radioimmunoassay and by a rapid-screening test

A radioimmunoassay (RIA) for the detection of human IgG antibodies to cytomegalovirus (CMV) which utilizes extracts of CMV-infected cells as antigen is described. Sera of 66 healthy adults were titered by the RIA and by the complement fixation (CF) test. These same subjects were also used in the evaluation of a rapid screening radioimmunoassay (RSRIA) developed to detect IgG antibodies to CMV in small serum samples. The specificity of the RIA was evaluated by testing sera of seven CMV patients and ten patients with heterotypic titer rises to herpes simplex virus or varicella zoster virus (HSV or VZV). The RIA proved to be sensitive and specific. Possible applications of the RSRIA modification are discussed.     

Detection of rotavirus-specific IgG antibodies by immunoperoxidase assay and enzyme-linked immunosorbent assay

An indirect immunoperoxidase assay (IPA) has been developed for determination of IgG antibodies to rotavirus. The technique employed as antigen, SA-11 infected MA 104 cells, which were air-dried on glass slides and acetone-fixed. In parallel, rota-specific IgG antibodies were determined by enzyme-linked immunosorbent assay (ELISA). Specific IgG antibodies to rotavirus were determined in sera of healthy children and in sera of patients suffering from gastroenteritis. A good correlation (r = 0.92) and (r = 0.98) for healthy children and patients, respectively, was found between IPA and ELISA techniques. The IPA technique is rapid and simple and positive results, because of the intensive staining, are easily read by low-power light microscope. The potential application of IPA and ELISA methods in serodiagnosis of rotavirus infections is discussed.     

Detection of human cytomegalovirus-specific iga antibodies in colostrum by enzyme-linked lmmunosorbent assay (elisa)

Fifty women were examined after delivery for the prevalence of antibodies to human cytomegalovirus in colostrum and sera. Eighty percent of them had specific CMV IgG antibodies in the sera, as determined by the immunoperoxidase antibody to membrane antigen (IPAMA) technique. Of the CMV-seropositive women, 60% had specific CMV IgA antibodies in high titer in the colostrum as determined by enzyme-linked immunosorbent assay (ELISA). In only two of the seropositive women were specific CMV IgA antibodies detected in the sera as well. The significance of specific CMV IgA antibodies in colostrum as protection against perinatal infection and the mechanism of this production will be discussed.     

Laboratory evaluation of automated enzyme linked fluorescent assay for detecting serum specific IgG antibodies against rubella virus

The VIDAS rubella IgG(RBG) is a new, automated, enzyme-linkad fluorescent assay(ELFA) for detecting IgG antibodies to rubella virus in serum. The purpose of this study was to evaluate the usefulness of the qualitative and quantitative VIDAS RBG as laboratory tests in the rapid detecting anti-rubella IgG antibodies. Simultaneous parallel testing was performed by using the qualitative and quantitative RBG along with RUBAZYME(ABBOTT LABORATORIES) and the standard hemagglutination-inhibition(HI) test(R-HI kit, SEIKEN) on 200 blood samples submitted for anti-rubella IgG antibodies testing from patients at the VGH-Kaohsiung(Mar.-Sep., 1993). The qualitative and quantitative VIDAS RBG and RUBAZYME assay were compared to the standard R-HI test. Significant differences among the sensitivities of these three methods were found (100% sensitivity for the qualitative VIDAS RBG, 88.89% for the quantitative VIDAS RBG and 97.67% for the RUBAZYME). However, the specificities of these three methods were all the same, 100%. With these results we can conclude that the qualitative VIDAS RBG will provide a very good, precise and reliable method to determine serum specific IgG antibodies against rubella virus. Furthermore, the VIDAS RBG is fully automated and time-saving procedures (for every batch of test, it takes 25 minutes with VIDAS RBG, 2.5 hours with RUBAZYME and 4.5 hours with R-HI) in clinical laboratories.     

Qualitative and quantitative evaluation of bird-specific IgG antibodies

BACKGROUND: Exposure to organic dust produced by birds often gives rise to an immune response, e.g. IgG antibodies, but intense exposure can lead to high concentrations of IgG antibodies and the development of allergic alveolitis, often known as "bird fancier's lung". The aim of this study was to establish the distribution of bird-specific IgG antibodies in exposed and nonexposed individuals and compare a nonquantitative and quantitative method in evaluating raised levels of IgG antibodies. METHODS: Sera were collected in Sweden and South Africa and levels of IgG antibodies specific to pigeon, budgerigar and parrot antigens were quantified using the UniCAP system. Results were compared to the precipitation in gel assay. The IgG antibody values of symptomatic patients without precipitating antibodies (non-PP group; n = 51) and patients with precipitating antibodies (PP group; n = 34) were analyzed and compared to nonexposed asymptomatic blood donors (BD group; n = 73) and environmentally exposed pigeon breeders (n = 11). RESULTS: The IgG antibody response of the analyzed groups in Sweden and South Africa did not vary significantly from each other. IgG antibody responses were the strongest to pigeon antigens with clear increased IgG antibody levels in the PP group [geometric mean (GM) 603 mg/l] compared to the non-PP (GM 6.9 mg/l) and BD group (GM 5.0 mg/l). Threshold values, calculated as the GM value from the BD group plus 3 standard deviations (99% confidence interval), were 9.8, 10.8 and 10.0 mg/l for pigeons, budgerigars and parrots, respectively. Comparison of the two methods resulted in a good concordance with a level of agreement of 94.1% (kappa statistic = 0.83). CONCLUSIONS: The UniCAP system for the detection of bird-specific IgG antibodies is a highly reproducible, generally available, quantitative method for routine diagnostic testing and monitoring of exposed subjects with a very high level of agreement to the precipitating gel assay.     

Enzyme-linked immunosorbent assay (ELISA) for IgG antibodies in farmers' lung disease

The ELISA was used for detection of specific IgG antibodies to Micropolyspora faeni antigens in 158 farmers with a history of exposure to mouldy hay, eighty-eight of whom had a diagnosis of farmers' lung. The farmers’ lung group had significantly higher values in the ELISA than both the seventy exposed but asymptomatic farmers (P < 0.001) and a group of thirty-one adult controls (P < 0.001). The asymptomatic farmers also had significantly higher values than the control group (P < 0.02). The ELISA correlated better with the clinical diagnosis than the Ouchterlony agar-gel double-diffusion (precipitin) test. None of the control group gave positive reactions in the ELISA or the precipitin tests. The ELISA is therefore a sensitive, specific and quantitative test which is readily available and widely applicable.     

Enzyme-linked immunosorbent assay for subclass distribution of human IgG and IgA antigen-specific antibodies

In this study we describe an ELISA using monoclonal antibodies to IgG 1, 2, 3, 4, IgA1 and IgA2 for determining the subclass distribution of human-specific antibodies. No cross-reactivity of the subclass-specific reagents under the conditions used was observed. The sensitivity was 0.5 ng/ml for IgG1, 3, 4; 1.5 ng/ml for IgG2 and 50 ng/ml for IgA1 and IgA2. The reproducibility as described by the coefficient of variation calculated on repeated runs was 8-26% if the data were obtained by relating the absorbance values to a positive serum run in the assay, 17-58% when relating the OD figures to those of a standard myeloma plate. The method may be considered semiquantitative with high sensitivity and specificity, easy to handle and with small day-to-day variation. The assay has been applied to a number of antigens of protein and polysaccharide nature.     

Good laboratory practice before and after blood transfusion

Among the general principles that govern good laboratory practice before and after blood transfusion, there are two aspects which receive little attention: the importance of transfusion medicine as a distinct discipline; and the interrelationships within the hospital needed to make the "Blood Bank" effective.     

A rapid immunoperoxidase assay for determination of IgG antibodies to measles virus

A new indirect peroxidase antibody to membrane antigen (IPAMA) technique for the detection of IgG specific antibodies against measles virus is described. The technique utilizes as antigen measles-infected Vero cells dried on glass slides and stored at −70°C. Sera of 50 healthy medical students and laboratory workers and 24 sera of measles, encephalitis, and subacute sclerosing panencephalitis (SSPE) patients were checked by IPAMA and the results have been compared with the results obtained by the hemaglutination-inhibition (HI) test. There is good agreement between the results of both techniques as to the presence or absence of antibody in 48 out of the 50 tested. The advantages of the techniques are discussed.     

Immunoperoxidase assay for the detection of specific IgG antibodies to Hantaan virus

A technique, using indirect immunoperoxidase antibody (IPA), was developed for the detection of IgG antibody to Hantaan virus. The same protein A-peroxidase conjugate was used with mouse, rat and human sera. The IPA technique employs glass slides with air-dried gamma-ray-inactivated and acetone-fixed Hantaan-infected Vero-E6 cells. Antibody titers detected by IPA was comparable to those detected by the indirect fluorescent antibody technique.     

An enzyme-linked immunosorbent assay for IgG antibodies to human herpes virus 6

An indirect enzyme-linked immunosorbent assay (ELISA) with human herpes virus 6 (HHV6) membrane antigen was compared with indirect immunofluorescence assay (IFA) for measurement of HHV6 IgG antibodies. Five hundred serum samples from 403 Swedish patients with suspected symptomatic Epstein-Barr virus (EBV) infections were examined. The specificity of the ELISA compared with IFA was 98.7% and the sensitivity was 98.4%. In 90% of the patients, IgG antibodies to HHV6 were detected with both assays. The highest HHV6 IgG titers were found mainly in patients with EBV or CMV infections, but HHV6 mononucleosis was not diagnosed. The same HHV6 antigen was assessed for IgM ELISA but was found to be of limited value due to high IgM reactivity with the control antigen. The HHV6 IgM ELISA requires further investigation. The IgG ELISA described is a reliable alternative to IFA for measurement of HHV6 IgG antibodies and for large scale epidemiological studies.     

A Three-Layer Immunoradiometric Assay for Determination of IgG Subclass Antibodies in Human Sera ("IgG Subclass RAST")

We report the development of a three-layer immunoradiometric assay (TIRA) for measurement of IgG antibodies of all four subclasses in human sera. The first layer consists of diluted human serum, the second layer is monoclonal mouse antibodies to human IgG subclasses., and the third layer is ¹²⁵I-labelled rabbit anti-mouse IgG. Monoclonal anti-IgG1. anti-IgC3 and anti-IgG4 reacted only with their complementary IgG subclass, whereas the anti-lgG2 showed slight cross-reactivity to immunoglobins of other subclasses and classes and to light chain proteins. The observed cross-reactivity was found to be without importance, when the TIRA was applied to measurement of IgG subclass antibodies. Equipotency was established by use of appropriate dilutions of the monoclonal antibodies, and the assay was calibrated by use of human reference serum. The TIRA therefore permits reliable inter-individual and intra-individual comparisons of the IgG antibody response in all four subclasses. Von-sped fie binding obtained with pooled normal human serum was below 0.33'#. Inter-assay coefficient of variation was between 18 and 27%, The TIRA was applied to measurement of IgG subclass antibodies to timothy grass pollen in sera from grass pollen allergies undergoing immunotherapy.     

Interference by iatrogenically induced anti-mouse IgG antibodies in a two-site immunometric assay for thyrotropin

Two-site immunometric assays using mouse monoclonal antibodies are gaining increasingly widespread popularity and use. Patients with circulating antimurine immunoglobulin antibodies capable of interfering in these assays have been encountered and described sporadically. Parenteral administration of murine monoclonal antibodies for imaging and therapeutic purposes is increasing and is known to induce human anti-murine antibodies frequently. We examined 60 serum samples from 48 individuals who received such murine immunoglobulin to determine whether iatrogenically induced anti-murine antibodies could interfere in a two-site (sandwich) immunoradiometric assay for serum thyrotropin. We found that these circulating antibodies can indeed interfere in an "unblocked" assay, but that the interference appears to be suppressed by including nonspecific IgG in the commercial version of the assay kit.