Single Serum Dilution Research Articles

Rotavirus-associated diarrhoea in buffalo calves in Sri Lanka

Faecal samples from 150 buffalo calves, one to 150 days old, located in various districts of Sri Lanka, were examined for group A rotavirus antigen by a screening enzyme linked immunosorbent assay ( ELISA). Positive samples were confirmed by the blocking ELISA. In the calves studied 27·3 per cent were diarrhoeic, and the rest were non-diarrhoeic but were in contact with the animals showing diarrhoea. Antigen was detected in 36·6 per cent of the diarrhoeic animals and in 11·9 per cent of the nondiarrhoeic animals. There was a strong association between the presence of antigen in faeces and diarrhoea in these animals ( ξ 2 = 46·98; P<0·001). Of the 146 serum samples examined for antirotaviral antibodies, by the blocking ELISA at a single serum dilution (1:20) against a constant dose of antigen (8 units), 68·5 per cent were positive indicating a widespread infection with the virus in the population studied. This is the first record of the detection of rotavirus and its association with diarrhoea in buffalo calves in Sri Lanka.

A New Combi Test for Simultaneous Detection of Antibodies to Viral Capsid, Early and EBNA Antigens of Epstein-Barr Virus

In order to facilitate the differentiation between a recent (acute) and a past Epstein-Barr virus (EBV) infection, the Combi test was developed. This test is an anticomplement immunofluorescence test (ACIF) requiring only a single serum dilution to be tested on a single cellular spot. The cell line used expresses viral capsid antigen (VCA) and early antigen (EA) in about 5 to 10 percent of the cells as well as EBV nuclear antigens (EBNA) in more than 90 percent of cells. A satisfactory agreement between the Combi test and other tests for antibodies to EBV was obtained (IgG and IgM antibodies to VCA by IFA and EIA and antibodies to EBNA by ACIF including tests for heterophile and complement-fixing antibodies). When the standard serological tests gave negative results, the Combi test was also negative (absence of any fluorescence in the cells). Serologically confirmed recent (acute) infections lead to specific fluorescence in only 5 to 10 percent of the cells, while past infections result in fluorescence in 90 percent or more of the cells. For the diagnosis of a reactivated EBV infection or of EBV-associated malignancies, other tests should be employed. The test is based on the measurement of the activation and specific distribution of the C3 component of complement; the antibody class differentiation is therefore not necessary. The presence of rheumatoid factor (RF) and the IgG competition phenomenon do not influence the results of the Combi test. An introduction of the Combi test will enable a simplified, less expensive and more reliable serodiagnosis of EBV infections.

Foot-and-mouth disease: detection of antibodies in cattle sera by blocking ELISA

A blocking ELISA was developed for the detection of antibodies to foot-and-mouth disease virus SAT1, SAT2 and SAT3 and for the quantification of antibodies on a single dilution of serum. The avidin-biotin system was used. The test was compared with the liquid-phase ELISA executed at the World Reference Laboratory for foot-and-mouth disease. It was found to have favourable logistics and combined high specificity with high sensitivity. The quantitative test using a single dilution of serum was resource saving and proved to be a reliable and precise method for the assessment of antibody levels.

Detection of tetanus antitoxin using Eu(3+)-labeled anti-human immunoglobulin G monoclonal antibodies in a time-resolved fluorescence immunoassay

Tetanus antitoxin in human sera was detected with solid-phase immunoassays in microtitration modules coated with tetanus toxoid by using Eu(3+)-labeled anti-human monoclonal antibodies on the basis of an exactly calibrated antibody standard. The use of a time-resolved fluorescence immunoassay (TR-FIA) significantly improved the quantitative detection of tetanus antitoxin over that of the enzyme-linked immunosorbent assay (ELISA) technique because of its high sensitivity and its wide measurement range, detecting antibody levels between 0.001 and 12.5 IU/ml with a single serum dilution of 1:100. For the same purpose, two different serum dilutions (1:100 and 1:1,000) were needed in the ELISA technique. TR-FIA is reproducible and can be performed in 3.5 h. A study of 2,630 serum samples was undertaken to examine the age-dependent distribution of titer levels, indicating the decline of sufficient protection in patients older than 60 years. The wide measurement range of TR-FIA enabled fast examination of large numbers of serum samples without the need for repetition, with further sample dilution, as was often necessary in the ELISA procedure.

Determination of anti-IgA antibodies with a flow cytometer-based microbead immunoassay (MIA)

A flow cytometer-based microbead immunoassay (MIA) was employed to detect anti-IgA antibodies in patients with IgA deficiency. 3 μm latex particles were coated with purified IgA and serum anti-IgA antibodies of the IgG class were detected with FITC-conjugated anti-human IgG. Antibodies against three different IgA preparations were tested from 22 patient samples as well as 20 controls and compared with a conventional enzyme-linked immunosorbent assay (ELISA) and a passive hemagglutination assay (HA). There was a very close correlation between the results obtained with the MIA and the ELISA assay and between MIA and the HA. Because of the low intra-assay variation and good linearity of the assay, the analysis of one single serum dilution was sufficient to determine the anti-IgA level of a patient and no titration series was required. we conclude that MIA is a satisfactory alternative method for routine anti-IgA antibody determinations. For laboratories already equipped with a flow cytometer the assay is cost effective.

Measurement of relative avidity of antibodies reactive with Porphyromonas (Bacteroides) gingivalis in the sera of subjects having adult periodontitis

Relative avidities of antibodies to Porphyromonas (Bacteroides) gingivalis in the sera of 15 patients having adult periodontitis and 15 healthy subjects were evaluated using an ammonium thiocyanate-dissociated ELISA. Graded concentrations of ammonium thiocyanate were added to a single dilution of serum in order to dissociate low avidity antibody binding to P. gingivalis. The concentration of thiocyanate resulting in 50% reduction in binding (absorbance) was termed the ID50 for that serum. When IgG-class antibodies were examined, the ID50 of anti-P. gingivalis antibodies in the sera of patients was significantly elevated (0.96M vs 0.71M; p less than 0.01, Student's t-test). In contrast, when IgM-class antibodies were examined no significant differences in ID50 between patients and controls were found for P. gingivalis (0.54M vs 0.53M). While the ID50 values of patient antibodies were found to be elevated relative to those of healthy controls, comparison with antibodies from rabbits immunized with P. gingivalis and with ID50 values from other human studies suggests that adult humans, in general, produce very low-avidity antibodies to P. gingivalis. It is suggested that the presence of low-avidity antibodies contributes to the pathology associated with periodontal disease.

Rubella-specific IgG 1 avidity: a comparison of methods

Two methods of determining the avidity of specific IgG 1 were compared with sera from different categories of rubella infection. Both methods were based on an antiglobulin enzyme-linked immunosorbent assay. In one method the absorbances were compared with and without diethylamine (DEA) in the serum diluent over a range of serum dilutions and the difference between the dilution curves measured (DEA-shift). In the other, the absorbances at a single serum dilution were compared with and without urea in the wash fluid used after the antigen/serum incubation (avidity index). Various concentrations of DEA were also assessed in the avidityindex method, as this method is simpler to perform. The DEA-shift method was shown to be more sensitive for diagnosing recent primary rubella or immunization by demonstrating specific IgG 1 of low avidity. The avidity-index method, however, was more specific when sera from cases of reinfection or non-specific rubella IgM reactivity were tested. 35 mM DEA was found to be the optimal concentration of DEA when DEA was substituted for urea in the avidity index method.

Studies on the avidity of IgG 1 subclass antibody specific for varicella-zoster virus

The avidity of varicella-zoster virus (VZV) specific IgG 1 has been assesed in sera from cases of VZV infection in the past and sequential sera collected after varicella and zoster infection. Two methods with a mild protein denaturant were used to determine avidity in an antiglobulin ELISA. One method compared the serum dilution curves obtained in the presence or absence of diethylamine (DEA) in the serum diluent (DEA shift). The other used a single serum dilution and the reactivity compared with or without the protein denaturant (DEA or urea) in the washing buffer after the antigen-serum incubation (avidity index (AI)). For up to two months after the onset of varicella the DEA shift method detected low avidity specific IgG 1 more frequently than the AI method. Both methods failed to detect low avidity specific antibody in sera from cases of zoster or varicella in the distant past. Thus the assessment of specific IgG 1 avidity can be of value in serologically discriminating between varicella and zoster.

Application of a rapid microplaque assay for determination of human immunodeficiency virus neutralizing antibody titers

To perform a human immunodeficiency virus (HIV) plaque assay in nonadherent host cells, we developed a novel technique in which HIV-infected MT-2 cells were formed into monolayers by centrifugation through molten agarose. Infection, formation of cell monolayers, and enumeration of plaques all took place in 96-well microtiter plates. When this process was preceded by 18 h of incubation of HIV with patient serum samples, neutralizing antibody titers between 1:10 and 1:5,000 could be accurately determined in patient serum samples. In addition to the determination of neutralizing antibody titers (with the use of various serum dilutions and a constant virus concentration), neutralization indices could also be determined with different virus dilutions and a single dilution of patient serum.

A quantitative enzyme-linked immunosorbent assay for bovine herpesvirus type 1 (BHV-1) antibody

A quantitative enzyme-linked immunosorbent assay (ELISA) was developed to detect and measure antibody to bovine herpesvirus type 1 (BHV-1) in cattle sera. The optical density produced from a single dilution of test serum was compared with a standard curve and the results were read and printed out from a computer interfaced to a multichannel ELISA reader. The printed results were expressed in ELISA units. The ELISA results obtained on 370 cattle sera were compared with those of the serum neutralisation test (SNT). An agreement of 90·5% was obtained when reciprocal SNT titres equal to or greater than 4 and IgG ELISA units equal to or greater than 50 were taken as indicative of a specific reaction. Of the 370 sera, 35 gave discrepant results of which 21 were SNT positive/IgG ELISA negative and 14 were SNT negative/IgG ELISA positive. When the SNT positive sera negative in the IgG ELISA were tested in an IgM ELISA, 19 were found to be positive. Thus, when the IgG and IgM ELISA results were combined the overall agreement between the ELISA and SNT increased to 95·7%. The IgG ELISA had a sensitivity of 82·4% and specificity of 94·4% relative to the SNT, whereas the combined IgG and IgM ELISA results gave a sensitivity and specificity of 98·3% and 94·4% respectively. There was a good positive correlation between the two tests ( r = 0·86).

Detection of Entamoeba histolytica immunoglobulins G and M to plasma membrane antigen by enzyme-linked immunosorbent assay

Sixty-one serum specimens from 22 patients with clinically diagnosed amoebic liver abscess (ALA), 10 hospitalized patients with a variety of diseases other than amoebiasis, 12 normal healthy controls, and 17 subjects from an amoebiasis-endemic area were assayed by enzyme-linked immunosorbent assay (ELISA). The plasma membrane fraction of axenic cultures of Entamoeba histolytica HK9 separated from other subcellular fractions by differential centrifugation was used as the antigen to detect specific immunoglobulin G (IgG) and IgM antibodies. Using a single serum dilution of 1/100 and optical densities at 492 nm of 0.200 and 0.250 as the cutoff values for the IgM and IgG ELISAs, their respective sensitivities in 22 ALA patients were 91% (20 of 22) and 95% (21 of 22). In 22 patients (10 hospitalized and 12 normal healthy controls), the specificities of the IgM and IgG ELISAs were 95% (21 of 22) and 91% (20 of 22), respectively. All five asymptomatic carriers of pathogenic E. histolytica were seropositive by the IgG ELISA and the amoebic gel diffusion test (AGDT). The AGDT was positive for three of six culture-negative controls, while the IgG ELISA was positive for all six. For six asymptomatic carriers of nonpathogenic zymodemes, the AGDT was positive for two, and the IgG ELISA was positive for three. There was an excellent correlation (r = 0.96) between the IgG ELISA and the AGDT. Only one of six culture-negative controls, none of the asymptomatic carriers of pathogenic E. histolytica, and one of six carriers of nonpathogenic E. histolytica were seropositive by the IgM ELISA, thus highlighting the specificity of the IgM ELISA in the diagnosis of ALA. It is believed that the use of plasma membrane fractions has improved the diagnostic potential of the IgM ELISA.

Time-resolved immunofluorometric assay (TR-IFMA) for the detection of the schistosome circulating anodic antigen

We report the development of a time-resolved immunofluorometric assay (TR-IFMA) for the quantitative determination of the schistosome circulating anodic antigen (CAA). A mouse monoclonal antibody (line 120-1B10-A), recognizing a repetitive epitope on CAA, was used as both antigen-capture antibody and as Europium-labelled antigen-detecting antibody. The lower detection limit of the assay was 20 pg of trichloroacetic acid soluble fraction of adult worm antigen (AWA-TCA) per ml, with a nearly linear measuring range from 20 pg to 130 ng AWA-TCA per ml. The TR-IFMA was compared with an enzyme-linked immunosorbent assay (ELISA), in which the same monoclonal antibody was used as antigen-capture antibody and as alkaline phosphataseconjugated antibody. The lower detection limit of the TR-IFMA was tenfold lower than that of the ELISA, while the linear range of the TR-IFMA exceeded that of the ELISA one hundred-fold. Serum samples of 80 Burundese individuals infected with Schistosoma mansoni (egg counts ranging from 4 to 2583 eggs per gram of faeces) were tested in both assays. Antigen concentrations in serum of individuals infected with S. mansoni ranged from 0–500 ng AWA-TCA per ml. The correlation between antigen levels measured by TR-IFMA and ELISA was good: Spearman's ϱ = 0·92. Whereas in the ELISA the samples had to be titrated, the wide linear range of the TR-IFMA allowed the assay to be performed at a single serum dilution, at which an exact estimation of the antigen concentration was possible.

Discrimination between sheep antibodies to Brucella melitensis and to Brucella ovis

When preparations containing smooth Brucella abortus lipopolysaccharide (LPS) were used as antigens in an ELISA, strong positive reactions were obtained with sera from sheep infected with Brucella melitensis or with Brucella ovis. Oxidation of the LPS with sodium metaperiodate greatly reduced the extent of the cross-reactions with antisera to B. ovis, with little effect on the reactions with antisera to smooth B. melitensis. Periodate oxidation of hot saline extract (HSX) antigen of B. ovis markedly reduced its reactivity in ELISA with anti- B. ovis sera and eliminated cross-reactivity with anti- B. melitensis sera. The reactivity of HSX was maintained after treatment with proteinase K. A simple ELISA system, in which replicate samples from a single serum dilution were tested in parallel against both B. ovis HSX antigen and periodate-oxidised smooth phase B. abortus LPS, was evaluated. It was found to discriminate well between antibodies induced by vaccination or virulent infection with B. melitensis strains and those induced by infection with B. ovis.

Simultaneous serological screening for congenital cytomegalovirus and toxoplasma infection

Sera from mother and baby matched by computer, were examined for evidence of infection with toxoplasma and cytomegalovirus (CMV) in a serological screening programme, based on simultaneous enzyme-linked immunosorbent assays of immunoglobulin G (IgG-ELISA) from a single master dilution of serum. There was evidence of primary CMV infection in 23 of 4717 pregnancies (13 by seroconversion in the last two trimesters: 10 by detection of specific IgM at antenatal booking) and primary toxoplasma infection in 10 of 4548 pregnancies (4 by seroconversion; 6 by detection of specific IgM). Detection of seroconversion depended on matching antenatal booking and cord specimen data. The best matching efficiency that could be achieved was 83%. The effectiveness of this approach to identifying neonates at risk of developmental abnormalities is discussed.

Improvement of a direct agglutination test for field studies of visceral leishmaniasis

To increase the potential for the wide-scale application of our direct agglutination test for visceral leishmaniasis, modifications in the components and procedures were introduced. Supplementation with 0.056 M citrate of the suspension medium stabilized the antigen for 9 weeks at 37 degrees C. To circumvent the need for cooling systems in the field, 0.2% (wt/vol) gelatin was added to the serum diluent instead of fetal bovine serum, with reliable results. Specificity and sensitivity were improved by the incorporation of 0.1 M 2-mercaptoethanol in samples with borderline titers. The test could be performed on samples of whole blood; thus the difficulties of preparation and storage of serum, plasma, or filter paper blood are avoided. For mass screening programs, a single serum dilution of 1:6,400 could be employed, contributing to a further reduction in test expenses. Sera from different geographical areas showed equal reactivities in this direct agglutination test despite the nonhomologous Leishmania donovani antigens used.

An enzyme‐linked immunosorbent assay for measuring antibody titre against avian reovirus using a single dilution of serum

An indirect enzyme-linked immunosorbent assay (ELISA) is described to measure antibody titres to avian reovirus in a single dilution of serum. Various methods of antigen coating were compared. Overnight treatment of plates at 4 C with antigen diluted in 0.01 M phosphate buffered saline, pH 7.5 appeared to be the most suitable as it increased the sensitivity and specificity of the test. Standard curve equations derived from linear regression analyses of predetermined end-point titres versus absorbances were employed to predict antibody titres from raw absorbances, corrected absorbances, specific absorbances, sample/positive ratios and sample/negative ratios measured in a single dilution of serum. The specific absorbance and sample/positive ratio appeared to be the most suitable because of their better performance and stability in predicting titres. In a trial a significant positive correlation was observed between the titres measured by ELISA and virus neutralisation test.

Studies on the establishment of a measles antibodies detection method using enzyme-linked immunosorbent assay (ELISA)

This study was performed to develop an improved enzyme-linked immunosorbent assay (ELISA) for the detection of measles antibodies.A highly purified virus antigen was prepared from Vero cells infected with measles virus by density gradient ultracentrifugation. The control antigen was prepared from uninfected Vero cells as well. Since antibodies to Vero cells, non-Forssman antibodies, were found in some sera of young age group, it was essential to absorb the test sera with Vero-cell antigen before the ELISA test. A cut-off level (0.18) was estimated after absorption of 157 sera with Vero-cell antigen. By making use of this cut-off level, a highly sensitive and specific ELISA test was established. A conversion table was prepared for the calculation of measles ELISA antibody titer defined as reciprocal of the highest serum dilution which showed a positive score from the measured absorbance of a single serum dilution of 1: 200. By this method, ELISA antibody titers of many samples can be recorded at once. After a measles AIK-C vaccine virus infection, infants developed ELISA antibodies earlier than HI and neutralizing (NT) antibodies. Measles ELISA antibodies persisted for as long as NT antibodies after natural measles infection.

ANOMALOUS BINDING CHARACTERISTICS OF HUMAN THYROXINE BINDING GLOBULIN DUE TO A DILUTION‐DEPENDENT SEPARATION ARTEFACT

Contrary to the accepted view, a recent study using Sephadex column separation suggested that thyroxine binding globulin (TBG) binds T4 and T3 with similar affinity, but with a much larger capacity for T4 than T3. We have evaluated this finding by comparing this separation method with equilibrium dialysis, taking account of the effect of serum dilution with each method. Estimates of free T4 fraction by equilibrium dialysis (with magnesium chloride precipitation) were valid over a wide range of serum dilutions. In contrast, Sephadex column separation gave a major overestimate of free hormone (underestimate of binding) in less diluted serum, indicating that this method cannot be used to establish a value for T4 affinity independent of serum dilution. Such a systematic error will result in a greater underestimate of affinity for the ligand with higher affinity when two ligands are compared at a single serum dilution. By equilibrium dialysis at 37 degrees C, the affinity of T4 for TBG was approximately 13-fold higher than that of T3, while the capacity of TBG for both T4 and T3 was close to the concentration of immunoreactive TBG. The previous report of similar T4 and T3 affinities appears to be due to a dilution-dependent underestimate of T4 affinity inherent in Sephadex column separation. Direct comparison of binding kinetics of various ligands requires a separation method that is valid over a wide range of binding protein concentrations.

Radioimmunoassay for the detection of IgG antibodies to herpes simplex virus and its use as a prognostic indicator of HSV excretion in transplant recipients

We report the development of a solid-phase radioimmunoassay for the detection of IgG antibodies to herpes simplex virus (HSV), using a mouse monoclonal antibody specific for the Fc portion of human IgG as the radiolabelled detecting antibody. The binding ratio to test antigen at a single serum dilution, 1:100, correlated significantly with the endpoint titre by radioimmunoassay and with neutralising antibody titre. When compared to neutralisation the radioimmunoassay had a sensitivity of 100% and a specificity of 93%. We believe that the anomalous results are not false positives but represent an increased sensitivity of the radioimmunoassay. We found, by either radioimmunoassay or neutralisation, that high levels of antibody prior to transplantation were associated with a significantly increased risk of HSV excretion post-transplantation in both renal and bone marrow transplant recipients. Thus the radioimmunoassay is a sensitive, specific, and rapid test that can be used as a prognostic indicator of HSV excretion in transplant recipients.

Detection of serum antibodies against [formula omitted] in Potomac horse fever by enzyme-linked immunosorbent assay

An indirect enzyme-linked immunosorbent assay (ELISA) was developed which was specific and sensitive in detecting antibodies to Ehrlichia risticii in Potomac horse fever (PHF). The ELISA antibody titers were correlated with the indirect fluorescent antibody (IFA) titers. E. risticii propagated in human histiocyte culture was purified on renografin gradient and the band of the organisms at a density of 1.182 g/ml was used as antigen. ELISA antibody titers were determined through computer assisted analysis, the observed antibody titers were derived by serial serum dilutions and using a resultant standard curve the predicted antibody titers were obtained from a single serum dilution. The standard curve had a correlation coefficient of 0.8975. The observed and predicted antibody titers were in good agreement, as the respective titers fell within a two-fold range. There was a good correlation between ELISA and IFA test results, but the ELISA titers were several times higher. In experimental infections of horses produced with the infected equine whole blood and the Ehrlichia infected macrophage culture, the antibodies were first detected in two weeks and one week postinoculation (PI), respectively. In both cases the titers reached a peak in about 4 weeks PI with a mean titer of 1:6558 and 1:4030, respectively. The antibody titers of the convalescent sera of field cases of PHF were comparatively lower than the experimentally infected horses.