Indirect Solid-phase Enzyme Immunoassay Research Articles

Improvement of lifetime and post-mortem diagnostics of trichinellosis in animals of the Suidae family (comparative study)

Trichinosis is one of the most dangerous helminthozoonoses. Methods of compressor trichinelloscopy and peptolysis of animal muscle tissue in artificial gastric juice used for postmortem diagnosis of trichinosis are reliable enough to identify sources of infection and prevent the development of helminthiasis in humans. In addition to these methods, indirect solid-phase enzyme immunoassay (ELISA) and crystalloscopy were used. Meat juice samples obtained from domestic pigs from various regions of the Russian Federation and wild boars were used as the tested biological material. The formulation of ELISA and crystalloscopy, accounting and interpretation of the results obtained were carried out according to the classical method: the result was considered positive, exceeding the negative control by 0.08 units or more in optical density. The specificity of ELISA in all animals with heterologous invasion was 98%, hydrosol-hydrogel reactions - 80% and crystalloscopy - 75%. These methods can be used for testing for trichinosis, but the examination should be comprehensive: immunological and crystallographic methods should be confirmed by compressor trichinelloscopy and peptolysis using artificial gastric juice. Currently, in the system of veterinary and sanitary measures for trichinosis, there is a change in the structure of the sources of invasion. The probability of human infection with hunting products is higher than when eating pork obtained from meat processing enterprises. Therefore, field trichinelloscopic control in the field (CT, HH etc.) occupies a leading place in diagnostics. Thus, the development of portable equipment for veterinary examination and the training of hunters in basic trichinelloscopy skills, as well as the improvement of the formulation and accounting of crystalloscopy and НН are becoming extremely relevant.

IL-22 and ACPA Levels and their Relationship to the Disease Activity in Rheumatoid Arthritis Patients

Aim: to determine ACPA IgG and IL-22 levels in RA patients and their relationship to the disease activity Place and duration of the study: A cross sectional study and prospective cohort study was performed from August 2020 to January 2021 in rheumatology outpatient clinic and laboratory of Al- Quwayiyah General hospital. Methodology: Forty five rheumatoid arthritis patients were included and 35 healthy participants free of any diseases considered as control group. The patients in this study met the American College of Rheumatology's 2010 guidelines. RA Disease activity was assessed for rheumatoid patients using DAS28 scoring. Serum samples collected from the patients and control to perform ESR, Hs-CRP, RF factors and also IL22 and ACPA IgG which were detected using sandwich ELISA and indirect solid phase enzyme immunoassay techniques respectively. Results: Out of the 45 RA patients, 34(75.6%) were females and 11(24.4%) were males aged from (28-67years) with median patient age 42 years. There was no statistically significant difference regarding age and sex between RA patients and control. Thirty (66.7%) of the 45 RA patients had low disease activity or remission, while 15 (33.3%) had moderate to extreme disease activity. Thirty two 32(71.1%) patients of the 45 RA patients had erosive disease. The level of ESR, hs-CRP and RF are increased in the patient group than control, in spite that there were significant differences in the Mean± SD among RA group and control group regarding RF, there was no significant statistical differences ESR, hs-CRP. in the study there was an increase in ACPA and IL-22 levels in patients suffering of RA; 21.52±1.29 U/ml and 71.22±10.63 pg\ml. respectively. While among control there was low serum levels; 14.06±2.01U/ml 33.25±2.41pg\ml and respectively. Significant statistical difference was observed regarding IL-22 and ACPA IgG levels among RA patients and control (P=0.038 and P=0.019 respectively). There is a significant positive relationship (positive correlation) detected between ACPA and IL-22 levels, (r=-0.810; p=0.597). The levels of IL-22 and ACPA were significantly associated with DAS 28. Their relationship was strong as the r value was 0.427 and 0.411 respectively. Conclusion: IL-22 and ACPA IgG levels were highly increased among RA patients in comparison to the control group. The IL-22 and ACPA IgG levels were strongly correlated with the rheumatoid disease activity, DAS 28. These results suggest that Il-22 can be used in association with ACPA IgG level as diagnostic and prognostic markers of rheumatoid arthritis

Comparative analysis of micronutrients status of HIV infected and HIV non- infected subjects attending three selected hospitals in Kano Metropolis, Kano State, Nigeria

Comparative studies on serum levels of micronutrients (iron, iodine, Vitamin A and β – carotene) were carried out among HIV negative and people living with HIV and AIDS attending three hospitals in Kano, namely Murtala Mohammed Specialist Hospital, Aminu Kano Teaching Hospital and Infectious Diseases Hospital. HIV infection was confirmed using indirect solid phase enzyme immunoassay technique of Immunocomb II HIV 1 and 2 Bi spot kits. Serum micronutrients levels of 270 subjects were detected using colorimetric and spectrophotometric techniques. Results obtained showed that serum vitamin A and β-carotene concentrations decrease with increase in age and were higher in males than females. The mean serum concentrations (in µg/dl) of iron, iodine, vitamin A and β- carotene were found to be 99, 1.53, 39.4, 45.98 for HIV negative subjects; 62.6, 1.54, 20.16, 19.2 for HIV positive subjects and 28.8, 0.9, 16.1 and 18.0 for AIDS subjects respectively. Deficiency of iron, vitamin A and β-carotene were found to be more common among people living with HIV and AIDS when compared with HIV negative subjects at 5% level of significance with AIDS patients having the lowest value (p<0.05). This could be due to malabsorption, altered metabolism, reduced food intake and/or infection in HIV infected persons. Iodine level of these patients was found to be normal (1.54 and 0.9 µg/dl). The results imply that HIV infection affects the serum levels of iron, vitamin A and β-carotene by reducing their levels; hence, dietary supplementation with them could be useful in maintaining good health in HIV infected subjects and reducing mortality.Key words- AIDS, HIV, Kano, Micronutrients, Serum.

Prevalence of antimitochondrial antibodies in autism spectrum subjects

ABSTRACT Objective: Autism is a neurodevelopmental disorder characterized by impairment in verbal and nonverbal communication, repetitive and stereotypic behavior. Dysregulated immune system has a role in the pathogenesis of Autism. This study was designed to measure the prevalence of antimitochondrial (AM) antibodies in a group of autistic children. Methods: AM antibodies subtype 2 (AMA-M2) were evaluated by indirect solid phase enzyme immunoassay in 62 autistic children and 14 age-matched healthy controls. Autistic activity was assessed by using the Childhood Autism Rating Scale. Results: Significantly elevated levels of AMA-M2 were observed in the sera of autistic children (n = 54, 0.221 ± 0.029 IU/ml [mean ± SEM]) compared with healthy controls (n = 14, 0.111 ± 0.010 IU/ml [mean ± SEM], p = 0.0008) and there was no significant difference in patients with moderate to severe autism (p = 0.49). AM antibodies in autistic patients have no correlation with Childhood Autism Rating Scale score. Conclusion: The current study demonstrated significantly high levels of AMA-M2 in autistic subjects when compared with healthy controls. Further large-scale studies are required to dissect any pathogenic role of these antibodies in the development of autism.

A8.25 The effect of abatacept therapy on granzyme B serum levels in patients with rheumatoid arthritis

Background and objectives Rheumatoid arthritis (RA) is characterised by increased numbers of circulating CD28neg T-cells, a population displaying functional characteristic of cytotoxic memory cells, including expression of granzymes, a family of serine proteases playing key roles in the induction of target-cell death. Soluble granzymes (as granzyme B (GrB)) are elevated in sera from patients with RA, in which are associated with development of radiographic erosions. Abatacept (CTLA4-Ig; ABA) competing with the engagement of CD28 on T-cells, influences the subsequent T-cell activation. In patients treated with ABA, the number of circulating CD28neg T-cells decreases, suggesting that the drug can prevent the generation of CD28neg T-cell populations. Since CD28neg T-cells may be an important source of GrB, we have evaluated the effect of ABA therapy on GrB serum levels in patients with RA. Materials and methods Fifty-three RA patients, treated for at least 3 months with ABA were evaluated. Disease activity and response to the treatment were measured with DAS28 (based on CRP) and EULAR Criteria. T-cell counts were determined by flow cytometry. Serum GrB samples were collected before the first administration of ABA (T0) and then after 6 months (T6), and measured by an indirect solid-phase enzyme immunoassay with a sensitivity limit of 20 pg/ml. Results The percentage and the absolute number of circulating CD4 + CD28-neg T-cells decreased after ABA therapy (T0 vs. T6: p = 0.018; p = 0.018, respectively), as well as those of CD8 + CD28-neg T cells (T0 vs. T6: p = 0.005; p = 0.008, respectively). At T0, GrB serum levels were detectable in all RA patients, and were correlated with disease activity (p = 0.0022), and percentages of circulating CD4 + CD28- (p = 0.007) and CD8 + CD28- T-cells (p = 0.031). In 25 patients serum level of GrB were evaluated at T6: in 18 patients with a moderate or good clinical response to ABA the levels of GrB significantly decreased from T0 (median: 62.8 pg/mL[10th-90th percentile:45.8–116]) to T6 (53.8 [46.4–96.6]; p = 0.023), whereas no variation was observed in 7 non responders. The variation of GrB levels was directly correlated with that of DAS28-PCR (p = 0.040), but not with those of circulating CD28-neg T-cell subsets. Conclusions Costimulation blockade by ABA can decrease the serum levels of GrB in RA patients responding to the treatment, suggesting that this might be one of the mechanism by which ABA can prevent radiographic erosions. However, the lack of correlation of such decrease with the numbers of circulating CD28-neg T-cells suggests that these cells are not the main source of serum GrB.

Seroprevalence of Chlamydia trachomatis in Enugu, Nigeria

Chlamydia infections in women cause pelvic inflammatory disease, which often results in devastating consequences of infertility, ectopic pregnancy, or chronic pelvic pain. The infection is largely asymptomatic. To determine the seroprevalence of Chlamydia trachomatis in Enugu, South Eastern Nigeria. A population-based prospective study comprising female residents of Enugu, South Eastern Nigeria. Indirect solid phase enzyme immunoassay of Chlamydia antibodies was done using ImmunoComb C0. Trachomatis IgG Kit (Orgenics). The population comprised 136 female undergraduate students and 150 non-student women. The overall prevalence of C. trachomatis in the population studied was 29.4%. The percentage of subjects who admitted to be having multiple sexual partners was higher among the student population (71.2%) compared to those from the non-student population (28.8%). The highest percentage of seroprevalence was 28 (33.3%) in the age group of 20-24 years for the student population and 18 (21.4%) in the age group of 25-29 years for the non-student population. The highest seroprevalence of C. trachomatis antibodies (69.0%) in both populations was observed in females without any history of infection. Females that had pelvic inflammatory disease, sexually transmitted infection, and secondary infertility assayed for C. trachomatis had seroprevalence levels of 19%, 9.5%, and 2.4%, respectively. There was a positive correlation between positive Chlamydia assay and the type of subject population (student or non-student) with r2 value of 1.55 at P < 0.01. C. trachomatis infection is largely underdiagnosed and remains a silent disease in the apparently healthy population of Enugu, South eastern Nigeria.

Comparison of Anti-mutated Citrullinated Vimentin with Anti-cyclic Citrullinated Peptide and Rheumatoid Factors for the Diagnostic Value of Rheumatoid Arthritis

Objective: Serologic support is important for the diagnosis of rheumatoid arthritis (RA). Anti-cyclic citrullinated peptide (anti-CCP) has relatively low sensitivity despite of high specificity for the diagnosis of RA. Anti-mutated citrullinated vimentin (anti-MCV) is an indirect solid phase enzyme immunoassay for the quantitative measeurement of IgG class autoantibodies against MCV, a novel serologic marker for an aid in the diagnosis of RA. We investigated a diagnostic value of anti-MCV in comparison with those of anti-CCP, and rheumatoid factors (IgM, IgG, and IgA RFs) in RA patients. Methods: Cohorts of 48 patients with RA and 50 patients with osteoarthritis (OA) were enrolled. All patients underwent standard examinations, including anti-MCV, anti-CCP, IgM RF, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP). We measured IgG RF and IgA RF titers in 21 RA and 50 OA patients. The sensitivities and specificities of anti-MCV, anti-CCP, RFs were determined. Correlations with these markers, ESR and CRP were analyzed.

Development of an enzyme immunoassay for poliovirus antigens

An indirect solid-phase enzyme immunoassay (EIA) was developed for the detection of poliovirus antigen. Virus antigen was obtained in LLC-MK2 cell cultures and used to prepare antibodies in rabbit and guinea pig. Antibodies were evaluated by double immunodiffusion and neutralization test. Optimal concentrations of guinea pig and rabbit immunoglobulins were determined by checkerboard titration. Microtitre plates were coated with 15.0 µg/ml guinea pig anti-polio immunoglobulin and rabbit anti-polio immunoglobulin at the concentration of 7.94 µg/ml was used as detecting antibody. The standard curve with eight different antigen concentrations in eight replicates resulted in a coefficient of variation (CV) between 2.1% to 7.8%. The dose-response relationship was determined by simple linear regression with a coefficient of correlation (R²) equal to 96.4%. The assay detected a minimum of 2.3 µg/ml poliovirus antigen.

Production of fumonisins by Fusarium moniliforme strains isolated from corn grain

Fusarium moniliforme is the predominant fusarium species in the grain mycoflora of corn grown in Northern Caucasus, accounting for 95% of fusarium isolates. Eighty-five Fusarium moniliforme strains were grown on grain substrate and checked for the presence of fumonisins (B1 + B2 + B3) by indirect solid-phase enzyme immunoassay (EIA). All strains were capable of producing fumonisins (0.95 to 32,000 mg/kg). Strains sampled in the Krasnodar krai produced the highest fumonisin levels (averaging 5490 mg/kg).

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.

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.

Comparison of two methods for the assessment of the levels of autoantibodies to thyroid tissue antigens

The levels of autoantibodies to total extract of the thyroid were assessed using passive hemagglutination test and of antibodies to thyroglobulin (TG) and thyrocyte microsomal fraction (TMF) by indirect solid-phase enzyme immunoassay in 62 adolescent girls with diffuse enlargement of the thyroid and autoimmune thyroiditis. The efficacies of the two methods were compared. Autoantibodies to thyroid tissue antigens were reliably better detected by enzyme immunoassay. The mean levels of autoantibodies to TG and TMF measured by enzyme immunoassay were reliably increased in patients with autoimmune thyroiditis, whereas in the group of adolescent girls with diffuse enlargement of the thyroid they did not differ from the norm.

Potential of monoclonal antibodies to improve therapeutic monitoring of cyclosporine.

We show that monitoring of cyclosporine by immunoassay could be improved by using monoclonal antibodies (MAbs) of restricted specificity instead of polyclonal antisera that recognize both unmodified cyclosporine and its metabolites. MAbs with high affinity for cyclosporine have been prepared and characterized. We tested their ability to discriminate between native cyclosporine and its metabolites in indirect solid-phase enzyme immunoassay with a set of cyclosporine metabolites modified at residues 1, 4, 6, and 9 (corresponding to the six known sites of metabolism of cyclosporine). All the metabolites tested were detected by MAb1 at least 15- to 1000-fold less well than unmodified cyclosporine. A second MAb recognized unmodified cyclosporine and most of its metabolites equally well. Both MAbs retained their activity when coupled to alkaline phosphatase and could therefore be used in a direct solid-phase enzyme immunoassay.

Solid-phase enzyme-immunoassay for the detection of herpes simplex virus antigens in clinical specimens

An indirect solid-phase enzyme-immunoassay (EIA) for the detection of herpes simplex virus (HSV) antigens in clinical specimens was developed. Rabbits and guinea pigs were hyperimmunized with highly purified nucleocapsids of HSV type 1. Microtitre plates were coated with 0.25 μg of guinea pig anti-herpes simplex type 1 immunoglobulins per well. Clinical specimens, diluted in phosphate buffered saline containing fetal calf serum and detergents, were sonicated and incubated in the test wells overnight at 37°C. Rabbit anti-HSV immunoglobulins were added as a secondary antibody at a concentration of 3.2 μg per well, and peroxidase conjugated swine antibodies against rabbit immunoglobulins, diluted 1:1,000, were used as a fourth layer. Clinical specimens which were sent for virus isolation or for isolation of Chlamydia trachomatis were tested by the developed assay and 20 out of 27 isolation positive specimens were found positive by EIA. Five out of 67 specimens negative by isolation gave positive results by EIA. The specificity of the results was confirmed by a control test using wells coated with normal guinea pig immunoglobulins. The test detected antigens from both serotypes of HSV. Cross reactions with varicella-zoster- or with cytomegalovirus were not found, and antigens from uninfected cells did not result in false positive results.

Specificity of IgM antibodies in acute human coxsackievirus B infections, analysed by indirect solid phase enzyme immunoassay and immunoblot technique.

The specificity of the IgM response in acute human coxsackievirus B infections was examined by indirect solid phase enzyme immunoassay and immunoblot techniques. IgM antibodies detected by ELISA were either strictly type-specific, type-predominant or group-reactive to coxsackieviruses B-1 to B-5. Homotypic and type-dominant responses were clearly correlated with the serotype isolated from the patient. Analysis of ELISA antigens by SDS-gel electrophoresis, protein transfer and subsequent immunodetection of individual virus polypeptides, revealed VP1 as the major antigen; it was detected by homotypic as well as heterotypic serum specimens.

Identification of parainfluenza virus serotypes by indirect solid-phase enzyme immunoassay.

An indirect enzyme immunoassay was used to detect and identify parainfluenza virus serotypes 1 and 3 in cell culture residuals from which infectious virus could no longer be recovered.