ELISA For Detection Of Antibodies Research Articles

Development of a novel monoclonal antibody-based competitive ELISA for antibody detection against bovine leukemia virus

Infection with bovine leukemia virus (BLV) leads to enzootic bovine leukosis, the most prevalent neoplastic disease in cattle. Due to the lack of commercially available vaccines, reliable eradication of the disease can be achieved through the testing and elimination of BLV antibody-positive animals. In this study, we developed a novel competitive ELISA (cELISA) to detect antibodies against BLV capsid protein p24. Recombinant p24 protein expressed by Escherichia coli, in combination with the monoclonal antibody 2G11 exhibiting exceptional performance, was used for the establishment of the cELISA. Receiver-operating characteristic curve analysis showed that the sensitivity and specificity of the assay were 98.85 % and 98.13 %, respectively. Furthermore, the established cELISA was specific for detecting BLV-specific antibodies, without cross-reactivity to antisera for six other bovine viruses. Significantly, experimental infection of cattle and sheep with BLV revealed that the cELISA accurately monitors seroconversion. In a performance evaluation, the established cELISA displayed a high agreement with Western blotting and the commercial BLV gp51 cELISA kit in the detection of 242 clinical samples, respectively. In conclusion, the novel p24 cELISA exhibited the potential to be a reliable and efficient diagnostic tool for BLV serological detection with a broad application prospect.

Comparative Analysis of Lateral Flow Assay with Indirect ELISA for Detection of Anti-NSP Antibodies of Foot and Mouth Disease

Foot-and-mouth disease (FMD) was an exceedingly infectious disease that spread to Indonesia in May 2022. A reliable diagnostic serologic test that can distinguish between infected and vaccinated animals was an important part of FMD (serotype O) control programs in affected areas in Indonesia. For this reason, a non-structural protein (NSP) serological test based on 3ABC proteins has been used. The indirect ELISA serological test requires time, skill, and specialized equipment. An alternative method that can be employed is the lateral flow assay (LFA), which offers the advantages of simplicity and portability, enabling rapid acquisition of results. The objective of this study was to validate the efficacy of a user-friendly anti-NSP antibody LFA for rapid diagnostic purposes. This was done by assessing its sensitivity and specificity in stored samples that had previously been tested using indirect ELISA. There were 32 preserved biological materials from dairy and beef cattle in three provinces in Indonesia that were examined with developed LFA. The results of each sample on LFA were compared to the ELISA result for its sensitivity and specificity according to positive and negative values on both tests. The test had a sensitivity of 95.2% and a specificity of 100%, compared to the indirect ELISA. The measured kappa value is also very good at 0.93, so LFA can be optionally used when examining anti-NSP FMD antibodies. Therefore, the LFA anti-NSP for detecting FMD is considered reliable because of its simplicity and the accuracy of the test results. Keywords: Antibody, Bovine serum, Foot and mouth disease, Indirect ELISA, Lateral flow assay

Development and application of an indirect ELISA for detection of antibodies against emerging atypical porcine pestivirus

BackgroundAtypical porcine pestivirus (APPV) is a newly discovered swine pestivirus, which can cause congenital tremor and high mortality in newborn piglets and subclinical infection in adult pigs, leading to significant impacts on the pig industry. Currently, there is no approved serological method to assess APPV infection status in pig farms.MethodsIn this study, the envelope glycoprotein E2 of APPV was highly expressed in suspension HEK293 cells, and further an indirect enzyme-linked immunosorbent assay based on the recombinant E2 protein (E2-iELISA) was developed and evaluated.ResultsThe reaction parameters of the E2-iELISA were optimized, and the cutoff value was determined to be 0.2 by analyzing S/P values of 165 negative sera against APPV that were confirmed by virus neutralization test (VNT). Specificity test showed that the method had no cross-reaction with other common swine viruses. The E2-iELISA was evaluated using a panel of swine sera, and showed high sensitivity (113/120, 94.2%) and specificity (65/70, 92.9%), and the agreement rate with VNT was 93.7% (178/190). Subsequently, the E2-iELISA was utilized to investigate the seroprevalence of APPV in pig herds of China. When detecting 1368 pig serum samples collected from nine provinces in China, the overall seroprevalence of APPV was 73.9% (1011/1368).ConclusionOur findings suggest that the E2-iELISA is specific and sensitive, and could be a valuable tool for serological surveillance of APPV infection in pigs.

Establishment and application of an indirect ELISA for Getah virus E2 antibody detection

Getah virus (GETV) is a mosquito-transmitted disease that affects animals, causing fever, aseptic meningitis, and abortion. Its prevalence in China poses risks to both animal health and public well-being. Currently, there is a scarcity of seroepidemiological data on GETV due to the absence of commercial antibody detection kits for pigs. The aim of this study is to develop a rapid, accurate, and sensitive ELISA, providing a reliable tool for GETV seroepidemiology and laying the foundation for future commercial assay development. In this study, we removed specific hydrophobic domains and intracellular structures from E2 proteins and constructed the recombinant plasmid pCold-TF-E2. The recombinant protein was expressed using a prokaryotic expression system, and efficient purification of the rE2 protein was achieved using a nickel affinity column. The purified rE2 protein is suitable for the development of an indirect ELISA (rE2 ELISA). Following the optimization of reaction conditions for the rE2-ELISA, the cut-off value was 0.356. Additionally, the rE2-ELISA method showed a positive rate of 37.1% for IgG antibodies against GETV when testing 986 pig clinical serum samples collected from pigs in Sichuan between May 2022 and September 2022. The rE2-ELISA method displayed a 95.1% overall agreement with VNT, boasting a sensitivity of 98.2% and a specificity of 92.6%. These results indicate that IgG ELISA based on rE2 protein is an efficient and economical method for the detection of GETV antibodies in pigs, facilitating the diagnosis and prevention of GETV.

Retrospective Multi-Locus Sequence Analysis of African Swine Fever Viruses by "PACT" Confirms Co-Circulation of Multiple Outbreak Strains in Uganda.

African swine fever (ASF) is a haemorrhagic fever of swine that severely constrains pig production, globally. In Uganda, at least 388 outbreaks of ASF were documented from 2001 to 2012. We undertook a retrospective serological and molecular survey of ASF virus (ASFV) using banked samples collected from seven districts (Pallisa, Lira, Abim, Nebbi, Kabarole, Kibaale, and Mukono) of Uganda. Six assays (ELISA for antibody detection, diagnostic p72 gene PCR and genomic amplification, and sequencing of four gene regions (p72 [P], p54 [A], CVR of the 9RL-ORF [C], and TK [T]), hereinafter referred to as P-A-C-T (PACT)) were evaluated. Antibodies to ASFV were detected in the Abim district (6/25; 24.0%), and the remainder of the serum samples were negative (187/193; 96.9%). For the tissue samples, ASFV detection by assay was 8.47% for P, 6.78% for A, 8.47% for C, and 16.95% for T. The diagnostic PCR (p72 gene) detected seven positive animals from four districts, whereas the TK assay detected ten positives from all seven districts. In addition to the superior detection capability of TK, two virus variants were discernible, whereas CVR recovered three variants, and p72 and p54 sequencing each identified a single variant belonging to genotype IX. Our results indicate that dependence on serology alone underestimates ASF positivity in any infected region, that multi-locus sequence analysis provides better estimates of outbreak strain diversity, and that the TK assay is superior to the WOAH-prescribed conventional p72 diagnostic PCR, and warrants further investigation.

Simple, Rapid, and Highly Sensitive Magnetic Beads ELISA for Detection of SARS CoV-2 Antibodies (IgG) in Human Plasma Samples as a Point of Care Assay

The pandemic outbreak of coronavirus (SARS CoV-2) has been going on over the last 3 years. The people are vaccinated with different vaccines targeting the S protein. Aim. Therefore, it is essential to have an assay that can detect different parts of the virus as a serological assay and can be performed as a point of care test. Hence, in this work, we decided to develop such an assay with the help of magnetic beads. Methods. The magnetic beads ELISA (MB ELISA) was developed in a microtube. The viral ligand-specific magnetic beads were used to detect the nucleoprotein (NP)-specific IgG antibodies in human plasma samples. The results were read with the naked eye as well as with professional ELISA readers. Results. 7 μL magnetic beads were suitable to detect the presence of NP-specific antibodies. The assay needs only a magnetic rack and a pipettor to be performed. The results were available within 30 min. The positive results were observed as yellow color visually but also read in ELISA reader as OD values. The sensitivity of this assay was 1:108 dilutions. The cross-reaction panel was negative with different pathogens and negative human plasma. Conclusions. This work may be the first report in literature about the development of a magnetic beads ELISA as a point of the care assay, which is reproducible, highly sensitive, robust, and easy to perform. It was used to detect the presence of NP-specific IgG antibodies in the plasma samples successfully. This assay can be used as a professional assay, where the results can be measured with an ELISA reader. This assay may be suitable in small clinics also under field conditions. It can be used to detect the SARS CoV-2 infection in vaccinated persons (S protein-based vaccines) along with non-vaccinated population in latent and active phase.

Development of a recombinant protein-based ELISA for detection of antibodies against bovine herpesvirus 6 (BoHV6).

Bovine herpesvirus 6 (BoHV6) belongs to the Herpesviridae family, Gammaherpesvirinae subfamily and Macavirus genus. It is common in cattle, but was also detected in American bison (Bison bison) and water buffalo (Bubalus bubalis). The aim of the experiment was to develop an ELISA for serological examination of cattle sera for the presence of anti-BoHV6 specific antibodies. Viral DNA from a BoHV6-positive cow was amplified by qPCR and the resulting fragments of the gB and gH genes encoding glycoproteins B and H (gB and gH) were cloned into the pLATE52 vector to express recombinant gB (rgB) and gH (rgH) in Rosetta (DE3) E. coli. The expressed recombinant proteins were used as antigens in the developed ELISA. The proteins expressed had the expected molecular weight. A total of 143 sera were examined, and 141 of them were positive, according to the chosen cut-off values of 9% and 10% for the sample-to-positive ratios of the rgB and rgH antigens, respectively. The rgB and rgH recombinant antigens of BoHV6 were successfully expressed in E. coli and successfully used in a newly developed ELISA.

2070. Outbreak of Crimean-Congo Hemorrhagic Fever in Kyzylorda region, Kazakhstan, March-July 2022

Abstract Background Crimean-Congo Hemorrhagic Fever (CCHF) is a viral tick-borne disease with a high mortality rate. It is endemic in Kazakhstan. The Kyzylorda region reported higher-than-expected cases from March to July 2022 (15 compared to 10 in the previous year). We conducted an investigation to identify additional cases, determine risk factors, and assess CCHF knowledge and practices. Methods We conducted a case-control study in July 2022. We defined cases as PCR-confirmed or hospitalized with CCHF symptoms. The control group was made up of regional contacts including neighbors and household members at a 1:2 ratio. We reviewed medical records from April to July. We conducted face-to-face interviews using structured questionnaires. Blood collected from the control group was tested with PCR and ELISA for detection of IgM and IgG antibodies. We also collected ticks from domestic animals for PCR-testing. We used logistic regression to assess factors associated with CCHF. Results We identified 38 cases (n=15 confirmed, n=7 probable and n=16 suspected cases) and 71 controls. Cases were mostly male (74%) with mean age of 45 (range 17-81); 27% died. Top patient symptoms (n=38) were weakness (100%), headache (97%), fever (84%) and loss of appetite (55%). Risk factors for CCHF were being male (odds ratio and 95% confidence interval: 5.5, CI: 2.3-13.1), animal husbandry (3.1, 1.3-7.2), crop production (4.0, 1.5-10.5), tick contact (262.5, CI: 31.4-2192.2), tick bite (70.0, 8.8-556.9), contact with tick blood (18.7, 2.2-155.9), ticks on the body or clothing (77.8, 9.8-618.9) and having been in places with high tick risk (15.1, CI: 4.6-49.7). The control group were more likely to have awareness of CCHF, know that they can protect themselves from CCHF, refrained from visiting forests and rural areas, used repellents, and taken protective measures to reduce ticks in domestic animals. Of 55 controls tested: one was IgG positive. Of 163 ticks tested, one was PCR-positive. Conclusion Exposure to activities that are high risk for exposure to ticks increased odds of CCHF. Improved awareness of prevention to exposure from ticks, CCHF associated risk factors, and the importance of seeking early treatment is needed. This can be achieved by increasing population awareness and educating medical providers. Disclosures All Authors: No reported disclosures

A Robust Quadruple Protein-Based Indirect ELISA for Detection of Antibodies to African Swine Fever Virus in Pigs.

African swine fever (ASF) emerged in domestic pigs and wild boars in China in 2018 and rapidly spread to neighboring Asian countries. Currently, no effective vaccine or diagnostic tests are available to prevent its spread. We developed a robust quadruple recombinant-protein-based indirect enzyme-linked immunosorbent assay (QrP-iELISA) using four antigenic proteins (CD2v, CAP80, p54, and p22) to detect ASF virus (ASFV) antibodies and compared it with a commercial kit (IDvet) using ASFV-positive and -negative serum samples. The maximum positive/negative value was 24.033 at a single antigen concentration of 0.25 μg/mL and quadruple ASFV antigen combination of 1 μg/mL at a 1:100 serum dilution. Among 70 ASFV-positive samples, 65, 67, 65, 70, 70, and 14 were positive above the cut-offs of 0.121, 0.121, 0.183, 0.065, 0.201, and 0.122, for CD2v, CAP80, p54, p22-iELISA, QrP-iELISA, and IDvet, respectively, with sensitivities of 92.9%, 95.7%, 92.9%, 100%, 100%, and 20%, respectively, all with 100% specificity. The antibody responses in QrP-iELISA and IDvet were similar in pigs infected with ASFV I. QrP-iELISA was more sensitive than IDvet for early antibody detection in pigs infected with ASFV II. These data provide a foundation for developing advanced ASF antibody detection kits critical for ASF surveillance and control.

Evaluation of the accuracy of the Asanté assay as a point-of-care rapid test for HIV-1 recent infections using serum bank specimens from blood donors in South Africa, July 2018 - August 2021.

Point-of-care (POC) rapid recency testing can be used as a cost-effective tool to identify recently infected individuals (i.e. infected within the last 12 months) in near-real time, support epidemic control and identify hotspots for transmission as part of recent infection surveillance. To evaluate the performance of the Asanté (HIV-1) rapid recency assay as a POC rapid test among blood donors in South Africa (SA). The study was a cross-sectional and validity study of the Asanté HIV-1 Rapid Recency Assay performed on 715 consecutively archived plasma donor specimens from the SA National Blood Services to determine their recency and established HIV infection status. ELISA and rapid assays for HIV antibody detection were used as the reference-testing standard for confirming an infection, while the Maxim HIV-1 limiting antigen (LAg) avidity assay was used as a reference for comparing HIV recency status. Validity tests (sensitivity, specificity, negative and positive predictive values) and Cohen-Kappa tests of the agreement were conducted to compare the Asanté HIV-1 rapid recency assay results with the reference tests. Of the 715 studied blood samples, 63.1% (n=451/715) were confirmed to be HIV-positive based on the reference standard. The sensitivity and specificity of the Asanté HIV-1 rapid recency assay in diagnosing established HIV infection compared to the ELISA were 98.4% (95% CI 96.7 - 99.3) and 99.6% (95% CI 97.6 - 100), respectively. Compared with HIV rapid assay, the sensitivity and specificity of the Asanté HIV-1 rapid recency assay was 98.7% (95% CI 97.0 - 99.4) and 99.2% (95% CI 97.1 - 100), respectively. Of the 451 HIV-positive blood samples, 43% were confirmed as recent HIV infections by the Maxim HIV-1 LAg avidity assay. There was high agreement between the Asanté HIV-1 rapid recency assay and the Maxim HIV-1 LAg avidity assay (94.1%, k=0.879, p<0.0001). The sensitivity and specificity of the Asante HIV-1 assay was 89.4% (95% CI 84.0 - 93.0) and 97.7% (95% CI 94.8 - 99.0), respectively. The Asanté HIV-1 rapid recency assay test results demonstrated high accuracy (>90%) compared with the HIV ELISA and rapid assays for determining established infection and the Maxim HIV-1 LAg avidity assay for classifying recent HIV-1 infections. The assay's sensitivity for established infections was below the World Health Organization criteria (<99%) for POC devices. The Asanté HIV-1 rapid recency assay can be used to distinguish between recent and long-term infections, but may not be considered a POC test for determining HIV infection.

Evaluation of an In-house Indirect ELISA for Differential Detection of IgM and IgG anti-Brucella Antibodies in Human Brucellosis

Brucellosis caused by various species of the genus Brucella is one of the most important zoonotic diseases of global importance with veterinary, public health, and economic concerns. The study aimed to standardize IgM and IgG-based iELISA to detect anti-Brucella antibodies for serodiagnosis of acute and chronic human brucellosis. The test was standardized using 1:320 dilution of smooth lipopolysaccharide (sLPS) antigen from B. abortus S99 strain, 1:80 serum dilution, 1:4000 anti-human IgM and IgG conjugates, respectively for both IgM and IgG iELISA. The cut-off using 50 each brucellosis positive and negative human sera panel samples was set at ≥ 42 for both IgM and IgG iELISA. A total of 700 human sera samples were evaluated (137 veterinary doctors, 157 artificial inseminators, and 406 veterinary assistants). Overall, the study detected 8.3%, 8.1%, 8%, and 6.1% positivity by in-house IgG iELISA, RBPT, IgM iELISA, and SAT tests, respectively. Considering commercial iELISA kit as a gold standard, the sensitivities of IgM and IgG iELISA were 90% and 97.9%, respectively, whereas, specificities were &gt;99%. The study established &gt;98% specificity and &gt;90% sensitivity for differential detection of immunoglobulin classes in the standardized iELISA. The developed assay outperformed the other evaluated tests with a shorter assay time and can be implemented in both endemic and non-endemic regions for surveillance and diagnosis of human brucellosis.

The effect of glycaemic control on neutralizing antibody response to COVID-19 among patients with Type 2 diabetes mellitus in the Kurunegala District of Sri Lanka; A prospective cohort study

Background: The antibody response following COVID-19 vaccination among patients with diabetes mellitus (DM) is of particular concern given the increased risk of severe disease in this population. The correlation between glycaemic control among persons with DM and the antibody response was not published in Asian populations. Hence, this study aimed to determine whether glycaemic control has an association with the development of an adequate antibody response for SARS-CoV-2 among patients with DM following the administration of two doses of the COVID-19 vaccine.Methods: A prospective cohort study was carried out at three vaccination centers in the Kurunegala district from November 2021 to January 2022. Seventy-one patients with type 2 diabetes were recruited for this study and followed up on vaccination with the Sinopharm COVID-19 vaccine. HbA1 c levels at the first dose and after 6-8 weeks from the second dose of vaccine were analyzed. The neutralizing antibodies (NAbs) were analyzed using C Pass™ neutralizing antibody detection ELISA Kit following 6-8 weeks of the 2nd dose.Results: The median (IQR) age of the total population (63.4% females) was 53 years (44.0-58.0) and they were diagnosed with diabetes for 6 years (3-11 years). The median first and second HbAlc values were 9.3% (7.2-10.7%) and 8.2% (7.1-10.2%) respectively. From the total population, only 66.2% developed protective levels of NAbs after 6-8 weeks of the second dose of the vaccine. The second HbAlc value was significantly lower compared to the first (z=-2.63, p=0.008). There was no significant difference in terms of sex, age, duration of diabetes, pre-vaccination HbAlc level, or HbAlc level 6-8 weeks after the vaccination among those who developed protective levels of antibodies and those who did not (p&gt;0.05). There was no difference in sero-conversion depending on the abnormal HbAlc value (.28%) (1st HbAlc p=0.957, 2nd HbAlc p=0.360).Conclusion: We did not detect an association between glycaemic control and sero-conversion. However, 1/3rd of patients with diabetes did not have a protective level of NAbs following 2 doses of Sinopharm COVID-19 vaccination. Furthermore, glycaemic control did not deteriorate with COVID-19 vaccination.

Development of conjugated secondary antibodies for wildlife disease surveillance.

Disease monitoring in free-ranging wildlife is a challenge and often relies on passive surveillance. Alternatively, proactive surveillance that relies on the detection of specific antibodies could give more reliable and timely insight into disease presence and prevalence in a population, especially if the evidence of disease occurs below detection thresholds for passive surveillance. Primary binding assays, like the indirect ELISA for antibody detection in wildlife, are hampered by a lack of species-specific conjugates. In this study, we developed anti-kudu (Tragelaphus strepsiceros) and anti-impala (Aepyceros melampus) immunoglobulin-specific conjugates in chickens and compared them to the binding of commercially available protein-G and protein-AG conjugates, using an ELISA-based avidity index. The conjugates were evaluated for cross-reaction with sera from other wild herbivores to assess future use in ELISAs. The developed conjugates had a high avidity of >70% against kudu and impala sera. The commercial conjugates (protein-G and protein-AG) had significantly low relative avidity (<20%) against these species. Eighteen other wildlife species demonstrated cross-reactivity with a mean relative avidity of >50% with the impala and kudu conjugates and <40% with the commercial conjugates. These results demonstrate that species-specific conjugates are important tools for the development and validation of immunoassays in wildlife and for the surveillance of zoonotic agents along the livestock-wildlife-human interface.

Development and Validation of Competitive ELISA for Detection of H5 Hemagglutinin Antibodies

Influenza A viruses (IAVs) belonging to the goose/Guangdong (Gs/GD)-lineage H5Nx remain a major concern for the global poultry industry, wildlife, and humans. The hemagglutinin (HA) protein is the dominant antigenic epitope carrier within IAV, which in turn triggers substantial immunogenic responses in the infected host. The current study describes the development and validation of a highly sensitive competitive H5 ELISA (cELISA) based on a novel monoclonal antibody developed in mice immunized with inactivated virus H5N1 (A/Turkey/ON/6213/1966). The cELISA is capable of detecting the H5 antibody response to a wider range of H5-subtype viruses belonging to both North American and Eurasia lineages, including the Gs/GD H5Nx from clade 2.3.4.4b that is currently causing the highly pathogenic avian influenza outbreaks in Eurasia, Africa, and Latin and North America. The developed H5 cELISA provides a specific, sensitive, and species-independent serological assay for the rapid detection of H5 antibodies. The assay is more robust and more sensitive than the hemagglutination inhibition assay, which is the “Gold standard”. The assay can be used in serological diagnosis, serosurveillance, and vaccine monitoring of serum samples collected from different species of animals.

Performance evaluation of newly developed surrogate virus neutralization tests for detecting neutralizing antibodies against SARS-CoV-2

We evaluated newly developed surrogate virus neutralization tests (sVNT) for detecting neutralizing antibodies (NAbs) against the receptor binding domain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). VERI-Q SARS-CoV-2 Neutralizing Antibody Detection ELISA Kit (MiCo BioMed, Gyeonggi-do, Republic of Korea, hereafter, “eCoV-CN”) is an enzyme-linked immunosorbent assay-based sVNT, and VERI-Q SARS-CoV-2 Neutralizing Antibody Rapid Test Kit (MiCo BioMed, hereafter, “rCoV-RN”) is a point-of-care lateral-flow immunochromatography test with auto-scanner. A total of 411 serum samples were evaluated. Both evaluations used a 50% plaque reduction neutralization test (PRNT50) as the gold standard. Compared with PRNT50, the eCoV-CN showed 98.7% positive percent agreement (PPA), 96.8% negative percent agreement (NPA), 97.4% total percent agreement (TPA), with kappa values of 0.942. The rCoV-RN showed 98.7% PPA, 97.4% NPA, 97.8% TPA, and kappa values of 0.951, comparing to PRNT50. Neither assay indicated cross-reactivity for other pathogens, and the signal indexes were statistically significantly correlated to the PRNT50 titer. The two evaluated sVNTs show comparable performances to the PRNT50 with the advantages of technical simplicity, speed, and do not require cell culture facilities.

SEROPREVALENCE OF DENGUE IN A TERTIARY CARE HOSPITAL: CROSS SECTIONAL STUDY

Background: Dengue is endemic and an important emerging public health problem in India. Dengue illness is major concern causing increased morbidity and mortality. Disease burden is highly variable between different places within a country. This study was conducted to determine the age wise seroprevalence of dengue and seasonal variation of dengue infection in tertiary care hospital in Mumbai. Materials And Methods: The present cross sectional study was conducted from January 2021 to March 2022 for a period of 15 months. Total 1550 serum samples were collected from suspected dengue cases. All the samples were subjected to ELISA for detection of dengue NS1 antigen and IgM antibody. Results: Out of 1550 suspected dengue patients 187 (12.06%) were seropositive for NS1antigen or IgM antibody or both NS1 antigen &amp; IgM antibody. Of 187 seropositive dengue cases 121 (64.71%) were male and 66 (35.29%) were female. The maximum dengue cases (31.55%) were from 31-40 years age group, followed by 21-30 (23.52%) years. Most dengue positive cases were seen in monsoon period. The monsoon peak was observed in September (22.00%) followed by August (17.11%). However, in May (dry season) few cases were reported. Conclusion: Dengue illness is a major public health concern. With early diagnosis and treatment, morbidity and mortality can be reduced. Knowledge of prior infection is also very important if vaccination strategy by WHO is implemented in future. Maximum cases were observed in monsoon period but dengue cases were seen throughout the year. Hence continuous surveillance of cases is required for early diagnosis and treatment, to know the prevalence throughout the year, for vector control measures &amp; administration of dengue vaccine as per WHO strategy if implemented in future in our country

Development, Validation and Application of a Bridging ELISA for Detection of Antibodies against GQ1001 in Cynomolgus Monkey Serum.

Immunogenicity is a major issue associated with the PK, efficacy, and safety evaluation of therapeutic protein products during pre-clinical and clinical studies. A multi-tiered approach consisting of screening, confirmatory, and titration assays has been widely adopted for anti-drug antibody testing. GQ1001, a recombinant humanized anti-human epidermal growth factor receptor 2 monoclonal antibody covalently linked to a cytotoxin of DM1, possesses a novel format of antibody-drug conjugates. In this study, we reported the development, validation, and application of an acid-dissociation bridging enzyme-linked immunosorbent assay for the detection of antibodies against GQ1001 in cynomolgus monkey serum. The sensitivity of the screening assay was 126.141 ng/mL in undiluted serum. The screening assay and confirmatory assay were neither affected by the naïve monkey serum nor by 2% and 5% (v/v) erythrocyte hemolysates. Moreover, the assay was not subject to interference by 2500 ng/mL of human IgG1 in the samples. Drug interference at low positive control (150 ng/mL) and high positive control (8000 ng/mL) of anti-GQ1001 antibodies was not observed when GQ1001 concentrations were below 3.125 μg/mL and 100 μg/mL, respectively. Furthermore, no hook effect was observed for the positive antibodies in the concentration range of 8 to 64 μg/mL. The validated assay was, thereafter, successfully applied to a single-dose toxicity study of GQ1001. Anti-drug antibody positive rates among dosing animals and testing samples were reported, and no significant impact was found on toxicokinetic outcomes.

Evaluation of an In-House ELISA for Detection of Antibodies Against the Lumpy Skin Disease Virus in Vaccinated Cattle

Evaluation of an In-House ELISA for Detection of Antibodies Against the Lumpy Skin Disease Virus in Vaccinated Cattle

Evaluation of Two Adjuvant Formulations for an Inactivated Yellow Fever 17DD Vaccine Candidate in Mice.

The attenuated yellow fever (YF) vaccine is one of the most successful vaccines ever developed. After a single dose administration YF vaccine can induce balanced Th1/Th2 immune responses and long-lasting neutralizing antibodies. These attributes endorsed it as a model of how to properly stimulate the innate response to target protective immune responses. Despite their longstanding success, attenuated YF vaccines can cause rare fatal adverse events and are contraindicated for persons with immunosuppression, egg allergy and age &lt; 6 months and &gt;60 years. These drawbacks have encouraged the development of a non-live vaccine. The aim of the present study is to characterize and compare the immunological profile of two adjuvant formulations of an inactivated YF 17DD vaccine candidate. Inactivated YF vaccine formulations based on alum (Al(OH)3) or squalene (AddaVax®) were investigated by immunization of C57BL/6 mice in 3-dose or 2-dose schedules, respectively, and compared with a single dose of attenuated YF virus 17DD. Sera were analyzed by ELISA and Plaque Reduction Neutralization Test (PRNT) for detection of total IgG and neutralizing antibodies against YF virus. In addition, splenocytes were collected to evaluate cellular responses by ELISpot. Both inactivated formulations were able to induce high titers of IgG against YF, although neutralizing antibodies levels were borderline on pre-challenge samples. Analysis of IgG subtypes revealed a predominance of IgG2a associated with improved neutralizing capacity in animals immunized with the attenuated YF vaccine, and a predominance of IgG1 in groups immunized with experimental non-live formulations (alum and AddaVax®). After intracerebral (IC) challenge, attenuated and inactivated vaccine formulations showed an increase in neutralizing antibodies. The AddaVax®-based inactivated vaccine and the attenuated vaccine achieved 100% protection, and alum-based equivalent formulation achieved 70% protection.

Diagnostic performance of different specimens in detecting enterovirus A71 in children with hand, foot and mouth disease

Hand, foot and mouth disease (HFMD) is a major public health problem among children in the Asia-Pacific region. The optimal specimen for HFMD virological diagnosis remains unclear. Enterovirus A71 (EV-A71) neutralizing antibody titres detected in paired sera were considered the reference standard for calculating the sensitivity, specificity, positive and negative predictive value of throat swabs, rectal swabs, stool, blood samples and cerebrospinal fluid (CSF) by RT-PCR or ELISA assay. In this study, clinical samples from 276 HFMD patients were collected for analysing the sensitivity of different kind of specimens. Our results showed that stool had the highest sensitivity (88%, 95% CI: 74%–96%) and agreement with the reference standard (91%). The order of diagnostic yield for EV-A71 infection was stool sample ​≥ ​rectal swab ​> ​throat swab ​> ​blood sample ​> ​CSF sample, and using a combination of clinical samples improved sensitivity for enterovirus detection. The sensitivity of ELISA for IgM antibody detection in sterile-site specimens was significantly higher than that of RT-PCR (serum/plasma: 62% vs. 2%, CSF: 47% vs. 0%) (P ​< ​0.002). In conclusion, our results suggest that stool has the highest diagnostic yield for EV-A71-infected HFMD. If stool is unavailable, rectal swabs can be collected to achieve a similar diagnostic yield. Otherwise, throat swabs may be useful in detecting positive samples. Although IgM in blood or CSF is diagnostically accurate, it lacks sensitivity, missing 40%–50% of cases. The higher proportion of severe cases and shorter interval between onset and sampling contributed to the increase in congruency between clinical testing and the serological reference standard.