Enzyme-linked Immunoassay Procedure Research Articles

Programming an antibody-peptide sandwich procedure for selectivity and ultrasensitive detection of fibrin using double-stranded DNA (dsDNA) hemp string probe

A novel, highly sensitive and selective sandwich-like enzyme-linked immunoassay procedure for the detection of fibrin was developed. Firstly the monoclonal anti-fibrinogen which was coated on the surface of microtiter plate can interact with fibrin, and the probe of double-stranded DNA (dsDNA) hemp string containing the homing peptide can secondarily recognize the fibrin. On the other hand, combined with the hybridization chain reaction (HCR), the terminal of dsDNA can be folded to a long G-quadruplex-hemin DNAzyme, which can catalyze luminol–H2O2 system to generate strong chemiluminescence signal. Under the optimal experimental conditions, the assay showed a linear toward fibrin concentration in the range of 0.14–72.5 pM with detection limit of 0.04 pM. Because of high selectivity and ultra-sensitivity, the designed procedure provides great potential for applications in medical diagnosis.

Development of a fluorescent microsphere immunoassay for cystatin B (CSTB) in serum of patients with hepatocellular carcinoma

Serum cystatin B (CSTB) concentrations have been reported to be increased in patients with hepatocellular carcinoma compared to concentrations seen in normal subjects. In this study, we developed a "fluorescent microsphere immunoassay" (FMI) capable of specifically detecting CSTB in serum. The FMI used a microparticle conjugated polyclonal antibody to CSTB and biotinylated monoclonal antibody as capture protein and probe protein, respectively. The results were obtained using the Bio-Plex(200) system. The dose-response relationship between CSTB and fluorescent intensity showed linearity in the range 0-1000 pg/mL and 7 pg/mL, sensitivity lower than 11.2 pg/mL. This result revealed that the FMI system was more sensitive than enzyme-linked immunoassay (ELISA). Additionally, the FMI system used smaller sample volumes compared to ELISA. We measured CSTB with both the FMI and an ELISA procedure and compared the two methods. The CSTB concentrations in serum specimens as measured with the FMI assay system were similar to those measured with ELISA. Thus, the new FMI using the Bio-Plex system may be useful for detection of CSTB in human serum.

Anticardiolipin antibodies in the sera of patients with diagnosed chronic fatigue syndrome

Examination of anticardiolipin antibodies (ACAs) in the sera of patients clinically diagnosed with chronic fatigue syndrome (CFS) using an enzyme-linked immunoassay procedure demonstrated the presence of immunoglobulin M isotypes in 95% of CFS serum samples tested. The presence of immunoglobulin G and immunoglobulin A isotypes were also detected in a subset of the samples. Future studies will focus on elucidating whether alterations to mitochondrial inner membranes and/or metabolic functions play a possible role in the expression of ACAs.

In vitro assessment of bee venom effects on matrix metalloproteinase activity and interferon production.

Controversial immunomodulatory properties of bee venom (BV) have provided an appropriate field for more investigation. The aim of present research was to verify the effects of honeybee venom on matrix metalloproteinase activity and interferon production as well as cell proliferation in monocyte and fibroblast cell lines.The monocyte and fibroblast cell lines (K562, HT-1080, WEHI-164) were used in order to assess proliferative response, interferon-1 production and matrix metalloproteinase-2 (MMP-2) activity. Australian BV (ABV) and Iranian BV (IBV) preparations at concentrations of 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, and 1microg/ml were added to each overnight cultured cells. In time course study, cells were treated each with ABV and IBV. In all cases supernatants were collected 24 hours after treatment. A sample of the each medium was used for zymography and interferons assay. Non-treated cells were used as controls.The production of IFN-a and IFN-b in supernatant of cell cultures was assessed using enzyme linked immunoassay procedure. MMP-2 activity, as an inflammatory index, was evaluated using zymoanalysis method.The results of this study showed that, there were no significant difference between two sources of honey bee venoms when they were added to an identical cell line, whereas, the responses of various cell lines against bee venom were different. The increasing amounts of bee venom to human monocyte cell line (K562) revealed a significant increase in proliferative response. Our findings showed that the bee venom had no influence on IFN-a production in cell culture media, whereas, adding the BV to K562 cell line could significantly increase the production level of IFN-b only on day 8 post-treatment. In addition the effect of bee venom on MMP-2 activity in both cell culture media, WEHI-164 and K562 was similar. The stimulatory effect of bee venom on MMP-2 activity occurred at low doses. In contrast, its inhibitory effect was seen at high concentrations.It is concluded that, honeybee venom affects on MMP-2 activity and interferon beta production as well as cell proliferation in a time and dose-dependent manner.

Bee Venom, Immunostimulant or Immunosuppressor? Insight into the Effect on Matrix Metalloproteinases and Interferons

The current evidence has defined conflicting properties for bee venom. The goal of this study was to determine whether bee venom (BV) is an immunosuppressor or immunostimulant. The WEHI-164, HT-1080, and K562 cell lines were used for assaying toxicity, proliferative response, matrix metalloproteinase-2 and -9 (MMP-2 and MMP-9) activity, and interferon production. The Australian and Iranian BV (ABV and IBV) were used at concentrations of 0.025–1 μg/ml in triplicate and 2-fold dilutions. MMP-2 and -9 activities were evaluated using the zymography method. The production of interferons (IFN-α and IFN-β) were assessed using enzyme-linked immunoassay procedure. Our results show no significant difference between two sources of honeybee venom (ABV and IBV) when they are added to an identified cell line, whereas the response of various cell lines against BV could be different. The increasing amounts of ABV and/or IBV (between doses 0.025–0.5 μg/ml) to human monocyte cell line (K562) exhibit a significant increase in proliferative response. Our data show that the immunomodulatory effect of ABV and/or IBV on MMP-2 and MMP-9 activity in both cell culture media, WEHI-164 and K562, is similar. The stimulatory effect of BV on MMP-2 and –9 activities is occurred between doses 0–0.05 μg/ml. In contrast, the inhibitory effect of BV on these two MMPs is seen at concentrations of >0.05 μg/ml. The ABV and/or IBV has no influence on IFN-α production in cell culture media, whereas adding the BV to K562 cell line could significantly increase the production level of IFN-β only on day 8 posttreatment. We conclude that time- and dose-dependent response as well as the type of treated cell line could determine the immunosuppressive and/or immunostimulant property of bee venom that could be effective in future therapeutic strategies.

The effects of aspartic acid-bond isomerization on in vitro properties of the amyloid beta-peptide as modeled with N-terminal decapeptide fragments.

The 42-amino acid A beta, the major constituent of the senile plaque deposits of the brains of Alzheimer's disease patients, exhibits a high degree of heterogeneity at its N-terminus. Isomerization of aspartic acid bonds at residues 1 and 7 renders A beta more prone to aggregate and form extended structure as it was shown by in vivo and in vitro studies. We recently demonstrated the ability of mid-chain aspartic acid-bond isomerization to break the dominant helical structure of the N-terminal decapeptide fragment by CD. In the current study we use molecular modeling to show that insertion of the extra -CH2-group into the decapeptide backbone results in the formation of stable reverse-turns and destabilizes the helical conformer that competes with the extended structure at the full-sized peptide level. The molecular modeling also reveals a limited propensity of the diisomerized peptide to form extended structure directly. Anti-A beta pAb 2332 is more sensitive for the non-isomerized status of the decapeptide than that of the full-sized peptide. mAb 6E10, raised against unmodified A beta recognizes only the unmodified decapeptide or the peptide isomerized at the first aspartic acid in a conformation-dependent manner, but does not recognize the mid-chain isomerized or diisomerized decapeptide in any circumstance. The diisomerized decapeptide was used as immunogen to generate polyclonal antibody 14943 that is not selective for the isomerized status of either the full-size peptide or the decapeptide, but recognizes the isomerized peptides preferentially when the peptide antigen structures are conserved during the enzyme-linked immunoassay procedure. Owing to the aberrant behavior of the full-sized A beta peptide during standard RP-HPLC, serum stability studies that indicate extracellular stability can be more effectively performed on the decapeptide fragments. Remarkably, the diisomerized peptide exhibits a significantly increased stability towards serum peptidases compared with the unmodified or monoisomerized peptides, suggesting a possible mechanism of the retention of the isomerized A beta peptide in the affected brains.

Colony immunoblot assay of botulinal toxin.

Botulinal neurotoxin in and around colonies of Clostridium botulinum types A, B, and E and of toxigenic Clostridium butyricum was detected by an enzyme-linked immunoassay procedure whereby the toxin was transferred from the agar medium to a nitrocellulose support and the immobilized toxin was probed with type-specific antibodies. The method identified the toxin types of the colonies grown from a mixed inoculum of C. botulinum serotypes. The specificity of the antitoxins for type A and B toxins was improved by adsorption of the antitoxins with the antigens of heterologous type cultures.

A novel covalent enzyme-linked immunoassay (CELIA) for simultaneously measuring free and immune complex bound antibodies of defined specificity I. Application to naturally occurring antipolyamine antibodies in human sera

A simple covalent enzyme-linked immunoassay procedure (CELIA) is described for the routine determination of free and immune complex-bound antibodies in sera. Assays for the latter could not have been performed by adsorption ELISA due to the high ionic strength of the reassociating buffer. For the measurement in human sera of free naturally occurring IgG and IgM antibody directed against the hapten spermine, polycarboxystyrene microtiter plates with covalently coupled spermine were used. For the determination of immune complex-bound antipolyamine IgG and IgM antibody titers, serum was first dissociated at pH 2.3 in tubes and then reassociated at pH 8.1 in the wells of a microtiter plate containing covalently bound spermine. The reactivity of anti-spermine antibodies was increased from 2- to 13-fold after dissociation and reassociation compared to that of non-dissociated area. The apparent reaction constant ( R app.) of free IgG antibodies to spermine in the sera of 19 bronchopulmonary patients with cancer differed significantly from the R app. values of IgG antibodies having this specificity in ten other patients with non-malignant disease.

Screening for hemoglobins S and C in newborn and adult blood with a monoclonal antibody in an ELISA procedure

To facilitate the screening of blood for the presence of hemoglobins S or C, we devised an enzyme-linked immunoassay (ELISA). The ELISA procedure incorporated a murine monoclonal antibody (mAb), beta s-1, which recognized both Hb variants but did not react with Hb A, Hb A2 or Hb F. Hemoglobins in cord or adult hemolysates were coated on the surface of wells of polystyrene microtiter plates and treated with beta s-1 mAb, followed by goat anti-mouse IgG conjugated with horseradish peroxidase. After addition of tetramethylbenzidine substrate solution, a deep blue color developed, signifying the presence of Hb S or Hb C. The beta s-1 mAb ascites fluid could detect purified Hb S and Hb C when diluted to over 1/512,000 and cord blood hemolysates containing Hb/S or Hb C when diluted to 1/128,000. Although maximal reactivity was achieved using undiluted hemolysates, the ELISA system could easily detect Hb S and Hb C in cord blood hemolysates when diluted 10(-4). The sensitivity of the ELISA was 1%, which exceeds the lowest quantities of these variants normally found in cord blood. In addition, we found that the ELISA procedure was suitable for detecting Hb S/Hb C in whole blood as well. The entire assay could be conducted on multiple samples in less than 1 h, thus providing a specific, sensitive, rapid and simple screening technique for Hb S and Hb C in cord or adult blood.

Measurement of Rheumatoid Factor Isotypes in the Clinical Laboratory

In this study we assessed the clinical utility of measuring all major rheumatoid factor (RF) isotypes (IgG, IgA, and IgM) in the diagnostic immunology laboratory using an enzyme-linked immunoassay (ELISA). An improved method for IgG-RF was tested which employed a commercially available monoclonal anti-human IgG Fd antibody and did not require pepsin digestion of samples. We detected elevated levels of all three RF isotypes in a population of hospitalized rheumatoid arthritis patients (n = 109). We demonstrated a significant association between IgM and IgA RF which occurred in 36% of our subjects, while less than 6% had IgM + IgG RF or IgG + IgA RF. A comparison of the IgM ELISA with the Rheumaton revealed a statistically significant correlation (r = 0.65, p = 0.001). In addition, the two methodologies were equivalent in sensitivity (ELISA: 76%, Rheumaton: 78%). However, the ELISA procedure was more time consuming, costly, and required greater technical expertise. The following clinical and laboratory findings were significantly associated with RF isotypes: IgG RF and the presence of rheumatoid nodules (p = 0.03), elevated erythrocyte sedimentation rate (ESR) and IgG RF (p = 0.007), and elevated ESR and IgM RF (p = 0.0009). Our ELISA methodology did not provide significant advantages over existing techniques to justify its use as part of the routine laboratory assessment of rheumatoid factor.

Use of a urease-antibody conjugate in an enzyme immunoassay for the detection of blastomycosis.

A urease-antibody conjugate was used in an enzyme-linked immunoassay (ELISA) for the detection of antibodies in blastomycosis. A Blastomyces dermatitidis immunodiffusion antigen was used as the reference antigen in a solid phase indirect ELISA procedure and the endpoints were determined visually. Urease ELISA results on serum specimens from patients with blastomycosis compared favorably (90%) with results obtained spectrophotometrically by the alkaline phosphatase enzyme system. Specificity was evaluated with assays on sera from patients with histoplasmosis (20% cross reactivity), coccidioidomycosis (0% cross reactivity) and on Histoplasma capsulatum skin-test positive individuals (0% cross reactivity). The ease of performance of the urease ELISA combined with no requirements for specialized spectrophometric equipment are factors that favor the continued development of the test as an alternative serodiagnostic method. The assay may prove to be useful and a valuable adjunct to fungal antibody screening procedures.

Quantification of measles virus antigens in infected CNS tissue: Development of an enzyme linked immunoassay

An enzyme linked immunoassay procedure has been developed which allows accurate quantification of low levels of measles virus antigens in infected central nervous system (CNS) tissues. In its development a systematic study was made of the effects of a large excess of nonviral proteins on the assay results. It was found that controls using single proteins such as bovine serum albumin were inadequate. Further, results indicate that experimental samples and viral antigen standards must be closely matched for their content of CNS tissue proteins since such proteins exert distinctive quantitative effects, both dramatic and subtle.

Plasminogen activator-anti-human fibrinogen conjugate

A covalent conjugate between the plasminogen activator urokinase and polyclonal rabbit anti-human fibrinogen has been formed using the heterobifunctional coupling reagent N-succinimidyl 3-(2-pyridyldithio) propionate. The resultant urokinase-anti-human fibrinogen conjugate was separated from unreacted material by gel filtration. The conjugate exhibited amidase activity against the small chromogenic substrate pyroglutamyl-glycyl-arginine-p-nitroanilide as well as plasminogen activator activity in an assay employing plasminogen and the plasmin substrate D-valyl-leucyl-lysine-p-nitroanilide. Retention of antibody specificity for fibrinogen was demonstrated using an enzyme linked immunoassay procedure. The conjugate was found to have greater stability in human plasma than unconjugated urokinase.