Preparations for serodiagnosis of diseases due to causative agents of ixode tick-borne borreliosis (Lyme disease). Communication 1. Comparative study of the 1st generation enzyme immunoassay test-systems for detection of antibodies to Borrelia burkdorferi sensu lato]

Four foreign and one Russian 1st generation test-systems for detecting class G antibodies or summary antibodies to Borrelia burkdorferi sensu lato were comparitively investigated with the use of the clinical material under conditions of an encoded experiment. Cross reactions with sera from patients with syphilis, Epstein-Barr infection, cytomegalovirus infection and systemic lupus erythematosus were observed. The best specificity and sensitivity parameters were provided by the Enzygnost Borreliosis test-system.     

Preparations for serodiagnosis of diseases due to causative agents of ixode tick-borne Borreliosis (Lyme disease). Communication 2. Comparative study of recombinant enzyme immunoassay test-systems (rELISA) for serological diagnosis of ixode tick-borne Borreliosis]

One foreign and two Russian recombinant enzyme immunoassay test-systems were comparatively investigated under conditions of an encoded experiment. Sensitivity in the experiment with the Russian test-systems Borreliosis-ELISA-IgG and Lyme Best was 63.8 and 68.8% respectively. As for the test-system Borrelia IgG Recombinant, it was 47.5%. All the test-systems were highly specific (94.4 to 99.5%). The test-systems Lyme Best and Borrelia IgG Recombinant revealed partial cross reactions with sera from patients with systemic lupus erythematosus and leptospirosis.     

Assessment of sensitivity of commercial test-systems for HBsAg immunodetection according to their ability to detect HBsAg-mutants of hepatitis B virus

Comparative study of widely distributed on Russian market commercial test-systems for HBsAg detection by enzyme immunoassay was performed. Panel of serum samples containing mutant forms of HBsAg was developed for the study. It showed that only 2 out of 7 studied test-systems are able to detect mutant forms of HBsAg with the same sensitivity as "wild-type" forms of HBsAg. Test-systems based only on monoclonal antibodies did not detect HBsAg with G145R substitution in concentration 5 ng/ml. The study confirms the relevance of problem of HBsAg-mutants detection for hepatitis B immunodiagnostics.     

Immunoenzyme test-systems for diagnostics of cytomegalovirus infection

Cytomegalovirus infection (CMVI) is becoming the serious medical-social problem both in clinical-epidemiological aspects and in connection with demographic consequences. One of the necessary conditions for solving this problem is the organization of regular and comprehensive epidemiological surveillance for CMVI. Thereby, improvement of methods of diagnostics is becoming the most actual issue. Immunoenzyme assay-based test-systems "ECOlab-CMV-IgM" and "ECOlab-CMV-IgG" which are able to detect IgM and IgG for CMVI diagnostics and assessment of IgG titers dynamics were presented. Their high specificity and sensitivity were shown.     

Single-serum diagnosis of recent rubella infection with the use of hemagglutination inhibition test and enzyme-linked immunosorbent assays

Single-serum diagnosis of recent rubella infection was attempted with the use of hemagglutination inhibition (HI) test and two commercially available enzyme-linked immunosorbent assays (ELISAs). The period during which IgM antibody was detectable by IgM capture ELISA was 4 to 30 days after the onset of rash. Rubella IgG ELISA values relative to HI titer were lower in the sera from the patients with recent infection than in the sera from the subjects with remote infection. IgM ELISA and the combined use of IgG ELISA and HI test are two useful methods of single-serum diagnosis of recent rubella infection.     

Statistical analysis of distribution of antibody level against Mycobacterium bovis antigens for bovine tuberculosis diagnostics

Antibody responses to purified protein derivate PPD of tuberculin and to antigens MPB63 and MPB83 of Mycobacterium bovis were determined in bovine herd (94 adult animals). Statistical approach based on approximation by multiple Gaussians with Levenberg-Marquardt algorithm for analysis of antibody level distribution against antigens examined was provided. Our results confirm that indirect ELISA with recombinant MPB83 and MPB63 as well as conventional PPD could be used for test-systems development for detection of cow tuberculosis infection at the herd level.     

Comparison of diagnostic performances of ten different immunoassays detecting anti-CCHFV IgM and IgG antibodies from acute to subsided phases of Crimean-Congo hemorrhagic fever

Crimean-Congo Hemorrhagic Fever Virus (CCHFV) is a geographically widespread tick-borne arbovirus that has been recognized by the WHO as an emerging pathogen needing urgent attention to ensure preparedness for potential outbreaks. Therefore, availability of accurate diagnostic tools for identification of acute cases is necessary.A panel comprising 121 sequential serum samples collected during acute, convalescent and subsided phase of PCR-proven CCHFV infection from 16 Kosovar patients was used to assess sensitivity. Serum samples from 60 healthy Kosovar blood donors were used to assess specificity. All samples were tested with two IgM/IgG immunofluorescence assays (IFA) from BNITM, the CCHFV Mosaic 2 IgG and IgM indirect immunofluorescence tests (IIFT) from EUROIMMUN, two BlackBox ELISAs for the detection of CCHFV-specific IgM and IgG antibodies (BNITM), two Anti-CCHFV ELISAs IgM and IgG from EUROIMMUN using recombinant structural proteins of CCHFV antigens, and two ELISAs from Vector-Best (IgM: μ-capture ELISA, IgG: indirect ELISA using immobilized CCHFV antigen). Diagnostic performances were compared between methods using sensitivity, specificity, concordance and degree of agreement with particular focus on the phase of the infection.In early and convalescent phases of infection, the sensitivities for detecting specific IgG antibodies differed for the ELISA test. The BlackBox IgG ELISA yielded the highest, followed by the EUROIMMUN IgG ELISA and finally the VectorBest IgG ELISA with the lowest sensitivities. In the subsided phase, the VectorBest IgM ELISA detected a high rate of samples that were positive for anti-CCHFV IgM antibodies. Both test systems based on immunofluorescence showed an identical sensitivity for detection of anti-CCHFV IgM antibodies in acute and convalescent phases of infection.Available serological test systems detect anti-CCHFV IgM and IgG antibodies accurately, but their diagnostic performances vary with respect to the phase of the infection.     

Dynamics of the formation of antibodies to surface and internal proteins of the herpes simplex virus in a model experiment on rabbits

It has been shown by immunoblotting that antibodies to HSV-1 nucleocapsid proteins (39-45K) predominate at the early stages of infection (up to day 21 after infection) in rabbits. At later stages (up to day 75) the intensity of bands corresponding to virus-specific glycoproteins (presumably gD, gE, gC) increases. At the same time the level of antibodies to nucleocapsid proteins diminishes. In ELISA the same pattern was obtained using envelope and nucleo-capsid proteins denatured by SDS and 2-mercaptoethanol. The possibility of using individual HSV antigens for the development of highly specific diagnostic ELISA test-systems is discussed.     

Naturally acquired IgM antibody response to the C-terminal region of the merozoite surface protein 1 of Plasmodium vivax in Korea: use for serodiagnosis of vivax malaria

The antibody levels against the C-terminal region of the merozoite surface protein 1 of Plasmodium vivax (PvMSP1c) were measured in 276 patients with P. vivax malaria (patient group), 320 malaria-na?ve healthy individuals (control group 1), and 70 malaria-na?ve individuals with various disorders (control group 2) using the immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (ELISA) and the direct sandwich ELISA. To evaluate the antibody response during relapse, 5 relapsed patients were tested using the IgM capture ELISA. The IgM antibodies were negative in 99.7% of control group 1 and in 100% of control group 2; they were positive in 90.6% of the patient group. The total antibody levels were positive in 88.4% of the patient group with the direct sandwich ELISA. The sera from the second malaria episode, i.e., relapsed patients, were 100% positive on the IgM capture ELISA. The results of this study suggest that the IgM capture ELISA may be a useful diagnostic method for P. vivax malaria for both primary infection and relapse.     

Haemorrhagic fever with renal syndrome: evaluation of ELISA for detection of Puumala-virus-specific IgG and IgM

IgM and IgG ELISA to Puumala virus were evaluated using sera from patients with haemorrhagic fever with renal syndrome (HFRS) from different geographical regions: Sweden, Denmark, Norway, Belgium and the European USSR.IgM ELISA proved useful in the diagnosis of HFRS in patients from all the regions mentioned above. Specific IgM could be detected as early as day 1 post onset of disease, and patients remained IgM-positive for several months. Specific IgG ELISA antibodies were also frequently detected in acute sera, and acute-convalescent serum pairs often failed to show a significant titre rise or increase in optical density (OD) values. This limits the use of IgG ELISA in patient diagnosis. Sera collected 2 years after infection revealed higher IgG ELISA OD readings than convalescent sera, and very high values were still detectable 10 to 20 years postinfection. IgG ELISA is therefore useful for the testing of immunity and in seroepidemiological studies.Acute and convalescent sera from HFRS patients in Korea and the Asian USSR showed no or only very weak reactivity in the Puumala virus IgG and IgM ELISA. These results are consistent with the “one-way” crossing described earlier.     

A shortened dengue IgM capture ELISA using simultaneous incubation of antigen and peroxidase-labeled monoclonal antibody

A shortened IgM capture ELISA for the detection of dengue IgM antibodies using simultaneous incubation of antigen and peroxidase-labeled monoclonal antibody was described. The shortened two-step assay was compared with the four-step IgM capture ELISA on sera from dengue patients confirmed by the hemagglutination inhibition (HI) test. When paired acute and convalescent sera were tested, the shortened ELISA showed 100% agreement with HI results. It detected dengue IgM antibodies in the acute sera of 66% of patients with a primary dengue infection, 60% of patients with a secondary infection, and 98% of patients with a presumptive secondary infection. When the results of 151 dengue patients were combined, 75% of the acute sera were positive by the shortened IgM capture ELISA.     

Detection of IgG binding to Schistosoma mansoni recombinant protein RP26 is a sensitive and specific method for acute schistosomiasis diagnosis

We recently described the first recombinant Schistosoma mansoni protein RP26, which was capable of acute infection diagnosis. The aim of the present work was to further characterize the RP26 diagnostic properties in immunoblot and enzyme-linked immunosorbent (ELISA) assays. Testing sera from uninfected donors and sera from patients with acute or chronic Schistosoma infection by Western blot immunoassay revealed 100% specificity and 100% sensitivity for acute infection identification. Sera from uninfected, acute, and chronic schistosomiasis were also probed for IgG, IgG4, IgA, and IgM reactivity to RP26 plus soluble egg antigens (SEA) in ELISA. The mean IgG reactivity to RP26 by sera from acute schistosomiasis patients was significantly higher than the chronic ones. The IgG4, IgA, and IgM reactivities to RP26 were low and similar in both infected groups. The mean IgA and IgM reactivities to SEA were significantly higher in the group of acute compared to chronic group, whereas mean IgG4 reactivity was higher in chronic group. To estimate the specificity of Schistosoma infection diagnosis sera from patients infected with other different parasites were tested to detect IgG reactivity to RP26 and IgA and IgM reactivity to SEA. For IgA against SEA detection, 72% of sera were positive and 48% of sera were positive for IgM detection. Based on these results we can suggest that detection of sera IgG binding to RP26 is a sensitive and specific method for acute schistosomiasis diagnosis. Therefore, RP26 is a candidate for immunodiagnostic kit development.     

Evaluation of the indirect and IgM‐capture anti‐human cytomegalovirus IgM ELISA methods as confirmed by cytomegalovirus IgG avidity

Primary cytomegalovirus (CMV) infection during pregnancy often results in congenital CMV infection with severe clinical complications. IgM antibodies are one of the indices of primary infection. The IgG avidity index (AI) is also known to remain low for 3 months after primary infection. Here, we evaluated and compared the performance of CMV IgM and IgG avidity assays. Because sensitivity and specificity reportedly differ between CMV IgM kits, CMV IgM detection was compared between the two commercially available ELISA kits that are most commonly used in Japan. Sera for CMV IgM were first screened using a traditional indirect ELISA kit. Selected samples were then tested for CMV IgM and CMV AI using a CMV IgM‐capture ELISA kit and a CMV IgG avidity assay, respectively. The rate of concordance between the IgM kits was 89% (42/47), indicating the absence of any significant difference. Most of the CMV IgM‐positive plasma samples showed high CMV IgG AI; however, 18 commercially available plasma samples with low CMV IgG AI were all CMV IgM‐positive. One plausible explanation for this discrepancy is that the duration of low IgG AI is shorter than that of IgM positivity. Alternatively, CMV IgM tests may generate pseudo‐positive readouts in cases of congenital infection. Nevertheless, our study confirms that CMV IgG AI can be a reliable indicator of CMV primary infection.     

The use of Bartonella henselae-specific age dependent IgG and IgM in diagnostic models to discriminate diseased from non-diseased in Cat Scratch Disease serology

Cat Scratch Disease (CSD) is caused by Bartonella henselae infection and is a common cause of regional lymphadenopathy. The diagnosis of CSD largely depends on serology, but is hampered by both low sensitivity and specificity of the applied IgG and IgM assays. Using an in-house ELISA, we detected a significant age-dependent increase in the IgG levels in the general population compared to CSD patients. With this knowledge, we developed diagnostic models to differentiate diseased from non-diseased persons. Evaluation of these models using samples from PCR-positive patients (n=155) and age-matched controls (n=244) showed an important increase in the assay performance if the combination of the IgG and IgM results were taken into account. If the specificity was set at 98% the sensitivity was only 45% and 32% for the IgM and IgG ELISA, respectively but increased to 59% when these results were combined. Also the use of age-dependent factors further improved the clinical relevance of the outcome raising the sensitivity to 64%. Although the sensitivity of the ELISA remains low we conclude that the use of models using the combination of both IgM and IgG test results and age-depending factors can be a useful diagnostic tool in the serodiagnosis of CSD.     

Study of two different enzyme immunoassays for the detection of Mayaro virus antibodies

This paper presents the evaluation of an enzyme immunoassay in which Mayaro virus-infected cultured cells are used as antigen (EIA-ICC) and an IgM antibody capture ELISA (MAC-ELISA) for Mayaro serologic diagnosis using 114 human sera obtained during a Mayaro outbreak occurred in Bolivia, in 1987. Results were compared with those obtained by haemagglutination-inhibition test (HAI). MAC-ELISA was the most sensitive technique for anti-Mayaro IgM detection. MAC-ELISA was twice as sensitive as IgM EIA-ICC. The data shows that MAC-ELISA is a practical and valid technique for diagnosis of recent Mayaro infection. IgG EIA-ICC showed high sensitivity and high specificity compared to HAI. The combination of anti-Mayaro IgG and IgM EIA-ICC results presented the highest sensitivity of the study. Anti-Mayaro IgG and IgM simultaneous detection by EIA-ICC can be used for recent infection diagnosis (in spite of a less sensitive IgM detection than by MAC-ELISA), for surveillance and sero-epidemiologic studies, and for studies of IgG and IgM responses to Mayaro infection.     

Detection of IgM Antibrucella Antibody in the Absence of IgGs: A Challenge for the Clinical Interpretation of Brucella Serology

The use of enzyme-linked immunosorbent assay (ELISA) for the detection of IgG and IgM antibodies antibrucella has become widespread in the diagnosis of human brucellosis. IgM anti-Brucella antibodies are indicative of acute infection. Between 2009–2013, 5307 patients were evaluated for serologic diagnosis at the Microbiology Laboratory of the Albacete General Hospital. A ELISA IgM-positive, IgG-negative anti-Brucella antibody serology pattern was detected in 17 of those patients. Epidemiology data, symptoms, laboratory data, treatment and outcome from these patients were reviewed. Sixteen patients presented with musculoskeletal pain, fatigue and/or fever and 1 was asymptomatic. Five patients received treatment with doxycycline combined with rifampin, gentamycin or streptomycin during 6–12 weeks, with no improvement. None of the 17 patients were finally diagnosed with brucellosis. Our results indicate that anti-Brucella IgM positive serology, per se, is not enough to diagnose acute brucellosis and other methods should be used for confirmation. Brucella serology data should be interpreted taking into account the patient''s clinical history and epidemiological context.     

A Novel IgM‐capture enzyme‐linked immunosorbent assay using recombinant Vag8 fusion protein for the accurate and early diagnosis of Bordetella pertussis infection

An ELISA that measures anti‐PT IgG antibody has been used widely for the serodiagnosis of pertussis; however, the IgG‐based ELISA is inadequate for patients during the acute phase of the disease because of the slow response of anti‐PT IgG antibodies. To solve this problem, we developed a novel IgM‐capture ELISA that measures serum anti‐Bordetella pertussis Vag8 IgM levels for the accurate and early diagnosis of pertussis. First, we confirmed that Vag8 was highly expressed in all B. pertussis isolates tested (n = 30), but little or none in other Bordetella species, and that DTaP vaccines did not induce anti‐Vag8 IgG antibodies in mice (i.e. the antibody level could be unaffected by the vaccination). To determine the immune response to Vag8 in B. pertussis infection, anti‐Vag8 IgM levels were compared between 38 patients (acute phase of pertussis) and 29 healthy individuals using the anti‐Vag8 IgM‐capture ELISA. The results revealed that the anti‐Vag8 IgM levels were significantly higher in the patients compared with the healthy individuals (P < 0.001). ROC analysis also showed that the anti‐Vag8 IgM‐capture ELISA has higher diagnostic accuracy (AUC, 0.92) than a commercial anti‐PT IgG ELISA kit. Moreover, it was shown that anti‐Vag8 IgM antibodies were induced earlier than anti‐PT IgG antibodies on sequential patients’ sera. These data indicate that our novel anti‐Vag8 IgM‐capture ELISA is a potentially useful tool for making the accurate and early diagnosis of B. pertussis infection.     

应用单克隆抗体测定人弓形虫IgM抗体的研究

为了检测人血清弓形虫ＩｇＭ抗体,采用抗人ＩｇＭ单克隆抗体和特异性抗弓形虫单克隆抗体建立捕获ＥＬＩＳＡ法,并与ＰＣＲ方法进行了比较。结果检测１０６５份献血员血清,检出阳性３例,用ＰＣＲ方法检测呈阳性结果;检测２３例类风湿病人血清及２份弓形虫ＩｇＧ抗体阳性血清均为阴性反应。说明该方法不受类风湿因子（ＲＦ）和特异性ＩｇＧ抗体的干扰,同时也表明捕获ＥＬＩＳＡ检测人血清中弓形虫ＩｇＭ抗体特异性,敏感性良好。     

Evaluation of the usefulness of commercial enzyme-linked immunosorbent assays for diagnosis of Mycoplasma pneumoniae infections

The usefulness of the ELISA distributed by BioChem ImmunoSystems, Medial Polska, Biomedica/Virotech and prepared in our laboratory (ELISA FH-K) for diagnosis of the M. pneumoniae infections was estimated. Eighty six serum samples obtained from 86 patients with respiratory tract infections were simultaneously tested by ELISA-IgM/-IgG and by complement fixation test which was accepted as a reference test. The highest sensitivity in relation to the CFT was displayed by the ELISA BioChem ImmunoSystems and Medial Polska (100%), slightly lower sensitivity by the ELISA Biomedica/Virotech--96.5% and ELISA FH-K--90.9% when determining mycoplasmal antibodies of IgM. The lowest sensitivity was displayed by the ELISA Biomedica/Virotech when determining antibodies of the IgG class (54.9%). The specificity of ELISA in relation to the CFT was generally higher when detecting mycoplasmal antibodies of the IgM class then of IgG class. The study demonstrated that all 4 ELISA may be used in routine serodiagnosis of M. pneumoniae infection. For the improve of sensitivity of ELISA it's recommended to measure simultaneously the level of mycoplasmal antibodies of IgM and IgG.     

Comparison of commercially available and novel West Nile virus immunoassays for detection of seroconversion in pig-tailed macaques (Macaca nemestrina)

We report the assessment and validation of an NS1 epitope-blocking enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to West Nile virus (WNV) in macaques. Sera from naturally infected Macaca nemestrina were tested by ELISA and plaque reduction neutralization test (PRNT). Results were correlated with hemagglutination inhibition (HAI) data. Our results demonstrate that the blocking ELISA rapidly and specifically detects WNV infection in M. nemestrina. In addition, the diagnostic value of 7 commercially available immunoassays (PanBio immunoglobulin [Ig] M ELISA, PanBio IgG ELISA, PanBio immunofluorescence assay (IFA), InBios IgG ELISA, InBios IgM ELISA, Focus Diagnostics IgG ELISA, and Focus Diagnostics IgM ELISA) in M. nemestrina was evaluated and compared with that of the epitope-blocking ELISA. The PanBio IgG ELISA was found to effectively diagnose WNV exposure in M. nemestrina. Further, PanBio IFA slides are fast and reliable screening tools for diagnosing flaviviral exposure in M. nemestrina.