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Paracoccidioidomycosis (PCM) is a systemic mycosis caused by fungi of the genus Paracoccidioides. Serological tests are auxiliary in the diagnosis of PCM. However, the lack of standardization is a central problem in serodiagnosis and antibody titration. The objective of this study was to propose a methodology based on Fourier transform infrared spectroscopy (FTIR) for predicting antibody titers in patients with PCM. A total of 118 serum samples from patients with PCM were included, for which antibody titration using double immunodiffusion (DID) was previously performed. Serum samples were analyzed by attenuated total reflection (ATR)-FTIR and a supervised analysis with partial least squares regression (PLS) was used to predict the antibody titers. The PLS model with two latent variables and with the use of one orthogonal signal correction (OSC) showed a determination coefficient (R2) higher than 0.9999 for both the calibration and prediction set. The model was able to predict the antibody titers from patients with PCM with a minimal error. Therefore, modeling with FTIR/ATR and multivariate calibration proved to be a fast and highly accurate method for antibody titration, replacing the need for antigen production and performance of traditional serological tests.

The implementation of animal health improvement and disease prevention activities with respect to bovine leukosis involves a need for timely detection of cattle infected with bovine leukaemia virus (BLV) on livestock farms. That is why early diagnosis using more sensitive and highly accurate methods is of particular importance. The paper presents the results of cattle serum tests for bovine leukosis with enzyme-linked immunosorbent assay (ELISA) and immunodiffusion (ID), as well as the comparative assessment of their effectiveness. A total of 440 cattle blood samples were subjected to serological testing with immunodiffusion; 37 (8.4%) of them tested positive for bovine leukaemia virus. The cattle blood samples were submitted from the Kumtorkalinsky (127), Karabudakhkentsky (122), Buynaksky (89) Raions, from Makhachkala (56) and Kaspiysk (46). Seropositivity was 17 (13.4%), 8 (6.6%), 5 (5.6%), 4 (7.1%) and 3 (6.5%), respectively. For the comparative assessment of the diagnostic tests, 100 (5 ID-positive and 95 ID-negative) serum samples were taken and tested with ELISA. As a result, specific antibodies against BLV gp51 antigen were detected in 4 ID-negative serum samples. All ID-positive serum samples also tested positive with ELISA. All in all 9 virus carriers were detected with ELISA, that is 44.4% more than with immunodiffusion. Thus, enzyme-linked immunosorbent assay is characterized by a higher sensitivity, as compared with immunodiffusion, and allows for improved detection of infected animals. However, alongside the advantages, this technique has certain disadvantages, one of which is the high price of the diagnostic test kit for anti-BLV antibody detection and the equipment required.

ABSTRACT The Ouchterlony double immunodiffusion assay is a serological technique used in the detection of antibodies and antigens for diagnostic purposes and also used in immunology laboratory courses as a common teaching assay where students observe the geometrical precipitation line patterns that form in the agarose, elucidating degrees of homology between antigens. In this classical technique, students must wait several hours to days to obtain results when protein antigens and antibodies are used. Furthermore, these proteins degrade over time if not frozen or stored in the refrigerator and are the most expensive consumables of the laboratory exercise. In this study, inexpensive and commonly used inorganic ionic salt solutions that are stable and can be stored at room temperature for several years were used to mimic antigens and antibodies. The precipitation lines started to form in the agarose plates after 15 min and fully developed within an hour, showing different geometrical precipitation patterns and spur formations that could be identified by students as full identity, partial identity, and nonidentity between the simulated (inorganic) antigens. Students conducting this exercise in a combined lecture and laboratory immunology course were able to finish the exercise as well as record and discuss results within class time, and tvhey showed increased interest in the laboratory exercise and had a better understanding of antibody-antigen reactions. Thus, this simulated laboratory experiment is an inexpensive, safe, and fast exercise that allows students to observe precipitations reactions of the Ouchterlony assay within the class session time.

Histoplasmosis is a condition generally characterized by constitutional and pulmonary symptoms. Clinical manifestations include vague constitutional and pulmonary symptoms. It is unusual for it to present as a neck mass. In this report, we present the case of a 13-year-old girl admitted because of a growing neck mass. Serum and urine Histoplasma were negative, but complement fixation positivity, Computer Tomography (CT) chest and histological analysis and history of recent travel pointed to the diagnosis of Histoplasma. The objective of this report is to highlight diagnostic testing that can be used to prevent such a case from being undiagnosed.

The article gives an account of certification performed for a new batch of a Reference standard to be used with the test system for determination of the fractional (antigenic) composition of human serum products by immunoelectrophoresis -industry reference standard (IRS) 42-28-77 - in accordance with existing requirements. A candidate reference standard was represented by a batch of normal human serum intended for diagnostic purposes (more than 500 donors) and a batch of immunoelectrophoresis serum against human serum proteins (antiserum against human serum proteins). In accordance with the certification programme, the fractional (antigenic) composition of the reference standard was determined by immunoelectrophoresis using «KliniTest-EF» buffer and 0.05 M borate buffer solution and the modes stipulated in the general monograph 1.8.2.0002.15 Agar gel immunoelectrophoresis. The reaction between the antiserum against human serum proteins and the normal human serum components of the reference standard produced at least 15 precipitation lines. The study demonstrated that the bromphenol blue dye could be used together with «KliniTest-EF» buffer to assess albumin migration, and the pyronin B dye could be used together with «KliniTest-EF» buffer and 0.05 M borate buffer solution to assess immunoglobulin migration. The study confirmed the applicability of the certified reference standard designed for the test system for determination of the fractional (antigenic) composition of human serum products by immunoelectrophoresis, and set the criteria for its use in the context of human serum products identification (species specificity) testing not only by immunoelectrophoresis, but also by agar-gel immunodiffusion.

Background: Blastomycosis can cause severe disease with progressive respiratory failure and dissemination even in immunocompetent individuals. We sought to evaluate risk factors for severe disease and mortality using clinical and laboratory data within a large health system in an endemic area. Methods: We performed a retrospective cohort study of patients diagnosed with blastomycosis at all Mayo Clinic sites from 1 January 2004 through 31 March 2020. Diagnosis was established by culture, histopathology/cytopathology, serology, antigen testing, or PCR. Disease was categorized as mild for patients treated in the outpatient setting, moderate for hospitalized patients who did not require intensive care, and severe for patients admitted to the intensive care unit. Logistic regression was used to evaluate risk factors for severe disease. A Cox proportional hazards model was constructed to evaluate mortality. Findings: We identified 210 patients diagnosed with blastomycosis. Mean age was 51 years (range, 6–84). Most subjects were male (71.0%). Extrapulmonary disease was confirmed in 24.8%. In this cohort, 40.5% of patients had mild disease, 37.6% had moderate disease, and 21.9% had severe disease. Independent risk factors for severe disease were neutrophilia (odds ratio (OR) 3.35 (95% CI 1.53–7.35), p = 0.002) and lymphopenia (OR 3.34 (95% CI 1.59–7.03), p = 0.001). Mortality at 90 days was 11.9%. Median time from diagnosis to death was 23 days (interquartile range 8–31 days). Independent risk factors for mortality were age (OR 1.04 (95% CI 1.01–1.08), p = 0.009), neutrophilia (OR 2.84 (95% CI 1.04–7.76), p = 0.041), and lymphopenia (OR 4.50 (95% CI 1.67–12.11), p = 0.003). Blastomyces immunodiffusion had an overall sensitivity of 39.6% (95% CI 30.1–49.8). Sensitivity was higher among those who were tested 4 weeks or longer after the onset of symptoms. Urine Blastomyces antigen had a significantly higher sensitivity of 80.8% (95% CI 68.1–89.2) compared to serology. There was a trend towards higher antigen concentration in patients with severe disease. The sensitivity of PCR from respiratory specimens was 67.6% (95% CI 50.1–85.5). Conclusion: In this cohort, we did not find an association between pharmacologic immunosuppression and disease severity. Lymphopenia at diagnosis was an independent risk factor for mortality. This simple marker may aid clinicians in determining disease prognosis.

Background: The influenza virus is one of the most important factors for higher morbidity and mortality in the world. Recently, researchers have been focused on influenza conserved antigenic proteins such as hemagglutinin stalk domain (HA2) for vaccine production and serological studies. The HA2 plays a major role in the fusion of the virus with host cells membrane. The immunity system enables to produce antibody against HA2. The aim of this study is polyclonal antibody production against influenza HA2. Methods: This study was done in the Influenza Research Lab, Pasteur Institute of Iran, Tehran for one year from September 2013 to October 2014. In the present study, recombinant HA2 protein was produced in prokaryotic system and purified using Nickel affinity chromatography. The purified HA2 was mixed with Freund’s adjuvant (complete and incomplete) and injected into two New Zealand white rabbits by intramuscularly and subcutaneously routes. Immunization was continued for several months with two weeks interval. Before each immunization, blood was drawn by venous puncture from the rabbit ear. Function of rabbit's sera was evaluated using radial immunodiffusion (RID) in both forms, Single RID (SRID) and Double RID (DRID). Finally, antiserum activity against HA2 was evaluated using western blotting as serological assay. Results: Sedimentary line and zone was observed in RID assays (SRID and DRID) represent interaction between HA2 protein and anti- HA2 antibody. As well as, western blotting results was positive for HA2 protein. Therefore, these results showed that polyclonal antibody produced against HA2 protein can identify HA2 protein antigenic sites. Conclusion: These findings show that humoral immune responses have properly been stimulated in rabbits and these antibodies can identify HA2 protein and may be suitable for other serological methods.

Antigenic proteins were used as markers for the study of relationships between three liverwort species from P. epiphylla complex. It has recently been shown that the electrophoretic phenotypes of this species suggested an amphiploid origin of P. borealis. Two sibling species: P. epiphylla -species S and -species N could have probably represented the parental species for P. borealis. We examined three clones of P. borealis from different localities using immunodiffusion. Then we compared them with P. epiphylla species S and N as well as with the mixture of proteins of P. epiphylla S and N samples. The results indicate that polyploid P. borealis shows an identical immunological pattern to that of the mixture of proteins of putative parental species. Only in one case the result resembled much more the pattern of P. epiphylla S proteins. The sibling species P. epiphylla S and N showed antigenic difference but the nature of the differences requires further studies. Antigenic pro-perties of proteins from P. epiphylla S and N and of their allopolyploid - P. borealis, indicated some specifity of the protein spectrum in each of the parental species and intermediate character of proteins in the polyploid forms.

The prevalence of anti-lentiviruses antibodies of small ruminants was investigated in goat herds in the city of Teresina, PI, Brazil. A seroepidemiological survey was conducted involving 480 animals, apparently healthy, belonging to six rural properties. The diagnostic test was the agar gel immunodiffusion (AGID), using antigens produced from cellular cultures infected with caprine arthritis encephalitis virus (CAEV Cork). Prevalences by gender and age were estimated considering sampling fractions for each farm. A general prevalence of 4.2%, was observerved, being 4.2% for females and 3.6% for males. Prevalences were higher among older goats. Regarding the breed standard, 23.5% were of the Anglo Nubian, 5.9% of the Boer, 35.3% Anglo Nubian x Boer crossbred, and 35.3% of undefined breed. It is concluded that small ruminant lentiviruses are endemic among goat herds of Teresina.

The purpose of this work was to evaluate two serological assays: double immunodiffusion (DI) and immunoblotting (IB) in immunodiagnosis of paracoccidioidomycosis (PCM). We evaluated by IB assay 23 sera samples from patients with clinical confirmation of PCM, all of them with negative DI results against culture filtrate from Paracoccidioides brasiliensis isolate 113. For IB, as well as for comparative DI assay, we employed soluble components of the cell wall outer surface (SCCWOS) from P. brasiliensis isolate 113 cultivated at 36°C in Fava-Neto's agar medium for 5 and 10 days. Among the 20 sera samples analyzed by DI, 13 (65%) were negative and 7 (35%) were positive against SCCWOS obtained on the 5th and 10th days. By IB assay, 95.4% and 100% of sera reacted against gp43 and gp70 present in SCCWOS from the 5th day and 95.6% recognized these fractions when evaluated against SCCWOS from the 10th day. Our results demonstrated that the use of an immunoenzymatic assay significantly improves the sensitivity of PCM immunodiagnosis and also suggests that at least two serological tests for antibody detection should be adopted in cases of questionable diagnosis.

Early immunological data, obtained by immunodiffusion and immunoelectrophoresis, on the whole-cell antigenicity of kinetoplastid protozoa were retrieved and used to construct a dendrogram of antigenic distances. Remarkably, they supported the same taxonomic conclusions as analyses based on DNA and protein sequence data.

Early immunological data, obtained by immunodiffusion and immunoelectrophoresis, on the whole-cell antigenicity of kinetoplastid protozoa were retrieved and used to construct a dendrogram of antigenic distances. Remarkably, they supported the same taxonomic conclusions as analyses based on DNA and protein sequence data.

Oral swab samples, serum and colostrum was taken from 15 mares and 14 of their foals, within 24 h of birth. The presence of antibody against Actinobacillus spp. isolated from the oral cavity was investigated using agar gel immunodiffusion. Antibodies against 48 out of the 77 Actinobacillus isolates from all horses in the study were present in the respective sera of 13 mares and 9 foals. In 11 mother-foal pairs, the antibody content of the foal serum was similar to that of the mare, and in 9 cases this was reflected in the antibody content of colostrum from the mare. The results indicate that an immune response to Actinobacillus spp. colonising the oral cavity is present in many adult horses and that this immune response can be transferred from mother to foal via colostrum.

The RID assay is one of the in vitro methods used for in-process control in the production of rabies vaccines for veterinary use. It has been shown to be very useful for determining antigen concentration in the final bulk product. The work presented in this paper, including the production and standardization of candidate standard reagents for use in the Radial Immunodiffusion Assay (RID) was carried out at the Pan American Institute for Food Protection and Zoonoses (INPPAZ/PAHO/WHO). The study was completed with the cooperation of the Faculty of Veterinary Sciences, National University of La Plata (NULP), Argentina, where the validation of the proposed standards and the quality control of samples from 28 different batches of rabies vaccines produced with Pasteur strain rabies virus (PV) in BHK cells were performed. The activity of the vaccines was determined by in vivo (NIH) and in vitro (RID)assays. The results of the candidate reagents for the reagent standardization tests showed stability, sensitivity and reproducibility. The Relative Potency the 1.2 between the problem vaccines and the reference vaccine was estimated by variance and regression analysis. The results of our validation study show that the INPPAZ (PAHO/WHO) is capable of producing and distributing the above-mentioned standard reagents, as well as of providing support for the incorporation of the RID technique (sensitive, rapid and inexpensive) to the laboratories that manufacture rabies vaccines in Latin America and the Caribbean.