Factors influencing the sensitivity of herpes simplex virus detection in clinical specimens in a simultaneous enzyme-linked immunosorbent assay using monoclonal antibodies

A rapid simultaneous enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies was investigated for herpes simplex virus (HSV) detection. All HSV isolated (n = 127) were detected, whereas no response was obtained with HSV negative preparations. Equivalent results were obtained from 275 of 277 clinical specimens in the monoclonal ELISA and in an ELISA using polyclonal antibodies, confirming that appropriately selected monoclonal antibodies may be as efficacious as polyclonal antibodies in antibody-based assays. In clinical specimens, the rate of HSV detection (sensitivity) relative to tissue culture isolation was low for both assays, and the major factor responsible for this was the low concentration of virus present in some specimens. The sensitivity of ELISA obtained in routine use varied with different panels of unselected specimens and was related to the speed of development of the cytopathic effect. These results emphasise the need for caution in assigning a definitive sensitivity level to ELISA tests evaluated on different panels of specimens.     

Production of subgroup-specific monoclonal antibodies against human rotaviruses and their application to an enzyme-linked immunosorbent assay for subgroup determination

Nonneutralizing monoclonal antibodies were prepared against two strains, S2 and YO, of human rotaviruses isolated in cell culture. S2-37 and YO-5 antibodies had subgroup I and subgroup II specificities, respectively. The remaining antibodies (S2-65, YO-71, YO-89, and YO-156) reacted commonly with all the rotaviruses examined. All of the monoclonal antibodies agglutinated exclusively single-shelled particles and immunoprecipitated 42,000-dalton protein, a major component of inner capsid. Using the three monoclonal antibodies (S2-37, YO-5, and YO-156), an enzyme-linked immunosorbent assay was developed for detecting and subgrouping human rotavirus isolates.     

Development of enzyme-linked immunosorbent assay for rapid determination of sildenafil in adulterated functional foods

To determine the sildenafil in adulterated functional foods, a rapid direct competitive enzyme-linked immunosorbent assay (ELISA) procedure employing a polyclonal antibody generated from sildenafil-KLH was established. The antibody showed high sensitivity and specificity in phosphate buffer, with an IC₅₀ of 6±0.1 ng/mL and the limit of detection was 0.6±0.02 ng/mL. The matrix effect of the functional food samples was easily eliminated by using methanol and distilled water for extraction and dilution with PBS, without any clean-up procedure such as solid phase extraction or liquid–liquid extraction, much more simple and rapid than the other reports. For the validation of the established SIL–ELISA, the functional food sample spiked at three different concentrations, analysed by HPLC. The results showed good correlation between the data of ELISA and HPLC (r ²=0.9832). It approved that HPLC methods show the same results with ELISA, and the developed immunoassay method is considered reliable for detection of sildenafil.     

The use of human antigen-specific monoclonal antibodies in an enzyme-linked immunosorbent assay for the determination of anti-HBsAg antibody subclasses

A human monoclonal anti-HBsAg antibody (IgGl, λ) was used as a reference for an ELISA determination of the serum levels of anti-HBsAg antibodies in human volunteers after vaccination with H-B vax. The IgG subclass distribution of specific antibodies showed a marked dominance of IgGl antibodies. In addition, small amounts of specific IgG4 antibodies were occasionally found, suggesting a different pattern from that found after natural disease. The novel use of a human monoclonal antibody to measure specific antibodies may give a more accurate determination of specific IgG subclass levels than previously available methods.     

Quantitative determination of staphylococcal enterotoxin A by an enzyme-linked immunosorbent assay using a combination of polyclonal and monoclonal antibodies and biotin-streptavidin interaction.

A sandwich enzyme-linked immunosorbent assay to detect staphylococcal enterotoxin A (SEA) was developed by using monoclonal antibodies (MAb) to SEA as primary capture antibodies. The antigen was detected with purified rabbit anti-SEA antibody as the secondary antibody. The secondary antibody was identified by direct conjugation with biotin or via biotinylated sheep F(ab')2 fragments to rabbit antibody. The biotin was then reacted with avidin-alkaline phosphatase (AP) conjugate, avidin-biotin-AP conjugated complex, or streptavidin-AP conjugate. The enzyme was identified by using p-nitrophenylphosphate. The incorporation of the avidin-biotin-AP conjugated complex or streptavidin-AP conjugate augmented the sensitivity 32-fold over that of the enzyme-linked immunosorbent assay without these reagents. Controls were run by substitution of the anti-SEA MAb with unrelated MAb of the same isotype. Sample values were considered positive when the A405 exceeded those of the negative controls by 3 standard deviations (greater than 99% confidence interval). The toxin could be quantitated with purified SEA standards through linear regression analysis with lower detection limits of 4 ng/ml (r = 0.99) and 0.25 ng/ml (r greater than or equal to 0.98). Concentrations of protein A up to 10 micrograms/ml did not cause interference. Analyses of crude growth extracts of SEA-secreting strains of Staphylococcus aureus were reproducible and were expressed in terms of 95% confidence intervals. Lack of cross-reactivity was seen with extracts of other toxigenic and nontoxigenic strains of S. aureus. The assay can be completed in one working day, provided that MAb-coated plates are available.     

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.     

Detection of human group C rotaviruses by an enzyme-linked immunosorbent assay using monoclonal antibodies.

An enzyme-linked immunosorbent assay using monoclonal antibodies was established for the detection of human group C rotaviruses. Seventeen clinical samples which were found to contain group C rotaviruses were all strongly positive, whereas 9 samples containing group A rotaviruses and 51 samples lacking rotaviruses were all negative with this test.     

Detection of IgE antibodies to larvae and adults of chironomids by enzyme-linked immunosorbent assay

Using the ELISA method, IgE antibodies to chironomid midges; Tokunagayusurika akamusi larva (TAL), T. akamusi adult (TAA), Chironomus yoshimatsui larva (CYL), C. yoshimatsui adult (CYA) and Chironomus plumosus adult (CPA), were measured in the sera of 36 children with bronchial asthma who visited Tamano City Hospital near Lake Kojiima in the Okayama prefecture. IgE antibodies to adult midges (TAA, CYA and CPA) were widely detected, but only few IgE antibodies to larvae (TAL and CYL). The amount of IgE antibody to CYA and to CPA correlated (P less than 0.001), but the amount of IgE antibody to TAA and to CYA or CPA did not. This suggests that the allergenicity of CYA and CPA is similar, but the allergenicity of TAA is independent of that of CYA or CPA. On the other hand, the ELISA inhibition test showed that TAA allergenicity was independent of that of TAL, CYL, CYA, CPA and the house-dust mite, Dermatophagoides farinae (Df). The inhibition test also showed that CYA and CPA had similar allergenicity, but differed from TAL, TAA, CYL and Df. This indicates that the allergenicities of chironomid midges change during the metamorphosis from larva to adult, and are not common among all species.     

A gel casting technique allowing rapid and sensitive quantitation of antibodies by diffusion-in-gel enzyme-linked immunosorbent assay

A technique is described which allows rapid and sensitive quantitation of antibodies by diffusion towards an antigen-coated surface through a gel of wedge-shaped profile. A sensitivity of 0.16 micrograms antibody per ml solution was obtained after 45 min incubation with stirring at 22 degrees C and 0.08 micrograms/ml could be detected after 90 min incubation with stirring or with incubation at 37 degrees C.     

Development and evaluation of a capture enzyme-linked immunosorbent assay for determination of rubella immunoglobulin M using monoclonal antibodies.

A capture enzyme-linked immunosorbent assay (ELISA) for detection of virus-specific immunoglobulin M (IgM) antibody was developed which used a panel of labeled monoclonal antibodies to rubella virus hemagglutinin. The rapidity of the test system was increased by using, after 1-h incubation of the test serum, a second 1-h incubation of the serum with a mixture of viral antigen and labeled monoclonal antibody. The new assay was tested for specificity on 371 human sera from people without any recent contact with rubella virus; of these, 66 were sera selected from people with rheumatoid factor or IgM antibody to human cytomegalovirus, Epstein-Barr virus, or other viruses. In parallel, the new assay was performed on 191 sera from patients having recent contact with rubella virus. Results were compared with those obtained by an indirect ELISA method on IgM serum fractions, using purified rubella virus as a solid phase. Of the 371 sera tested for specificity, 5 (1.3%) gave false-positive results with indirect ELISA (1 rheumatoid factor, 2 heterophil antibody, and 2 human cytomegalovirus sera positive for IgM), and none were false-positive with the capture assay. Two sera from a patient with primary cytomegalovirus infection, which were positive for rubella IgM antibody with both methods and were initially interpreted as false-positive, were finally considered to be true-positive, since they were reactive only in the presence of IgM antibody and viral antigen. Of the 191 sera from 92 patients (84 patients with acute rubella, four newborns from mothers with rubella during pregnancy, and four vaccinees), 136 (71.2%) were found to be positive for IgM by direct ELISA, and 128 (67.0%) were positive by capture ELISA; 12 sera drawn during the first 2 days of disease, or at least 40 days after onset (or after vaccination), were detected only by indirect ELISA, and 4 sera were detected only by capture ELISA. Thus, specificity and sensitivity, respectively, were 100 and 91.4% for capture ELISA and 98.6 and 97.1% for indirect ELISA. However, when the number of patients was considered, 86 were detected as IgM positive by indirect ELISA, and 87 were detected positive by capture ELISA. The overall agreement between the two assays was 96.2%. Capture ELISA using monoclonal antibody appears preferable over indirect ELISA on IgM serum fractions because of its higher specificity and shorter time for test performance; furthermore, there is no need for serum fractionation or virus purification for the capture ELISA.     

QuEChERS extraction followed by enzyme-linked immunosorbent assay for determination of deoxynivalenol and zearalenone in cereals

A simple, rapid and cost-effective QuEChERS method for the determination of ZEN and DON by ELISA in cereals was developed. The single factor experiment was used to ?nd the optimal QuEChERS parameters (extraction solvent, extraction method, extraction time, the QuEChERS composition, sorbent and clarification time) and extracts were analysed by ELISA. The optimised method was validated for selectivity, linearity, limits of detection, matrix effect and precision. The recovery values of ZEN and DON by ELISA were compared with a standard HPLC method and the results showed good coefficient with HPLC, which proved to be suitable for the screening of cereal samples for the presence of ZEN or DON. This is the ?rst report of the determination of ZEN and DON in cereals after QuEChERS extraction combined with the enzyme-linked immunosorbent assay method.     

Simultaneous determination of the level of antibodies to influenza virus surface and internal proteins by enzyme-linked immunosorbent assay

Enzyme-linked immunosorbent assay (ELISA) has been adopted for simultaneous determination of the levels of antibodies to different influenza virus proteins in human sera with known haemagglutination-inhibition (HI) titre. Whole virus of serotypes H1N1 and H3N2, haemagglutinin (HA), matrix (M) and nucleoprotein (NP) proteins have been used as antigens. For detection of antibodies bound to the antigen, peroxidase labelled Staphylococcus protein A conjugate has been used. Correlation of the ELISA and HI titres of anti-HA antibody has been demonstrated. The use of isolated HA as antigen increased the specificity of ELISA. The analysis of human reconvalescent sera has shown that increase in the titre of antibodies to internal proteins does not always coincide with the increase of antibody level to HA. Out of 8 sera with significant increase of the HI titre to the H3 subtype 5 specimens showed 4-fold increase of antibody titre to NP protein. The antibody titre to M protein was elevated in 2 sera only, while 1 serum showed no rise of antibody response to the tested viral proteins.     

Rapid detection of human rotavirus strains in stools by single-sandwich enzyme-linked immunosorbent assay systems using monoclonal antibodies

Using murine monoclonal antibodies (MAbs) raised against the common antigen of group A rotavirus (RV), two single-sandwich ELISA systems were developed for detection of RV in stools: one using polyclonal antibody (PAb) as capture and a MAb as detector antibody (referred to as PAb-MAb assay); and the other based on the use of two different MAbs as capture and detector antibodies (referred to as MAb-MAb assay). In each single-sandwich ELISA system, samples and peroxidase-labeled MAb were incubated sequentially (two-step method) or simultaneously (one-step method). Using the two-step procedure on purified RV, 50 pg of protein was detected in the PAb-MAb as well as in the MAb-MAb assay, whereas the one-step method detected 0.4 ng and a conventional double-sandwich ELISA detected 3.2 ng of viral protein. Titration of RV samples from stools and cell cultures showed that single-sandwich ELISA titers were, on the average, 10-100-fold higher than those obtained by electron microscopy (EM), but 10-100-fold lower than those obtained by solid-phase immune EM (SPIEM). However, when 200 stool samples previously examined by EM or SPIEM were tested by the single-sandwich ELISA systems, specificity and sensitivity of these assays were 100%, and comparable to SPIEM. No false positive results were obtained when 54 samples of meconium and 91 stools from newborns in the first five days of life were tested. The two-step procedure appeared to be somewhat preferable over the one-step method, which, although faster, gave a marked prozone with a few samples in the MAb-MAb assay. The use of MAbs in rapid single-sandwich ELISA systems for RV detection in stools appears highly convenient, due to reliable results and short test performance times.     

Development and optimization of an indirect enzyme-linked immunosorbent assay for the determination of Hexoestrol

An indirect enzyme-linked immunosorbent assay (ELISA) for the detection of hexoestrol (HES) was developed, optimized and validated for the analysis of HES in pork. Many parameters, such as the volume ratio of solution A and solution B, colour developing time, pH value, incubation time, the volume ratio of the standard solution and diluted antiserum, blocking solution, blocking condition, coating solution and coating condition were studied and optimized in the paper. The regression equation of the final inhibition curve is y = ? 0.3345x + 1.4955, R²=0.9913. The linear range is 0.1–8.1 ng/ml, and the IC₅₀ is 0.671 ng/ml. The specificity of the assay was evaluated by the cross-reactivity rates of six compounds, of which two structurally related compounds had a relatively higher cross-reactivity rate of 25% and 6%. HES was added into a pork sample at 5 ng/g level and then the sample was extracted. The recovery is between 49.6% and 79.2%, and the variation coefficient is 0.164.     

Detection of antibodies against circulating cathodic antigen ofSchistosoma mansoni using the enzyme-linked immunosorbent assay

The applicability of aSchistosoma mansoni polysaccharide antigen, circulating cathodic antigen (CCA), for the detection of antibodies inS. mansoni infection was tested in the enzyme-linked immunosorbent assay (ELISA). Antibodies against this secretory antigen were detected in hamster infections after three weeks, while in infected humans anti-CCA antibodies could be demonstrated eight weeks after infection. Antibodies could be demonstrated both in recent and chronic infections in man, but more false-negative results were observed in chronic infections. The antibody response was composed of both IgM and IgG antibodies.This investigation received financial support from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases     

Quantitative determination of pegylated consensus interferon in rhesus monkey serum using a competitive enzyme-linked immunosorbent assay

A competitive enzyme-linked immunosorbent assay (ELISA) with generated mouse anti-consensus interferon (CIFN) antibody was developed for quantitative determination of pegylated consensus interferon (PEG-CIFN) in rhesus monkey serum. The operational concentrations of the original assay were determined using the chessboard method. ELISA working range was 10–5000?ng/ml, corresponding to a limit of quantification of 8.4?ng/ml in rhesus monkey serum. In a precision test, intra-assay CV ranged 1.8–9.6% and inter-assay CV ranged 3.5–12.7%. Relative recovery rate of this ELISA assay ranged from 102.65–115.77%, with RSD values ranging from 2.26–5.44%. Three groups of rhesus monkeys received 1250μg/kg, 300μg/kg, 150μg/kg PEG-CIFN by subcutaneous administration, and blood samples were drawn via the femoral vein at the specified time points. PEG-CIFN in rhesus monkey serum was determined using the competitive ELISA, and the results were compared with antiviral activity assay. In conclusion, the competitive ELISA assay we developed has sufficient sensitivity, precision, and accuracy for the analysis of a rhesus monkey serum sample.     

Diagnosis of human coronavirus infections in children using enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) was developed for diagnosing human coronavirus (HCV) infections in children. One hundred and seventy seven nose swabs, throat swabs, and nasopharyngeal aspirates were collected from 30 children suffering from acute respiratory infections. These samples were tested for HCV antigens by ELISA and 28.2% of the samples were shown to be HCV positive. These results indicate that our ELISA should prove useful in the diagnosis of HCV infections in children. Further studies are in progress to extend the ELISA to detect HCVs in experimentally and naturally acquired infections in adults.     

Detection of antibodies specific for sheeppox and goatpox viruses using recombinant capripoxvirus antigens in an indirect enzyme-linked immunosorbent assay

Viruses in the genus Capripoxvirus, family Poxviridae, cause sheeppox, goatpox and lumpy skin disease, which are the most serious poxvirus diseases of production animals. Despite the considerable threat that these viruses pose to livestock production and global trade in sheep, goats, cattle and their products, convenient and effective serodiagnostic tools are not readily available. To develop a more effective antibody detection capability, selected open reading frames from capripoxvirus DNA were amplified and expressed in Escherichia coli as His-tagged fusion proteins. By screening 42 candidate antigens, two sheeppox virus virion core proteins that were expressed efficiently, purified readily using affinity chromatography and reactive against capripoxvirus immune sera in an indirect enzyme-linked immunosorbent assay (ELISA) were identified. The ELISA performed favourably when sera from sheep and goats infected experimentally with virulent capripoxvirus isolates were tested, with sensitivity and diagnostic specificity ranging between 95 and 97%, but it was unable to detect antibodies reliably in vaccinated sheep or goats. Furthermore, no cross-reactivity with antibodies against orf virus was detected. This assay offers the prospect of a convenient and standardised ELISA-based serodiagnostic test, with no requirement for infectious reagents, that is well suited to high-throughput capripoxvirus surveillance on a flock or herd basis.     

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.     

An enzyme-linked immunosorbent assay using monoclonal antibodies for the detection of respiratory syncytial virus in clinical specimens

Summary An enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of respiratory syncytial virus in nasopharyngeal secretions. This assay employed as immunoreagents two monoclonal antibodies directed against two distinct epitopes of the viral nucleocapsid. One of them (RSV4) was used for antigen capture and the other (NC4) was labelled with N-hydroxy-succinimide-epsilon-caproil biotin and used for antigen detection. Streptavidin biotin-peroxydase complexes were employed as amplification mode. The immunoassay was performed in 6 hours and was able to detect as little as 1 ng/ml of purified nucleocapsid. When 87 nasopharyngeal secretions were analyzed by an indirect immunofluorescence assay using commercial reagents and by the newly developed ELISA, the sensitivity and the specificity of the two assays were found to be very similar.