Detection of early antibodies in human immunodeficiency virus infection by enzyme-linked immunosorbent assay, Western blot, and radioimmunoprecipitation.

A current concept of the serological response to human immunodeficiency virus (HIV) infection in humans is that antibodies to core antigens (p55, p24, and p15) are detectable earlier during initial stages of antibody production than antibodies against envelope antigens (gp160, gp120, and gp41). Comparative studies of Western blot (immunoblot), radioimmunoprecipitation assay (RIPA), and enzyme-linked immunosorbent assay (ELISA) during initial antibody production are limited to case reports and have not resolved the issue. Thirty of the 37 participants who are part of a prospective study had at least one specimen that was negative for anti-gp41 but had one or more other bands on Western blot. Twenty-seven of these 30 specimens were reactive for anti-gp120/160 in the RIPA. Of the same 30 specimens, kits from Bionetics identified 2 (7%), ElectroNucleonics 4 (13%), Abbott 13 (43%), Du Pont 25 (83%), and Genetic Systems 25 (83%). All participants had evidence of serological progression by Western blot, including a gp41 band, on subsequent visits; the ELISA kits of all manufacturers identified these later specimens with greater accuracy. These data show that the RIPA detects anti-envelope antibodies that may be not detectable by Western blot and that the production of anti-envelope antibodies approximately parallels the production of anti-core antibodies. The false-negative results by ELISA would permit transmission of HIV by blood transfusion from donors in early stages of infection. The sensitivity of licensed ELISA kits should be improved to identify antibody as soon as possible after infection.     

Passive hemagglutination test for detection of antibodies to human immunodeficiency virus type 1 and comparison of the test with enzyme-linked immunosorbent assay and Western blot (immunoblot) analysis.

A passive hemagglutination test (PHA) was developed for detecting antibodies to human immunodeficiency virus type 1 (HIV-1) utilizing sheep erythrocytes cross-linked with purified envelope glycoprotein (gp160) of HIV-1. In an analysis of 216 human serum samples, 100% correlation was observed in 86 reactive and 124 nonreactive serum samples between PHA and commercial enzyme-linked immunosorbent assays and Western blot (immunoblot) analysis. Serum samples from gp160-immunized chimpanzees also reacted equally well in PHA. The test is simple, rapid, and inexpensive, thus providing an alternate, quick method of detecting HIV antibodies. These advantages and the thermal stability of the reagents that are used make this an attractive alternative for detecting prior exposure of individuals to HIV-1.     

Rapid enzyme-linked immunosorbent assay (ELISA) for Aspergillus fumigatus antibodies.

A rapid enzyme-linked immunosorbent assay (ELISA) where component incubation periods were shortened to one hour, was compared with agar gel double diffusion (AGDD) and a standard ELISA procedure for detecting antibodies to Aspergillus fumigatus in 28 asthmatic patients with suspected allergic aspergillosis. Using two A fumigatus antigens the rapid ELISA compared well with AGDD and the standard ELISA method. Eleven sera that reacted with both antigens in AGDD were all positive against antigen 1 in both forms of ELISA, but two failed to react with antigen 2 in the standard ELISA and three failed to react with this antigen in the rapid method. Thirteen AGDD-negative sera were also negative in both forms of ELISA. The rapid ELISA provides a sensitive and reproducible test for routine serological investigation of allergic aspergillosis.     

Rapid enzyme-linked immunosorbent assay (ELISA) for the detection of IgG antibodies against

A rapid ELISA was developed for the detection of specific IgG against Micropolyspora faeni and Thermoactinomyces vulgaris and compared with counterimmunoelectrophoresis (CIE) in twenty-seven patients with suspected farmer's lung disease (FLD). Seventeen patients had precipitins to M. faeni or T. vulgaris or both, and ten had no precipitins. The optimum conditions for each step in the ELISA were determined: pre-equilibration of reagents at 37⁰C and vigorous agitation during incubation enabled the total test time required for the procedure to he reduced to 4 hr. A serum dilution of 10-fold produced good differentiation between CIE-positive and -negative sera. Little correlation was seen between CIE and ELISA for either M. faeni or T. vulgaris antigens in tests with sera from patients with precipitins: high readings were often recorded in ELISA where no precipitins had been detected with the same antigen. Positive- negative discrimination of unknown sera in ELISA was achieved through the inclusion of CIE-positive and -negative reference sera in each test run. Thirteen CIE-positive sera were classed as positive in ELISA with the M. faeni antigen while eight of thirteen CIE-positive sera were positive in ELISA with the T. vulgaris antigen. For both antigens, four CIE-negative sera were recorded as positive in ELISA.     

A novel enzyme-linked immunosorbent assay (ELISA) for the detection of beryllium antibodies

A novel immunological method has been developed for detecting antibodies (IgG molecules) specific to beryllium, a light metal used in industry and capable of causing chronic beryllium disease. Beryllium metal was vacuum deposited onto commercially available immunological microsticks, which were then exposed to test plasma containing the putative antibodies. Antigen-antibody complexes were located using a biotin-avidin amplification method. One employee diagnosed with chronic beryllium disease and one diagnosed as "sensitized" (lymphocyte transformation positive) exhibited antibody titers graphically and statistically different and higher than a pooled baseline control population. Plasma from these two employees (former beryllium workers) was used in four different approaches to validate the presence of beryllium antibodies. The assay proved to be reproducible.     

Amplification of the enzyme-linked immunosorbent assay (ELISA) in the detection of class-specific antibodies

A modification of the standard enzyme-linked immunosorbent assay (ELISA) is described which circumvents the requirement for specifically purified antibodies from which antibody-enzyme complexes are made. The assay utilizes the principle of a soluble anti-alkaline phosphatase immune complex (AP-A-AP) and has been called the amplified ELISA. Methods for preparing and evidence for the specificity of rabbit anti-rat γ-FC, IgM (μ) and IgA (α) are presented. These reagents are used to measure anti-DNP antibodies belonging to classes IgG, IgM and IgA in rat serum. Using antiglobulin and anti-enzyme reagents prepared in guinea pigs, anti-ovalbumin antibodies are measured in rabbit serum. Titration curves are similar when the amplified ELISA is compared to the standard ELISA. A change in slope suggesting an effect of saturation of antigen sites, occurs at the same input antibody concentration for both assays. Determination of the anti-DNP concentration of unknown sera by extrapolation from titration graphs of a known serum suggests that the value is overestimated, i.e., amplified when the amplified ELISA is used. In addition, the amplified ELISA has an improved ability to detect low levels of antibody. Evidence is presented which illustrates how the use of optimally conjugated DNP-proteins, age of conjugates, and optimal dilutions of secondary antiglobulins and the AP-A-AP reduce non-specific binding in the amplified ELISA. The amplified ELISA is capable of detecting 2.4 ng of antibody to ovalbumin in a one : one million dilution of rabbit serum with high reproducibility and low background.     

Development of a semi-quantitative enzyme-linked immunosorbent assay (ELISA) for detection of human IgG subclass antibodies

We have developed a sensitive enzyme-linked immunosorbent assay (ELISA) which measures antibodies to bee venom phospholipase A2 (PLA2) and hyaluronidase (HYAL), horse IgG, bovine casein, and the bacterium Streptococcus mutans in each of the four human IgG subclasses. For this purpose, we have used mouse monoclonal antibodies (McAb) specific for each subclass and one which showed 'pan-IgG' reactivity. Binding to human IgG was similar for all the McAb and dilution of human IgG resulted in similar dilution curves for each subclass. Results were expressed as arbitrary U ml-1 by comparing the optical density obtained with each subclass-specific McAb to a reference curve for total IgG antibody constructed using the 'pan-IgG' McAb. Close agreement was found between the total amount of IgG antibody and the sum of the antibody in each of the four subclasses (PLA2 r = 0.90, horse IgG r = 0.98, bovine casein r = 0.84, S. mutans r = 0.85), confirming that these assays provide semi-quantitative measurements of the amount of subclass-specific antibody.     

Determination of IgA antibodies to human cytomegalovirus by enzyme-linked immunosorbent assay (ELISA)

A sensitive enzyme-linked immunosorbent assay (ELISA) is described for detection of IgA antibodies cytomegalovirus (CMV). The antigen consisted of a sonicated extract of CMV infected human embryo cells. The tested sera were absorbed with staphylococcus aureus (strain Cowan 1) before analysis. Rabbit antihuman IgA peroxidase conjugate was used to detect human IgA bound to viral antigen. In parallel, Igm and IgG antibodies to CMV were studied by ELISA and by the immunoperoxidase antibody to membrane antigen (IPAMA) technique, respectively. CMV IgA was detected in high titers by ELISA in eight of nine CMV patients. The maximal IgA titers were generally lower than the IgM titers detected by ELISA. No CMV IgA antibodies (titer less than 20) were detected by ELISA in 57 control sera (healthy adults, hospitalized patients with various other diseases), paired sera of five patients with acute herpes simplex, infection, two patients with Epstein-Barr infections, one patients with varicella, and one with zoster infections. The potential application of the ELISA CMV IgA technique in serodiagnosis of CMV infections is discussed.     

A solid-phase enzyme-linked immunosorbent assay (ELISA) for detection of antibody to rubella virus.

An enzyme-linked immunosorbent assay was established for detection of antibodies to rubella virus. In this system commercially available rubella antigen was attached to the wells of polystyrene microtitre plates after which sera were added and incubated to allow the formation of antigen-antibody complexes. The presence of bound antibody was detected by adding anti-human globulin coupled to horseradish peroxidase and visually observing the colour change produced after addition of an appropriate substrate. The test was reproducible and simple to perform and had a similar sensitivity to the widely used haemagglutination inhibition system.     

Detection of antibodies to Sendai virus by enzyme-linked immunosorbent assay (ELISA).

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to Sendai virus, a paramyxovirus, is described. The assay was found to be about 20-fold more sensitive than the hemagglutination inhibition assay. Differentiation between virus specific IgG and IgM is possible. The test appears to be especially useful in the study of early events in antibody formation in vivo as well as in vitro.     

The evaluation of a quantitative enzyme-linked immunosorbent assay (ELISA) for anti-Aspergillus fumigatus IgG

A standard enzyme-linked immunosorbent assay method for anti-Aspergillus fumigatus IgG in human serum was modified to produce a quantitative assay. The resulting assay was reproducible and capable of separating individual precipitin line groups and provided a means of monitoring the variation in antibody levels over long periods in patients with pulmonary aspergillosis.     

Detection of neutralizing antibodies to Japanese encephalitis virus by enzyme-linked immunosorbent assay (ELISA)

Competitive ELISA was used for the detection of neutralizing antibody to JE. Based on the principle that human serum JE antibody competed with JE monoclonal antibody (MAb) for JE antigen, it was found that 3 JE MAbs (E3-3, NPF-5 and NNN-5) were suitable for competitive ELISA for the detection of JE neutralizing antibody. The sensitivity of cometitive ELISA for 29 JE confirmed serum specimens with titer of plaque reduction neutralization test (PRNT) was checked to be 82.1% (23/28). The specificity of E3-3 MAb to JE used in competitive ELISA was 100%. Correlation coefficient of JE confirmed cases of 57 hemagglutination inhibition (HI) titers in 1995 and 37 PRNT titers in 1994 compared with competitive ELISA were 0.744 and 0.732, respectively. Compared the competitive ELISA titers of 154 sera of healthy people with PRNT titers, the results showed that 70% of the sera could be detected by competitive ELISA which saved a lot of time and manpower.     

Indirect enzyme-linked immunosorbent assay (ELISA) for the detection of Coxsackievirus group B antibodies

An indirect, solid phase, microplate enzyme-linked immunosorbent assay (ELISA) was found to be highly sensitive and reliable for detecting antibodies to the group B Coxsackieviruses and other picornaviruses. Conditions for obtaining maximum sensitivity and reproducibility of the indirect ELISA are described. Antibody titres were comparable to those obtained by the virus neutralization test and over 50 times higher than those obtained by the complement-fixation test. Purified virions used in the indirect ELISA reacted with low levels of cross-reacting heterotypic antibodies elicited by each of the six group B Coxsackieviruses, although homotypic reactions resulted in highest titres.     

Comparative evaluation of an enzyme-linked immunosorbent assay (ELISA) to detect antibodies directed against glycoprotein I of pseudorabies virus and a conventional ELISA and neutralization tests.

To determine whether a newly developed enzyme-linked immunosorbent assay (ELISA), which detects antibodies directed against glycoprotein I (gI) of pseudorabies virus, is suitable for serodiagnosis, it was compared with a conventional ELISA and two neutralization tests. The gI ELISA was 99.2% as sensitive and 100% as specific as the conventional ELISA. Antibody titers measured by the gI ELISA were lower than those measured by the conventional ELISA, comparable to those measured by the 24-h neutralization test, and higher than those measured by the 1-h neutralization test. After experimental infections in pigs, antibodies were first detected by the gI ELISA on postinoculation day (PID) 14, by both neutralization tests on PID 7, and by the ELISA on PID 10. All four tests showed the same rate of decline of maternal antibodies in pigs born to sows that had been vaccinated with inactivated vaccine. Thus, not only is the gI ELISA unique in being able to differentiate infected pigs from those vaccinated with gI-negative vaccines, but it is also as useful as a conventional ELISA and as neutralization tests for serodiagnosis.     

A new enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against foot-and-mouth disease virus: II. Application

The liquid-phase blocking sandwich ELISA has been evaluated for the serological study of antibodies against foot-and-mouth disease virus (FMDV). The titres recorded for sera from a population of more than 300 British uninfected, unvaccinated cattle which were examined against each of the seven immunologically distinct FMDV types were less than 1 in 40. alternative to the existing VN test for the quantification of antibodies against FMD virus.     

Markers of human immunodeficiency virus infection in high-risk individuals seronegative by first generation enzyme-linked immunosorbent assay

A total of 228 stored serum samples from 140 high risk individuals was examined for serological markers of human immunodeficiency virus (HIV) infection by second generation enzyme-linked immunosorbent assay, immunoblot, and HIV antigen assay. All the samples were negative in first generation enzyme-linked immunoassay (ELISA). Seventy-four of the serum samples had been obtained from 40 sexual partners of HIV antibody positive individuals. Two of the samples were reactive for p24 in immunoblot, but no other markers of HIV infection were found. From 80 sexually active male homosexuals, 117 serum samples were obtained. They were all negative by the tests employed. Further, 37 serum samples from 20 seroconverters were studied. Four patients had antigenaemia 6-12 months before seroconversion was detected by first generation ELISA. Our data do not support the notion that serological signs of HIV infection are common in high risk individuals seronegative by first generation ELISA. However, HIV infection do occur in subjects negative by first generation ELISA, which emphasises the need for more sensitive screening assays and/or the use of antigen detection as part of screening in high risk individuals. The advent of second generation ELISAs has not in a substantial way reduced this demand.     

A new enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against foot-and-mouth disease virus. I. Development and method of ELISA

A liquid-phase blocking sandwich ELISA has been developed for the quantification of antibodies against foot-and-mouth disease virus which may replace the virus neutralisation (VN) test. This test employs the incubation of a constant amount of antigen with a range of test serum dilutions in the liquid-phase before being assayed using a trapping ELISA. Thus it does not rely on the availability or growth of tissue culture cells. The assay is rapid and relatively simple to perform, reagents are used economically and results may be recorded within 24 h. The ELISA is sensitive and results are more specific and more reproducible than those obtained by VN. Results are expressed as reciprocal antibody titres which are analogous and of a similar order to those recorded by VN. Individual titres, therefore, may be easily assessed by workers in the field who are already familiar with VN.     

Multicenter evaluation of the novel ABN Western blot (immunoblot) system in comparison with an enzyme-linked immunosorbent assay and a different Western blot.

A new, modular Western blot (immunoblot) system for human immunodeficiency virus (HIV) antibodies (ABN WesPage; Wellcome) was compared with enzyme immunoassays (Wellcome, Behringwerke, and Abbott) and with a U.S. Food and Drug Administration (FDA)-licensed Western blot (DuPont) in a multicenter study. A total of 649 serum samples from HIV patients at different stages of the disease, as well as from high-risk patients, from patients with conditions unrelated to AIDS, and from healthy blood donors, were used in the evaluation along with nine seroconversion panels. For evaluation of Western blot reactivity, both Centers for Disease Control (CDC) and FDA criteria were used. With the DuPont Western blot as the reference assay, the overall sensitivity and specificity of the ABN WesPage were 100 and 99.1%, respectively, when indeterminate results were not taken into account and when both tests were interpreted in accordance with CDC criteria. The DuPont Western blot detected significantly more antibodies to pol and gag gene products than the ABN WesPage. The ABN WesPage showed a higher positive rate of detection of viral envelope band gp160. When both Western blots were interpreted in accordance with CDC criteria, the ABN WesPage and the DuPont Western blot yielded 9.3 and 10.4% indeterminate results, respectively. When the DuPont Western blot was interpreted in accordance with the manufacturer's instructions (FDA criteria), 25.7% of the samples tested were regarded as indeterminate. The choice of interpretation criteria is of paramount importance for the evaluation of HIV Western blot patterns.     

Indirect enzyme-linked immunosorbent assay (ELISA) for detection of IgG antibodies against Coxsackie B viruses

In tests for IgG antibodies against Coxsackie B viruses in man, the enzyme-linked immunosorbent assay (ELISA) was essentially group-specific and, unlike the type-specific neutralisation test, usually failed to detect rises in antibody titre in paired, acute and convalescent, sera. However, in rabbits immunised against Coxsackie B viruses, ELISA demonstrated both group- and type-specific antibody responses. The lack of type-specificity of ELISA in man is probably because repeated infection with enteroviruses--echoviruses and Coxsackie A as well as Coxsackie B--results in masking of the type-specific antibody response by group-specific antibody.