Classical Serological Methods Research Articles

Challenges for Precise Subtyping and Sequencing of a H5N1 Clade 2.3.4.4b Highly Pathogenic Avian Influenza Virus Isolated in Japan in the 2022-2023 Season Using Classical Serological and Molecular Methods.

The continuous evolution of H5Nx highly pathogenic avian influenza viruses (HPAIVs) is a major concern for accurate diagnosis. We encountered some challenges in subtyping and sequencing a recently isolated H5N1 HPAIV strain using classical diagnostic methods. Oropharyngeal, conjunctival, and cloacal swabs collected from a dead white-tailed eagle (Haliaeetus albicilla albicilla) were screened via real-time RT-PCR targeting the influenza A virus matrix (M) gene, followed by virus isolation. The hemagglutination inhibition test was applied in order to subtype and antigenically characterize the isolate using anti-A/duck/Hong Kong/820/80 (H5N3) reference serum or anti-H5N1 cross-clade monoclonal antibodies (mAbs). Sequencing using previously reported universal primers was attempted in order to analyze the full-length hemagglutinin (HA) gene. Oropharyngeal and conjunctival samples were positive for the M gene, and high hemagglutination titers were detected in inoculated eggs. However, its hemagglutination activity was not inhibited by the reference serum or mAbs. The antiserum to a recently isolated H5N1 clade 2.3.4.4b strain inhibited our isolate but not older strains. A homologous sequence in the previously reported forward primer and HA2 region in our isolate led to partial HA gene amplification. Finally, next-generation sequencing confirmed the isolate as H5N1 clade 2.3.4.4b HPAIV, with genetic similarity to H5N1 strains circulating in Japan since November 2021.

Analysis of frequency and molecular genetics of Jk (a-b-) phenotype among blood donors from Jining area

To screen for Jk(a-b-) phenotype among blood donors from Jining area and explore its molecular basis to enrich the rare blood group bank for the region. The population who donated blood gratuitously at Jining Blood Center from July 2019 to January 2021 were selected as the study subjects. The Jk(a-b-) phenotype was screened with the 2 mol/L urea lysis method, and the result was confirmed by using classical serological methods. Exons 3 to 10 of the SLC14A1 gene and its flanking regions were subjected to Sanger sequencing. Among 95 500 donors, urea hemolysis test has identified three without hemolysis, which was verified by serological method as the Jk(a-b-) phenotype and demonstrated no anti-Jk3 antibody. The frequency of the Jk(a-b-) phenotype in Jining area is therefore 0.0031%. Gene sequencing and haplotype analysis showed that the genotypes of the three samples were JK*02N.01/JK*02N.01, JK*02N.01/JK-02-230A and JK*02N.20/JK-02-230A, respectively. The splicing variant of c.342-1G>A in intron 4, missense variants of c.230G>A in exon 4, and c.647_ 648delAC in exon 6 probably underlay the Jk(a-b-) phenotype in the local population, which is different from other regions in China. The c.230G>A variant was unreported previously.

Putative Novel Avian Paramyxovirus (AMPV) and Reidentification of APMV-2 and APMV-6 to the Species Level Based on Wild Bird Surveillance (United States, 2016-2018).

Avian paramyxoviruses (APMVs) (subfamily Avulavirinae) have been isolated from over 200 species of wild and domestic birds around the world. The International Committee on Taxonomy of Viruses (ICTV) currently defines 22 different APMV species, with Avian orthoavulavirus 1 (whose viruses are designated APMV-1) being the most frequently studied due to its economic burden to the poultry industry. Less is known about other APMV species, including limited knowledge on the genetic diversity in wild birds, and there is a paucity of public whole-genome sequences for APMV-2 to -22. The goal of this study was to use MinION sequencing to genetically characterize APMVs isolated from wild bird swab samples collected during 2016 to 2018 in the United States. Multiplexed MinION libraries were prepared using a random strand-switching approach using 37 egg-cultured, influenza-negative, hemagglutination-positive samples. Forty-one APMVs were detected, with 37 APMVs having complete polymerase coding sequences allowing for species identification using ICTV's current Paramyxoviridae phylogenetic methodology. APMV-1, -4, -6, and -8 viruses were classified, one putative novel species (Avian orthoavulavirus 23) was identified from viruses isolated in this study, two putative new APMV species (Avian metaavulavirus 24 and 27) were identified from viruses isolated in this study and from retrospective GenBank sequences, and two putative new APMV species (Avian metaavulavirus 25 and 26) were identified solely from retrospective GenBank sequences. Furthermore, coinfections of APMVs were identified in four samples. The potential limitations of the branch length being the only species identification criterion and the potential benefit of a group pairwise distance analysis are discussed. IMPORTANCE Most species of APMVs are understudied and/or underreported, and many species were incidentally identified from asymptomatic wild birds; however, the disease significance of APMVs in wild birds is not fully determined. The rapid rise in high-throughput sequencing coupled with avian influenza surveillance programs have identified 12 different APMV species in the last decade and have challenged the resolution of classical serological methods to identify new viral species. Currently, ICTV's only criterion for Paramyxoviridae species classification is the requirement of a branch length of >0.03 using a phylogenetic tree constructed from polymerase (L) amino acid sequences. The results from this study identify one new APMV species, propose four additional new APMV species, and highlight that the criterion may have insufficient resolution for APMV species demarcation and that refinement or expansion of this criterion may need to be established for Paramyxoviridae species identification.

Значение иммуноферментного анализа в серологической диагностике бруцеллеза крупного рогатого скота

For the serological diagnosis of brucellosis in cattle in veterinary laboratories of the Russian Federation, one or several tests are used simultaneously. The sensitivity and specificity of the determination of antibodies to the pathogens of this disease using various methods varies significantly, none of the serological tests is suitable for all stages of the infectious process in brucellosis. ELISA is a modern serological test that has not found wide application in the state veterinary laboratories of the Russian Federation, although the number of studies conducted by this method increases annually. The use of enzyme-linked immunosorbent assay makes it possible to confirm the diagnosis of brucellosis at all stages of the infectious process and to identify more seropositive animals than classical serological methods. Among serological methods, enzyme-linked immunosorbent assay (hereinafter referred to as ELISA) is the most sensitive, specific and easy-to-perform test. In accordance with the Order of the Ministry of Agriculture of the Russian Federation dated 08.09.2020 No. 533 «On approval of Veterinary rules for the implementation of preventive, diagnostic, restrictive and other measures, the establishment and cancellation of quarantine and other restrictions aimed at preventing the spread and elimination of foci of brucellosis (including infectious epididymitis of sheep)» the ELISA method is one of the confirmatory in the diagnosis of brucellosis. The data obtained as a result of the studies indicates that the results of ELISA coincide with the complex of classical serological methods (BRS – Bengal roses sample; AR – agglutination reaction; CFR – complement fixation reaction; RIH – reaction of indirect hemaggtination; IDR – immunodiffusion reaction) and a higher diagnostic sensitivity of the test.

THE IMPORTANCE OF BRUCELLIN ALGERIC SKIN TEST FOR DIAGNOSIS OF BOVINE BRUCELLOSIS

Infection with Brucella results in the induction of both humoral andcellular immune responses. Humoral immune resposne is based on monitoringthe occurrence of specific antibodies against smooth lipopolysaccharide (S-LPS)of Brucella. However, in cattle, classical serological methods can detect antigenicdeterminants for other types of microorganisms (cross reactivity) such as Escherichiacoli 0:157, Yersinia enterocolitica 0:9, Salmonella urban, Pseudomonas malthopilia andPasteurella. The aim of our work was to determine the immunological responsebased on the use of standardized and purified allergen in which lypopolysaharid hasbeen removed and doesn’t induce humoral immune response. A total of 16 dairycattle previously tested positive using RBT (Rose Bengal test) and CFT (complementfixation test) were tested for confirmation with BST (brucelline skin test) accordingto the instructions of the producer. B. melitensis B115 (Synbiotics BrucellergeneOCB) was used in the test. 14 of 16 cattle reacted with skin thickening >1 mm after72 hours from the application of brucellin. 2 animals with no skin thickening orthickening <1mm also reacted negative in CFT. This outcome can be attributed tocross reactions with other antigens than Brucella that commonly occurs in RoseBengal test.Brucellin allergic skin test is not recommended as a standalone diagnostic toolbecause all infected animals do not react therefore this test cannot be recommendedas a self-sufficient diagnostic test or for the purpose of international trade.However, due to high specificity and adequate sensitivity at the herd level, it can berecommended for the control of herds in areas free of brucellosis.

A new platform for serological analysis based on porous 3-dimensional polyethylene sinter bodies

A new sensitive and selective platform, three-dimensional immunosensor, has been developed for a rapid serological diagnosis; detection of a Borrelia infection was considered as a model assay. The immunosensor is based on a 3-dimensional (3D) porous solid surface (sinter body) with dimensions of 2×2.5mm where a recombinant variable lipoprotein surface-exposed protein (VlsE; Borrelia-antigen) is immobilized by different techniques. The sinter body served as a robust and inexpensive carrier, which facilitated a successful hydrophobic adsorption as well as covalent immobilization of the antigen with sufficient amounts of on the surface. Because of sinter body’s porosity, the detection could be performed in an immune affinity flow system based on a little disposable plastic column. The flow of reagents through the column is advantageous in terms of reducing the non-specific interaction and shortening the test time. Furthermore, three labels were tested for a colorimetric detection: i) a horseradish peroxidase (HRP) labeled secondary antibody, ii) nanoparticles based on Sudan IV, and iii) gold nanoparticles modified with protein A. HRP secondary labeled antibody provides the most sensitive test, 1000 fold dilution of serum sample can be clearly detected in only 20min. Gold nanoparticles modified with protein A were used as a direct label or as a catalyst for reduction of silver ions. Direct detection with gold nanoparticles provides short time of analysis (5min) while detection of metallic silver required longer time (12min) but with improved sensitivity. Nanoparticles based on Sudan IV showed high background and were less favorable. The assay is distinctive because of the rapid analysis time with all used labels, longest 20min. Compared to classical serological methods for Borrelia diagnosis, the developed method offers a simple, rapid and reliable tool of analysis with minimal cost and can be easily transferred to other infectious diseases.

A loop-mediated isothermal amplification (LAMP) assay for early detection of Schistosoma mansoni in stool samples: a diagnostic approach in a murine model.

BackgroundHuman schistosomiasis, mainly due to Schistosoma mansoni species, is one of the most prevalent parasitic diseases worldwide. To overcome the drawbacks of classical parasitological and serological methods in detecting S. mansoni infections, especially in acute stage of the disease, development of cost-effective, simple and rapid molecular methods is still needed for the diagnosis of schistosomiasis. A promising approach is the loop-mediated isothermal amplification (LAMP) technology. Compared to PCR-based assays, LAMP has the advantages of reaction simplicity, rapidity, specificity, cost-effectiveness and higher amplification efficiency. Additionally, as results can be inspected by the naked eye, the technique has great potential for use in low-income countries.Methodology/Principal findingsA sequence corresponding to a mitochondrial S. mansoni minisatellite DNA region was selected as a target for designing a LAMP-based method to detect S. mansoni DNA in stool samples. We used a S. mansoni murine model to obtain well defined stool and sera samples from infected mice with S. mansoni cercariae. Samples were taken weekly from week 0 to 8 post-infection and the Kato-Katz and ELISA techniques were used for monitoring the infection. Primer set designed were tested using a commercial reaction mixture for LAMP assay and an in house mixture to compare results. Specificity of LAMP was tested using 16 DNA samples from different parasites, including several Schistosoma species, and no cross-reactions were found. The detection limit of our LAMP assay (SmMIT-LAMP) was 1 fg of S. mansoni DNA. When testing stool samples from infected mice the SmMIT-LAMP detected S. mansoni DNA as soon as 1 week post-infection.Conclusions/SignificanceWe have developed, for the first time, a cost-effective, easy to perform, specific and sensitive LAMP assay for early detection of S. mansoni in stool samples. The method is potentially and readily adaptable for field diagnosis and disease surveillance in schistosomiasis-endemic areas.

DIAGNOSTIC VIROLOGIQUE DE LA GRIPPE

The authors review data concerning the classical and modern virological techniques allowing the diagnosis of influenza virus infection in laboratory. Traditional isolation methods of influenza virus in cell culture, direct and rapid detection of viral antigen by immunoassay tests, molecular biology techniques based on the detection of specific viral nucleic acids by reverse transcription polymerase chain reaction, and classical serological methods are described and their preferential indications specified.

Preliminary report on the use of biotechnology in sweet and sour cherry research

The goal of this investigation was to determine genetic differences between autochthonous and introduced cultivars of sweet cherry and between cultivars and types of sour cherry, and to find and optimize a method for the rapid recovery of clonal material. A great number of cherry cultivars and types within the population of cv. Oblačinska sour cherry exist in Croatia and the selection of autochthonous cultivars based on special visible properties for further investigation has been done in previous research. Differences were found in a number of important agronomic traits within the populations of cv. Oblacinska sour cherry. It is suspected that autochthonous sweet cherry cultivars are synonyms for known old cultivars, which were introduced randomly and naturalized under local names. In this approach difficulties arise from the effect of non-genetic factors on the expression of certain traits. The genetic/physiological problem of S allele autoincompatibility exists within sweet cherry cultivars. The detection of S alleles is required to determine compatible cultivar pairs in the orchard. Biotechnological methods based on the polymerase chain reaction (PCR) facilitate faster virus detection compared with classical serological methods and indexing. Thermotherapy and tissue culture make it possible to recover valuable clone material for introduction in the premultiplication process.

Brucellosis complicating chronic non-infectious disorders: diagnostic and therapeutic dilemmas

There is little information in the literature on the clinical progress of brucellosis in patients affected by other non-infectious diseases; however, the infection can often trigger an exacerbation of existing underlying conditions in certain target organs. In this report we present four cases of brucellosis complicating previous diseases, and the difficulties in relation to their diagnosis and treatment. The study involved four patients with the following disorders: polycythaemia vera, pulmonary fibrosis, cirrhosis of the liver and arthritis of the knee. Brucellosis was diagnosed by classical serological and bacteriological methods. The strains involved could be isolated only in three of the four patients: two strains were Brucella abortus biovar 1 and one was Brucella suis biovar 1. Two patients relapsed 10 and 7 months after admission, another presented chronic brucellosis and received various therapy schemes, and one died. Since the best selection of antibiotics and the optimal duration of therapy remain unknown for patients having brucellosis complicated by previous pathologies, these remain at the discretion of the attending physician. Management of our patients was controversial in terms of the selection of antibiotics, duration of treatment and decision regarding surgery.

Novel Dombrock Blood Group Genetic Variants in Different Ethnic Groups Identified by High Throughput Beadchip DNA Analysis.

Background : Modern bioarray chip technology enables rapid allele determination in a large number of donors by DNA typing when compared to classical serological methods. We analyzed five single nucleotide polymorphisms (SNPs) associated with Dombrock (DO) alleles in blood samples from donors of different ethnic groups, and found new genetic variants.Method : The BeadChip™ technology used primers for a single multiplex PCR reaction, and allele-specific oligonucleotides with variable 3′-terminal nucleotide for elongation-mediated multiplex analysis (eMAPTM) of the DO polymorphisms. Color-encoded beads displaying the elongation probes were assembled into planar arrays of small footprint on semiconductor chips, permitting the instant imaging of fluorescent elongation products from the entire array. Five SNPs of DO blood group antigens DOA/DOB (nt 378 C>T, 624 T>C, 793 A>G), Hy+/Hy− (nt 323 G>T) and Jo(a+)/Jo(a−)(nt 350 C>T) were analyzed simultaneously. Blood samples from a total of 445 donors, comprising of 100 Thai, 69 Jewish, 58 Chinese and 218 random New York City donors were analyzed.Results : As shown in the table below, the predominant diploid allelic combinations observed in all the groups were DOB/DOB followed by DOA/DOB and then DOA/DOA. In the random donor group, we also observed several less prevalent allele combinations: DOB/HY (14.2%), DOB/JO (1.4%), DOA/HY (0.5%), DOA/JO (2.3%), HY/HY (1.4%), JO/JO(1.8%), and HY/JO (0.9%). Interestingly, two novel alleles in trans with the known alleles: DOB/HA (11% in Thai, 2.9% in Jewish, 6.9% in Chinese and 2.8% in random donors), DOA/HA (1.8% in random donors, 1.7% in Chinese and 1.4% in Jewish), DOB/SH (0.5% in random donors) and HY/SH (1.4 % in random donors) were also detected. HA allele is 378T, 624T, 793A (like JO but with 350C). Hemagglutination studies showed that HA, as predicted from the nucleotide at position 793, encoded Doa antigen. SH allele is 378C, 624C, 793C (like HY but with 323G) and is predicted to encode Dob antigen.Conclusion: The BeadChip™ technology facilitated the identification of novel genetic variants, in addition to accelerating blood group typing.Most prevalent DO allele combinations (in %)Ethnic GroupDOB/DOBDOA/DOBDOA/DOAThai73142Jewish52.230.49Chinese79.312.10Random38.521.611

Comparison of serological and molecular techniques for detection of Tomato yellow leaf curl begomovirus*

Tomato yellow leaf curl begomovirus (TYLCV) causes severe damage to tomato crops. The virus is not considered as established in France, although a single outbreak (subjected to eradication measures) occurred in 1999. Since the vector of TYLCV, the whitefly Bemisia tabaci, is already present in France and the virus is present in several neighbouring Mediterranean countries, there is a high risk of introduction into France. To prevent spread of the disease, systematic detection is essential. Samples of tomato and Eustoma grandiflorum were tested, and different methods were compared to determine which is the best for regular tests. Classical serological methods (TAS‐ELISA and immuno‐printing) were compared with molecular hybridization (which is specially suitable for the single‐stranded DNA genome of Geminiviridae), and PCR. Tissue‐printing methods, which avoid time‐consuming sample preparation, gave consistent results with hybridization. In contrast, immunoprinting detection confirmed only 61 % of samples found positive with TAS‐ELISA. Furthermore, a comparison of signal intensities obtained with TYLCV‐IL or TYLCV‐Sar probes provided indications on viral strain(s) detected. The efficiency, reliability and feasibility of serological and molecular techniques for routine official testing are discussed.

Detection of bovine leukaemia virus (BLV) infection by DNA probe technology

Classical serological methods and Southern blot hybridization for the diagnosis of bovine leukaemia virus (BLV) infection have been compared during the first nine months of life of offspring from BLV serum-negative and serum-positive dams belonging to a Friesian dairy herd in Italy. At birth, 9/13 calves analysed showed serum positivity for anti-gp60 BLV antibodies by agar immunodiffusion and/or by ELISA. However, only two calves were positive for BLV integrated proviruses in their lymphocyte DNA. At six months of age, anti-gp60 BLV antibodies and proviral DNA positivities were simultaneously shown only by the two cattle identified as DNA-positive at birth. This pattern remained constant up to nine months of age. Furthermore, analysis of the molecular characteristics of BLV integrated proviruses, carried out by using, as probes, the almost complete proviral genome (Belgian isolate) or a subclone of the env gene radioactively labelled or chemically modified, revealed that the calves under study were infected by a different isolate (Japanese isolate) and that, in one of the cattle, the majority of integrated proviruses was characterized by deletions probably located in the 5' half of the proviral genome.

Anti-HIV antibodies in the CSF of AIDS patients: a serological and immunoblotting study.

CSF and serum samples from 16 AIDS patients were tested for the presence of anti-HIV antibodies either by classical serological methods or by an immunoblot technique based on agarose gel isoelectric focusing and transfer of the specific IgG antibodies onto HIV antigens-loaded nitrocellulose sheets. This method enabled the demonstration of an intrathecal synthesis of anti-HIV oligoclonal IgG antibodies, often superimposed on diffuse polyclonal production, in 14 patients. The two negative cases were devoid of neurological signs or symptoms. However, two patients classified in stage II of the disease (asymptomatic infection) displayed an intrathecal synthesis of anti-HIV antibodies.

Method for rapid quantitation and characterization of insulin antibodies.

Nearly all insulin-treated diabetic patients develop antibodies to insulin. The clinical importance of these antibodies is not exactly known regarding their general influence on the therapeutic control of the diabetic and the development of secondary complications. There is no doubt, however, about the role of the antibody titer in some forms of insulin resistance. Besides cases of hypoglycemia caused by insulin injections and the rare forms of diabetes caused by autoimmune insulin antibodies, insulin resistance is the main indication for measuring insulin antibodies. On the other hand, measurement of free and bound insulin in insulin-treated patients and the characterization of the antibodies is of broad scientific interest. The classical serological methods such as immunoprecipitation, complement binding, agglutination, and hemagglutination have proved not to be applicable to the insulin antibody, or too insensitive (see e.g., 1). The introduction of radiolabeled insulin by Kallee (2) made possible direct determination of antibody-bound insulin. In recent years several such methods have been described. Free and antibody-bound insulin are separated by either electrophoresis on various supporting materials (3-9); ultracentrifugation (1, 10, 11); adsorption by dextran-charcoal (12, 13), ion-exchange resin (14), or cellulose (15); gel filtration (16); or by precipitation methods (17-20). In some of these methods the equilibrium between free and antibody bound insulin is influenced during the procedure; moreover, most of them are too laborious for routine use. In 1971, Desbuquois and Aurbach (21) described how free and antibody-bound peptide hormones could be separated by use of polyethylene glycol (PEG). In the following we describe a method involving PEG for rapidly measuring and characterizing insulin antibodies.

Diagnostic Serologique de la Syphilis Par Immunofluorescence

SummaryThe author has studied 478 sera with the indirect immunofluorescence method and the classical serological methods (Bordet-Wasserman, Reiter, Kahn, V.D.R.L.) and Nelson’s test (TIT). The results of all these agreed for 442 sera (92,4 %), i.e. for 109 posittive and 334 negative sera. But in 36 cases (7,6 %) the results of the immunofluorescence disagree with those of the classical serology or TIT : the IF results were positive 24 times, negative 12 times. But in 33 cases, the IF raction agreed with the clinical diagnosis or with the TIT.In three sera only, the IF reactions seemed to be wrong. They were sera no. 7 (IF negative), no. 27 (IF positive ++) and no. 23 (IF positive + [+]). Taking into account the TIT and eventually the clinical symptoms, the results of the IF test are correct in 475 cases out of 478, and may be false in the 3 other Cases (i.e. 0,3 % of the total).Among the tests of the classical serology, when the IF test is positive, it is the V.D.R.L. test which is most often also positive.In the routine analysis, the IF test can economize material, personnel and time. But its chief advantage is surely a very great certainty, because it is a very sensitive and specific test and it closely agrees with the TIT and the clinical picture.