Indirect Enzyme Immunoassay Research Articles

Derivation and Properties of Recombinant Fab Antibodies to Coplanar Polychlorinated Biphenyls

Recombinant Fab antibodies (rFabs) specific for coplanar polychlorinated biphenyls (PCBs) were derived from a hybridoma cell line (Chiu et al. Anal. Chem. 1995, 67, 3829-3839). Immunoglobulin V(H)-C(H1) and V(L)-C(L) sequences from S2B1 messenger RNA were amplified by PCR and cloned into the M13 phagemid vector pComb3H. Phage displaying rFab were enriched by panning on a PCB hapten conjugate and expressed as soluble rFabs in Escherichia coli XL-1 Blue. Two rFab clones competitively bound PCBs 77 and 126 with half-maximal inhibition (I(50)) of 10-13 ppb in indirect and direct enzyme immunoassays (EIAs), with selectivity nearly identical to that of whole S2B1 IgG and its Fab fragments prepared by papain digestion. These results, and comparison of N-terminal amino acid sequences of MAb S2B1 and the rFab, indicated that rFab S2B1 is a functional copy of the MAb. The rFab S2B1 sequences have 75-89% sequence identity with antibodies that bind nitrophenyl haptens and are being used to construct a three-dimensional computational model of the PCB binding site.

Ochratoxin A: Contamination of grain

Ochratoxin A was quantitatively monitored in grain extracts by indirect solid-phase enzyme immunoassay with the use of an immobilized conjugate of the toxin with gelatin and polyclonal rabbit antibodies raised against the ochratoxin A-BSA conjugate. This monitoring found that 1.7 to 18.5% of the samples were contaminated with the toxin at a concentration of 25.9–291.7 μg/kg. An analysis of forage grain found ochratoxin A at concentrations of 440-3250 μg/kg.

Validation of enzyme-linked immunosorbent assays for the diagnosis of bovine brucellosis

The purpose of this study was to evaluate the performance of the indirect enzyme immunoassay (IELISA) and the competitive enzyme immunoassay (CELISA) for the diagnosis of bovine brucellosis in comparison to conventional serological tests routinely used in Argentina. Serum samples ( n = 3500), from Brucella-free herds, from vaccinated cattle and from naturally infected cattle, were tested by the following tests: buffered antigen agglutination test (BPAT), rose bengal test (RBT), 2-mercaptoethanol test (2-ME), complement fixation test (CFT), IELISA and CELISA. Sensitivity and specificity of the BPAT, RBT, IELISA and CELISA were determined relative to the 2-ME and the CFT. The CELISA was considered suitable for eliminating most serological reactions of vaccinated animals and was more specific than the other tests. The results indicate the potential use of the CELISA as a complementary assay in the brucellosis control and eradication program in Argentina and other countries, where Brucella abortusstrain 19 vaccination is mandatory.

Rapid Determination of Citrinin in Corn by Fluorescence Liquid Chromatography and Enzyme Immunoassay

A rapid assay procedure was developed for mycotoxin citrinin in corn using liquid-liquid extraction (LLE) cartridges. Ground corn was extracted with methylene chloride and 0.5 N phosphoric acid. The extract was added to an LLE cartridge containing a diatomaceous-earth adsorbant, previously impregnated with sodium bicarbonate solution. After aspiration to dryness, the cartridge was eluted with methanol-water (4 + 1), and aliquots were taken for quantitation by reversed-phase liquid chromatography with fluorescence detection. Recoveries of citrinin added to ground corn at 200-1600 ng/g ranged from 71.2 to 86.3%, with coefficients of variation between 4.1 and 10.6%. An indirect enzyme immunoassay was also evaluated, using sodium carbonate solution for extraction. Recoveries of citrinin added to ground corn at 200-2000 ng/g ranged from 53.2 to 67.2%, but the coefficients of variation varied between 18.4 and 51.5%. The LLE cartridge procedure offers the advantages of low solvent consumption and speed, and is amenable to automation.

Production and characterization of a monoclonal antibody against domoic acid and its application to enzyme immunoassay

For production of monoclonal antibodies against domoic acid, a causative agent of amnesic shellfish poisoning, three immunogens, domoic acid conjugated with bovine serum albumin (BSA), ovalbumin (OVA) and human gamma globulin (HGG), were prepared. The antiserum obtained from BALB/c mice immunized with domoic acid–BSA showed the highest affinity for domoic acid. The monoclonal antibody, DA-3, obtained from the mice was highly specific for domoic acid and showed a minor cross-reactivity with the isomers of domoic acid (isodomoic acids B, E, F, G and H), except for isodomoic acid A. Using DA-3 antibody, an indirect competitive enzyme immunoassay (idc-EIA) was developed for measurement of domoic acid. The working range for quantitative measurement of domoic acid and the quantification limit for domoic acid in shellfish were estimated to be 0.15–10 ng/ml and less than 0.04 μg/g, respectively. The mean recovery of domoic acid added to extracts of shellfish at toxin levels of 0.02 to 0.2 μg/ml was 103% with a coefficient of variation of 4.5%. The newly developed idc-EIA seems to be a useful method for monitoring domoic acid in shellfish.

Detection of diphtheria antitoxin by four different methods.

To investigate the reliability of the different methods used in Norway and Russia for detection of diphtheria antitoxin. One hundred and twenty-two sera were selected among Russian serum samples previously collected for seroepidemiologic studies of diphtheria antitoxin. The sera were selected to cover the total antitoxin range and were analyzed by four different antidiphtheria toxin assays: an in vitro toxin neutralization test using Vero cells (in vitro NT), an in vivo neutralization test using rabbit skin inoculation (in vivo NT), an indirect enzyme immunoassay (EIA) and a passive hemagglutination assay (PHA). The results were expressed according to the international standard as: not protected (<0.01 IU/mL), relatively protected (0.01-0.1 IU/mL) or protected (≥0.1 IU/mL). The sensitivity, specificity and inter-rater agreement (K or Kw) of each method were related to the in vitro NT selected as the reference method. The in vivo NT test corresponded very well with the in vitro NT in its ability to differentiate between protection/relative protection and no protection (sensitivity 97%, specificity 87% and K=0.84). The EIA test showed a high sensitivity (96%), but since many sera were categorized as protected rather than not protected, the specificity (30%) and inter-rater agreement (K=0.29) were low. The PHA test had a very high specificity (100%) but a low sensitivity (86%). The agreement between the two neutralization tests was high. If none of the neutralization assays is routinely available, the PHA test can be used to predict the need for vaccination on an individual basis but should not be used for seroepidemiologic studies, since the protection rate for diphtheria would be falsely too low, due to the lower sensitivity. The indirect EIA test used in this study should not be used routinely.

Production and characterization of monoclonal antibodies against the hemolysin BL enterotoxin complex produced by Bacillus cereus.

A total of five hybridoma cell lines that produced monoclonal antibodies against the components of the hemolysin BL (HBL) enterotoxin complex and sphingomyelinase produced by Bacillus cereus were established and characterized. Monoclonal antibody 2A3 was specific for the B component, antibodies 1A12 and 8B12 were specific for the L(2) component, and antibody 1C2 was specific for the L(1) protein of the HBL enterotoxin complex. No cross-reactivity with other proteins produced by different strains of B. cereus was observed for monoclonal antibodies 2A3, 1A12, and 8B12, whereas antibody 1C2 cross-reacted with an uncharacterized protein of approximately 93 kDa and with a 39-kDa protein, which possibly represents one component of the nonhemolytic enterotoxin complex. Antibody 2A12 finally showed a distinct reactivity with B. cereus sphingomyelinase. The monoclonal antibodies developed in this study were also successfully applied in indirect enzyme immunoassays for the characterization of the enterotoxic activity of B. cereus strains. About 50% of the strains tested were capable of producing the HBL enterotoxin complex, and it could be demonstrated that all strains producing HBL were also highly cytotoxic.

Validation of the fluorescence polarization assay as a serological test for the presumptive diagnosis of porcine brucellosis

Sera from Canadian pigs (brucellosis free, n = 14 037) and sera from pigs infected with Brucella suis ( n = 401) were tested by the buffered antigen plate agglutination test, the complement fixation test, an indirect and a competitive enzyme immunoassay and a fluorescence polarization assay. The results were analysed and assay sensitivity and specificity estimates were calculated. The sensitivity and specificity of the tests were as follows: the buffered antigen plate agglutination test, 77.1 and 96.9%; the complement fixation test (considering anticomplementary sera as negative), 93.3 and 95.5%; the complement fixation test (considering anticomplementary sera as positive), 58.1 and 99.9%; the indirect enzyme immunoassay, 94.0 and 97.9%; the competitive enzyme immunoassay, 90.8 and 96.6%; and the fluorescence polarization assay, 93.5 and 97.2%; respectively. It was concluded that the fluorescence polarization assay was a valuable asset to the diagnosis of porcine brucellosis because of its accuracy, ease of performance and relative cost.

Identification of a Chemical Marker of Environmental Exposure to Formaldehyde

Formaldehyde (F) binds human serum albumin (HSA) covalently, giving rise to a molecular adduct F-HSA having the F as hapten. The humoral immune response to the adduct provides a biological marker of F exposure. In order to titrate serum anti-F-HSA antibodies, a new indirect competitive enzyme immunoassay (displacement assay) was developed. Two groups of about 90 heterogeneous healthy subjects were examined using twoin vitroconjugated F-HSA adducts with different ratios between F and HSA (5:1 and 10:1). Contingency table analysis showed a greater sensitivity (97%) and specificity (92%) of the test with the 10:1 F-HSA adduct than with the 5:1. Data examination using multivariate analysis of variance revealed that in both groups the smoking variable significantly explains (P<0.01) the values of the F exposure marker. A significant association with immunological response was obtained only in male smokers, using 5:1 F-HSA adduct, while with 10:1 ratio, a good association in male and female smokers was found. Results confirm that the immunological assay developed (displacement assay) could be a useful method for evaluating F exposure, especially for public health monitoring on a large scale.

Sestavení kompetitivní enzymové imunoanalýzy pro stanovení α-laktalbuminu a β-laktoglobulinů kravského mléka

Polyclonal antibodies were raised against three immunogens- a-lactalbumin (LA), -lactoglobulin A (LGA) and B (LGB). Using these antibodies the procedures of an indirect competitive enzyme immunoassay (ELISA) were constructed, optimized a nd characterized for determination of indi vidual immunogens. It was found that ELISA of LA is very specific without any inter­ ferences of other whey proteins. However, in ELlSAs of both lactoglobulins A and B were demonstrated very high interferences of the other genetic varian t (cross-reactivities 20-280% depending on antibody and immu nogen). An excellent sensitivity of ELISA for all proteins (detection limits for LA, LGA and LGB were 13, 0.4 and 54 ng/ml, respectively) makes it possible to analyze milk samples diluted more than 1000 times. Average values of variation coefficient were in the range 16-27%. A compari­ son of whey protein determinations in raw cow's milk by ELISA and by capillary electrophoresis resulted in the best similarit y in results of LA concentration. The decrease of LA, LGA and LGB concen trations was detected by using capillary electrophoresis for an analysis of whey from heat-treated milk, while ELISA of the same milk sample showed the increase of LGB immunoreac­ tivity to 700%.

Relationships among deoxynivalenol, ergosterol and Fusarium exoantigens in Canadian hard and soft wheat

Soluble extracellular components (exoantigens) from cultures of Fusarium graminearum and F. sporotrichioides were used to produce antisera from chickens for an indirect enzyme immunoassay. This immunoassay was used to estimate Fusarium exoantigen levels in 40 samples of fusarium head blight-infected hard red spring wheat from Manitoba, and in 50 samples of infected soft white winter wheat from Ontario. These wheat samples were also assayed for deoxynivalenol (DON), the predominant Fusarium mycotoxin, and for ergosterol, a metabolite reflecting fungal biomass. Using F. sporotrichioides antisera, the linear correlations between exoantigen level and DON content for the hard and soft wheats had coefficients of 0.80 and 0.76, respectively. With the same antisera, linear correlations between exoantigen level and total ergosterol concentration for the hard and soft wheats had coefficients of 0.66 and 0.81, respectively.

Development of anti-phenylurea antibody purification techniques for improved environmental applications

The non-uniform affinity of polyclonal antibodies is a major problem when these antibodies are used in immunochemical-based environmental applications. A specific affinity chromatography procedure was developed to extract selectively anti-isoproturon antibodies from a crude polyclonal antiserum. These antibodies were fully characterized and helped to improve the performances of immunoaffinity extraction and an indirect enzyme immunoassay.

Standardization of Smooth Lipopolysaccharide Preparations for use in Diagnostic Serological Tests for Bovine Antibody Brucella abortus.

A procedure for standardizing Brucella abortus smooth lipopolysaccharide used in diagnostic tests for brucellosis is proposed. The procedure is based on the reactivity of antigen preparations with a panel of sera with or without antibody to B. abortus using a set of parameters established with 13 antigen preparations. For each serum dilution, a mean and two standard deviations were calculated in the indirect and competitive enzyme immunoassays. If data obtained with an antigen preparation, using the same serum dilutions, falls within the range established using two standard deviations, the antigen would be considered acceptable for diagnostic use.

Prevalence ofMycoplasma pneumoniae andChlamydia pneumoniae in children with community acquired pneumonia

A prospective one year study was performed on 62 children admitted at the All India Institute of Medical Sciences with community acquired pneumonia (CAP) for the prevalence of Mycoplasma pneumoniae and Chlamydia pneumoniae. Diagnosis of infection with M. pneumoniae was based on serological tests viz microparticle agglutination test for detection of IgM antibodies and indirect immunofluorescence test for antigen detection from throat swabs (sensitivity 85.7%, specificity 93.3%). The indirect solid-phase enzyme immunoassay for detection of IgG antibodies was used to determine the prevalence of C. pneumoniae (sensitivity 88.8%, specificity 75.8%). Seventeen patients (27.4%) were found to have serological evidence of M. pneumoniae infection whereas only 4 (6.4%) patients were seropositive for C. pneumoniae. Results of this study indicate that M. Pneumoniae plays a significant role in CAP in infants and young children. Thus specialized laboratory testing for these agents should be more widely used thereby affecting empiric antibiotic regimens.

Manganese superoxide dismutase (MnSOD) and autoantibodies against MnSOD in acute viral infections

Sera of 146 patients with acute EBV, HAV, HBV, CMV, HSV, and rubella virus infections, and sera from 35 healthy controls were tested for the antioxidant enzyme manganese superoxide dismutase (MnSOD). An enzyme immunoassay that detects all isomeres of the enzyme was developed. The mean MnSOD value of healthy controls was 107 ng/ml. In HAV, HBV and EBV infections characterized by viral replication in internal organs, there was an average 5-fold rise of serum MnSOD, whereas in viral infections with low direct cytopathogenity, such as rubella, CMV and HSV, the MnSOD levels showed only minor rises. These sera were also tested for autoantibodies against MnSOD using a novel sensitive indirect enzyme immunoassay. The average IgM anti-MnSOD concentration in sera of healthy controls was 112 GU. In sera of patients with acute HBV, CMV, HSV or rubella virus infections IgM anti-MnSOD values were only slightly raised above the cut-off level. In contrast, in some patients with acute EBV infections anti-MnSOD concentrations rose up to 20-fold of normal values. In HAV infections the same phenomenon was observed in patients who had reactivated EBV infections. These findings indicate that EBV may facilitate the B-cell response to MnSOD. These autoantibodies may inhibit the protective function of MnSOD and prolong the disease by oxygen injury. Our concept on the pathogenic effect of the autoantibodies against MnSOD emphasizes the importance of the antioxidant enzyme in viral infections.

Potential Role of Abscisic Acid in Cotton Fiber and Ovule Development

Fibers and ovules of a cotton cultivar (Gossypium hirsutum L. Trambak-108) were analyzed for growth and free abscisic acid (ABA) content by indirect enzyme immunoassay. An inverse correlation between fiber elongation and ABA content was observed. In the seed, accumulation of ABA was observed during secondary thickening and the maturation phase. The potential role of ABA in fiber and seed development is discussed.

Ligand Choice Strategy for the Purification of Polyclonal Antibodies Used for Improved Immunochemical-Based Analytical Methods for the Herbicide Isoproturon

A common problem in developing immunochemical-based analytical methods is that the desired antibodies represent only a small portion of the heterogeneous antibody population. Affinity purification of polyclonal serum requires the correct choice of ligands used to extract antibodies featuring similar characteristics in their recognition properties (similar paratop group and dissociation constants). This paper describes several strategies of ligand design and synthesis used to purify polyclonal anti-serum generated against the herbicide isoproturon. A synthetic ligand mimicking the analyte isoproturon produced a 2.6 fold increase in isoproturon antibody concentration compared to an extraction using an IgG specific commercial gel. Another strategy involving the use of herbicide-protein conjugates resulted in affinity gels efficient in extracting 6 times more specific antibodies than the IgG commercial gel. The performances of immunochemical-based analytical methods using these refined antibodies were significantly improved. An indirect enzyme immunoassay gave an IC90 of 0.01 ng/mL, when assayed with river water samples and a better correlation with HPLC analyses was also reached (r = 0.998) for those samples. Immobilization of such antibodies on activated silica afforded a high capacity immunoaffinity column, which retained an average of 8.2 μg of isoproturon for 20 mg of immobilized antibodies per gram of silica.

Development and Validation of an Indirect Enzyme Immunoassay for the Detection of the Herbicide Isoproturon in Water Matrices

An indirect enzyme immunoassay (EIA) for the detection of the phenylurea herbicide isoproturon is described. The specific antibodies did not cross-react with other structurally related compounds. The concentration of isoproturon that inhibits 50% of antibody-antigen binding (IC50) was 0.64 ng/mL. The sensitivities were 0.07 ng/mL (IC80) and 0.02 ng/mL (IC90) respectively, when the crude serum was used in the assay. Matrix effects were observed when river water samples were analyzed showing recoveries as high as 150%. The IC50 was increased to 0.81 ng/mL. To overcome these difficulties, a novel method of anatibody purification was developed to reduce the heterogeneity of the medium when the test was performed with complex surface water matrices. This technique involved the extraction of the specific anti-isoproturon antibodies from the crude anti-serum. The refined fraction gave an IC50 not higher than 0.29 ng/mL and an IC90 of 0.01 ng/mL, when assayed with river water samples. The method was validated using a HPLC procedure with a clean up step involving an immuno-affinity column using the same antibodies. Excellent correlation (r = 0.998) was obtained between HPLC and the EIA results when the refined antibody was used in the assay. The use of affinity purified antibodies as an effective procedure in reducing matrix effects was demonstrated.

Determination of ginsenoside Rf and Rg2 from Panax ginseng using enzyme immunoassay.

We have developed an enzyme immunoassay (EIA) to quantify trace amounts of ginsenoside Rf (Rf), one of the glycosides of protopanaxatriol from Panax ginseng. A carrier protein of bovine serum albumin (BSA) was coupled to the carbohydrate component of Rf using the periodate oxidation method. Antibodies were raised in rabbits using Rf-BSA conjugate as the immunogen and competitive indirect EIA was used for the determination of Rf. The working range was 0.01-10 ng per assay. The anti-Rf antiserum cross-reacted with ginsenoside Rg2 (105%), which is also a component of Panax ginseng and has a very similar chemical structure to Rf. These results suggest that the anti-Rf antiserum could also be used for the quantitation of ginsenoside Rg2 as well as ginsenoside Rf. In a comparison of EIA and HPLC the linear regression equation and correlation coefficient for the two methods were y(EIA) = 1.31x (HPLC)-11.48 and 0.98, respectively.

Seroprevalence of parvovirus B19 antibody in HIV positive asymptomatic persons

Objective: Parvovirus B19 is an important cause of chronic anemia in human immunodeficiency virus (HIV)-positive patients. Extensive seroprevalence studies for parvovirus B19 in HIV-positive individuals have not been carried out in the United States. The authors compared the seroprevalence for parvovirus B19 among patients with asymptomatic HIV infection and healthy blood donors. Methods: The seroprevalence of IgG antibodies to VP-1, a parvovirus B19 structural protein, was determined using an indirect enzyme immunoassay (EIA) and a Western blot assay in 72 HIV-positive adults without prior opportunistic infection or acquired immunodeficiency syndrome (AIDS)-related malignancy and results were compared to those of 134 healthy blood donors. Results: There was a significantly higher seroprevalence for parvovirus 1319 in HIV-positive subjects (57/72, 79%) than in the controls (58/134, 43%) (P < 0.001). Analysis by indirect EIA of the HIV-positive subjects 1 year later showed no significant change in seropositivity (48/70, 69%). For HIV-positive subjects, B19 seropositivity was not significantly related to age, sex, or CD4 count, but the parvovirus index did correlate with the total IgG level at both time points (P = 0.014 at the first estimation and P = 0.045 1 year later). Western blot analysis of IgG antibody to the VP-1 protein showed that 49 of 71 (69%) of the HIV-positive subjects were positive at the beginning of the study, and 50 of 71 (70%) were positive 1 year later. Conclusions: These results suggest an increased seropositivity to parvovirus B19 among HIV-positive individuals compared to healthy controls.