ACE configurator for ELISpot: optimizing combinatorial design of pooled ELISpot assays with an epitope similarity model.

The enzyme-linked immunosorbent spot (ELISpot) assay is a powerful in vitro immunoassay that enables cost-effective quantification of antigen-specific T-cell reactivity. It is used widely in the context of cancer and infectious diseases to validate the immunogenicity of predicted epitopes. While technological advances have kept pace with the demand for increased throughput, efforts to increase scale are bottlenecked by current assay design and deconvolution methods, which have remained largely unchanged. Current methods for designing pooled ELISpot experiments offer limited flexibility of assay parameters, lack support for high-throughput scenarios and do not consider peptide identity during pool assignment. We introduce the ACE Configurator for ELISpot (ACE) to address these gaps. ACE generates optimized peptide-pool assignments from highly customizable user inputs and handles the deconvolution of positive peptides using assay readouts. In this study, we present a novel sequence-aware pooling strategy, powered by a fine-tuned ESM-2 model that groups immunologically similar peptides, reducing the number of false positives and subsequent confirmatory assays compared to existing combinatorial approaches. To validate ACE's performance on real-world datasets, we conducted a comprehensive benchmark study, contextualizing design choices with their impact on prediction quality. Our results demonstrate ACE's capacity to further increase precision of identified immunogenic peptides, directly optimizing experimental efficiency. ACE is freely available as an executable with a graphical user interface and command-line interfaces at https://github.com/pirl-unc/ace.

Immunological evidence of an early seroconversion to oncogenic Merkel cell polyomavirus in healthy children and young adults.

Oncogenic Merkel cell polyomavirus (MCPyV) provokes a widespread and asymptomatic infection in humans. Herein, sera from healthy children and young adults (HC, n = 344) aged 0-20 years old were evaluated for anti-MCPyV immunoglobulin G (IgG) and IgM antibodies employing a recently developed immunoassay. Serum MCPyV IgG data from healthy subjects (HS, n = 510) and elderlies (ES, n = 226), aged 21-65/66-100 years old, from our previous studies, were included. The anti-MCPyV IgG and IgM rates in HC sera were 40.7% and 29.7%, respectively. A lower prevalence of anti-MCPyV IgGs was found in HC aged 0-5 years old (13%) compared to 6-10 (52.3%), 11-15 (60.5%) and 16-20 years old (61.6%) cohorts. Age-stratified HCs exhibited similar anti-MCPyV IgM rates (27.9%-32.9%). Serological profiles indicated that anti-MCPyV IgGs and IgMs had low optical densities (ODs) during the first years of life, while IgM ODs appeared to decrease throughout young adulthood. A lower anti-MCPyV IgGs rate was found in HC (40.7%) than HS (61.8%) and ES (63.7%). Upon the 5-years range age-stratification, a lower anti-MCPyV IgGs rate was found in the younger HC cohort aged 0-5 years old compared to the remaining older HC/HS/ES cohorts (52.3%-72%). The younger HC cohort exhibited the lowest anti-MCPyV IgG ODs than the older cohorts. Low anti-MCPyV IgMs rates and ODs were found in the 21-25 (17.5%) and 26-30 (7.7%) years old cohorts. Our data indicate that, upon an early-in-life seroconversion, the seropositivity for oncogenic MCPyV peaks in late childhood/young adulthood and remains at high prevalence and relatively stable throughout life.

Validation of Luminex immunological and competitive Luminex immunological assays for clinical immunogenicity assessment of a 14-valent recombinant human papillomavirus vaccine.

A novel virus-like particle (VLP)-based multivalent recombinant human papillomavirus (HPV) vaccine was developed and evaluated in human, including 14 HPV-type specific VLP antigens (HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59). The pseudovirus-based neutralizing assay (PBNA) method is widely used for immunogenicity assessment of HPV vaccine in clinical trials. However, as many as 14 antigen-specific antibody levels need be determined, PBNA is, for many reasons, challenging and time-consuming. In this study, we developed a Luminex immunological assay (LIA) and a competitive Luminex immunological assay (cLIA). These methods increase the throughput, reproducibility and precision, as well as reduce the complexity. All assay parameters showed good characteristics in the validation of both methods, benefiting from highly purified and structurally correct VLPs, high specific antibodies, standard VLP-microspheres and PE-mAbs conjugating process, adequate assay development and stable system. Validation data support the use of both methods for immunogenicity assessment in clinical trials. LIA showed higher sensitivity than cLIA, and due to limited epitopes of mAb, cLIA detected lower antibody responses, and therefore, fewer antibodies. This work not only supports clinical trials of 14-valent HPV vaccines more efficiently and reliably, but also provides a set of validation strategies and usable standards for general vaccine immunogenicity testing.

Identification of chicken-derived scFv against N-glycolylneuraminic acid retrieved from an immune library by phage display.

N- glycolylneuraminic acid (Neu5Gc) is a type of sialic acid, it can be synthesized by a range of mammals except chickens and healthy human. After entering human body, Neu5Gc in foods such as red meat and milk can cause chronic inflammation, thus promoting the development of cancer and related diseases. In this study, we identified a gene sequence of Neu5Gc-specific single-chain variable fragment (ScFv) by phage display from a primary chicken antibodies library. Then the gene sequence was used to express a 29 kDa anti-Neu5Gc ScFv protein as detection probe in competitive inhibition ELISA (IC-ELISA). The linear regression equation of the IC-ELISA was y = 23.12x+33.19 (R = 0.980), and the half-maximal inhibitory concentration (IC50) and the limit of detection (LOD) was 5.333 and 0.66 µg/mL. The mean recovery of the spiked samples was 83.04%, and the intra-assay and inter-assay coefficients of variation (CVs) were both 5.59%. The results suggested that the specific anti-Neu5Gc ScFv is a promising probe for the development of IC-ELISA and test strip in order to detect the presence of Neu5Gc in red meat, milk, and tumor tissues.

Evaluation of a new multi-epitope sequence of eight known Leishmania infantum antigens for HVL diagnosis by ELISA and Western blot.

BACKGROUND & OBJECTIVES: Leishmaniasis, known as a disease with high prevalence proportion throughout the world, is caused by protozoan parasites. Visceral leishmaniasis is the most severe form of this condition reported sporadically from all regions in Iran. Between different diagnostic tests, serodiagnosis of this infection is of utmost importance in both humans and dogs. Although rK39 ELISA test has been extensively validated in endemic areas, there are currently challenges regarding a more appropriate serodiagnostic test. METHODS: A novel multi-epitope construct was designed consisting of highexposedB cell epitopes using eight important antigens of Leishmania infantum (Gp63, KMP-11, HSP70, CPA, H2A, H3, LACK and TRYP). Our artificial sequence, a Multi-epitope Recombinant Protein (MRP), was consequently produced and purified. Then, immunoreactivity was investigated by ELISA test and western blotting as well. RESULTS: In the present study, the cutoff value (1.052) for the new MRP-ELISA was determined by receiver operator characteristic (ROC) curve analysis using 35 known positive and 20 known negative HVL sera previously tested for antibodies to L. infantum by DAT, showing a sensitivity of 93.1% and a specificity of 77.4%. The blotting test also showed a favorable band to detect visceral leishmaniasis. INTERPRETATION & CONCLUSION: According to the results, this new antigen had acceptable potential in detecting VL positive cases once western blotting was utilized, but the ELISA test did not proceed as expected for detecting true negative cases, probably due to some optimization issues.The present study is a promising start.

The Impact of Composites with Silicate-Based Glasses and Gold Nanoparticles on Skin Wound Regeneration.

The silver content of the skin regeneration ointments can influence its regeneration process but in the meantime, it can take the benefit of the antibacterial properties of silver by avoiding the bacterial infection of an open wound. In the current study, the skin healing and regeneration capacity of bioactive glass with spherical gold nanocages (BGAuIND) in the Vaseline ointments were evaluated in vivo comparing the bioactive glass (BG)-Vaseline and bioactive glass with spherical gold (BGAuSP)-Vaseline ointments. Spherical gold nanocages are stabilized with silver and as a consequence the BGAuIND exhibits great antibacterial activity. Histological examination of the cutaneous tissue performed on day 8 indicates a more advanced regeneration process in rats treated with BGAuSP-Vaseline. The histopathological examination also confirms the results obtained after 11 days post-intervention, when the skin is completely regenerated at rats treated with BGAuSP-Vaseline compared with the others groups where the healing was incomplete. This result is also confirmed by the macroscopic images of the evolution of wounds healing. As expected, the silver content influences the wound healing process but after two weeks, for all of the post-interventional trials from the groups of rats, the skin healing was completely.

Humoural immune response and pathological analysis in patients with false immune diagnosis of cystic echinococcosis.

The patients with false immune diagnosis of hydatid disease were investigated for the humoural immune response to analyse the possible reasons and mechanism leading to false immune diagnosis. Two hundred and thirty-nine patients with nature-unknown cysts and 30 healthy controls were detected by immunological assays (four hydatid antigen-based immunogold filtration assay and enzyme-linked immune absorbent assay) and ultrasound. Sensitivity of and specificity of immunological assay and ultrasound were calculated, respectively. The serological diagnosis was compared with surgical pathology to screen the patients with false immune diagnosis for the immunoglobulin measurement and pathological analysis. The history and cyst characteristics were also reviewed. The results indicate the immunoglobulin has little influence on false immunodiagnosis. The false-negative immunodiagnosis was caused by the cysts' inactive status while the false positive caused by previous rupture, antigen cross-reaction. The clinical diagnosis of cystic echinococcosis requires a combination of immunodiagnosis and ultrasonography, which is the necessary complementary confirmation.

Investigating outlier rates of cardiac troponin I and troponin T assays: A systematic review.

Objectives: This review aimed to harmoniously summarize and compare outlier rates for various cardiac troponin (cTn) assays, including high-sensitivity-cTn (hs-cTn) assays and contemporary cTn (generation of assays prior to hs-cTn ones) assays, from the published studies. Methods: The PRISMA guidelines were utilized to perform this systematic review. Five databases, including PubMed, Scopus, Embase, Cochrane Library, and Web of Science, were searched using specific keywords up to June 30th, 2023. Studies reporting specifically calculated outlier rates for cTn assays when conducting in-vitro diagnosis in human samples were included. Selected studies were then further assessed using the GRADE tool. Results: Thirteen studies were included. The data from the studies were summarized statistically in this review. The results showed substantial evidence of improved analytical robustness or reduced respective mean rates of outliers, critical outliers, and analytical outliers for hs-cTn assays (0.14 %, 0.18 %, and 0.18 %) compared to contemporary cTn assays (0.63 %, 0.71 %, and 0.50 %). Conclusion: The findings offer promisingly provide a comprehensive reference for laboratory scientists and clinical staff in choosing the most suitable cTn assay for patient care regrading outlier rates. Besides, this review reveals the advancements of hs-cTn assays with lower outlier rates than contemporary cTn assays. The emerging challenges for continuously improving analytical robustness of cTn assays are also elaborated.

A taxonomy of multiple regression methods for immunologists.

Multiple regression is a powerful tool in the immunologist's toolbox. This paper defines multiple regression, discusses availability and accessibility, provides some additional helpful definitions, treats the topics of transformation and extreme value screening, and establishes the paper's scope and philosophy. Then eleven methods of multiple regression are detailed, giving strengths and limitations. Throughout an emphasis is placed on application to immunological assays. A flowchart to guide selection of multiple regression methods is provided.

Comparison of diagnostic performances of different serological tests for SARS-CoV-2 antibody detection in cats and dogs.

Serosurveillance among animals, including pets, plays an important role in the current coronavirus disease 2019 (COVID-19) pandemic, because severe acute respiratory coronavirus 2 (SARS-CoV-2) infections in animal populations could result in the establishment of new virus reservoirs. Serological assays that offer the required sensitivity and specificity are essential. In this study, we evaluated the diagnostic performance of three different commercially available immunoassays for the detection of SARS-CoV-2 antibodies in pets, namely two ELISA tests for the detection of antibodies against SARS-CoV-2 nucleocapsid [ID Screen SARS CoV-2 double antigen multispecies (Double antigen) and ID Screen® SARS-CoV-2-N IgG indirect ELISA (Indirect)] and one test for the detection of neutralizing antibodies against SARS-CoV-2 receptor-binding-domain [surrogate virus neutralization test (sVNT)]. The obtained results were compared with those of conventional virus neutralization test (VNT), which was regarded as reference method. A total of 191 serum samples were analysed. Thirteen (6.8%) samples showed VNT-positive results. The overall sensitivity was higher for sVNT (100%) compared to nucleocapsid-based ELISA assays (23% for Double antigen and 60% for Indirect). The specificity was 100% for Indirect ELISA and sVNT, when a higher cut-off (>30%) was used compared to the one previously defined by the manufacturer (>20%), whereas the other test showed lower value (99%). The sVNT test showed the highest accuracy and agreement with VNT, with a perfect agreement when the higher cut-off was applied. The agreement between each nucleocapsid-based ELISA test and VNT was 96% for Indirect and 94% for Double antigen. Our findings showed that some commercially available serological tests may lead to a high rate of false-negative results, highlighting the importance of assays validation for the detection of SARS-CoV-2 antibodies in domestic animals.

Decreased IgG Antibody Response to Viral Protein Mimotopes of Oncogenic Merkel Cell Polyomavirus in Sera From Healthy Elderly Subjects.

Merkel cell polyomavirus (MCPyV) is the main causative agent of Merkel cell carcinoma (MCC), a rare but aggressive skin tumor with a typical presentation age >60 years. MCPyV is ubiquitous in humans. After an early-age primary infection, MCPyV establishes a clinically asymptomatic lifelong infection. In immunocompromised patients/individuals, including elders, MCC can arise following an increase in MCPyV replication events. Elders are prone to develop immunesenescence and therefore represent an important group to investigate. In addition, detailed information on MCPyV serology in elders has been debated. These findings cumulatively indicate the need for new research verifying the impact of MCPyV infection in elderly subjects (ES). Herein, sera from 226 ES, aged 66-100 years, were analyzed for anti-MCPyV IgGs with an indirect ELISA using peptides mimicking epitopes from the MCPyV capsid proteins VP1-2. Immunological data from sera belonging to a cohort of healthy subjects (HS) (n = 548) aged 18-65 years, reported in our previous study, were also included for comparisons. Age-/gender-specific seroprevalence and serological profiles were investigated. MCPyV seroprevalence in ES was 63.7% (144/226). Age-specific MCPyV seroprevalence resulted as 62.5% (25/40), 71.7% (33/46), 64.9% (37/57), 63.8% (30/47), and 52.8% (19/36) in ES aged 66-70, 71-75, 76-80, 81-85, and 86-100 years, respectively (p > 0.05). MCPyV seroprevalence was 67% (71/106) and 61% (73/120) in ES males and females, respectively (p > 0.05). Lack of age-/gender-related variations in terms of MCPyV serological profiles was found in ES (p > 0.05). Notably, serological profile analyses indicated lower optical densities (ODs) in ES compared with HS (p < 0.05), while lower ODs were also determined in ES males compared with HS males (p < 0.05). Our data cumulatively suggest that oncogenic MCPyV circulates in elders asymptomatically at a relatively high prevalence, while immunesenescence might be responsible for a decreased IgG antibody response to MCPyV, thereby potentially leading to an increase in MCPyV replication levels. In the worse scenario, alongside other factors, MCPyV might drive MCC carcinogenesis, as described in elders with over 60 years of age.

Diagnostic performance of two specific schistosoma japonicum immunological tests for screening schistosoma haematobium in school children in Zambia.

Dipstick Dye Immunoassay (DDIA) and Indirect Haemagglutination Assay (IHA), are two commercially available kits which have been widely used for screening Schistosoma japonicum in P.R. China. Whether they can be used for screening of Schistosoma haematobium are not clear. In order to evaluate the diagnostic efficiency of DDIA and IHA for screening Schistosoma haematobium, serum samples were collected from pupils in endemic areas in Zambia, Southern Africa, and tested by DDIA and IHA by single-blind manner. Meanwhile, the pupils were microscopically examined by infection with Schistosoma and soil-transmitted helminths, visually observed for parasite eggs. Of the enrolled 148 pupils, 61% tested positive for S. haematobium infection, while 31% and 36% of pupils were infected with hookworm and Ascaris respectively. Regarding the parasitological tests as reference standard, for the diagnosis of S. haematobium infection, IHA performed higher sensitivity (74%, 95% CI: 65%-83%) than that of DDIA (60%, 95%CI: 49%-70%). The sensitivities of IHA and DDIA are significant higher in 10-14 years old students than those of 7-9 years old group. The specificity of DDIA and IHA were 61% (95%CI: 49%-74%) and 72% (95%CI: 60%-84%), respectively. The co-infection with STHs decreased the specificity of DDIA but had no impact on that of IHA. Our study indicated that IHA has more potential as an alternative diagnostic tool for identifying schistosomiasis haematobium but need further improvement.

Generalized mathematical model for immunoassay interference.

As part of a landmark review of the antigen excess, Jacobs et al. (Autoimmunity Reviews 2015;14:160-167) contrast how immunoassay "interference" by non-target biomolecules can cause spurious readings for clinical diagnostic tests. The purpose of the present Letter is to complement and analytically extend this description by Jacobs et al. by briefly presenting a generalized mathematical model of immunoassay interference that is derived from a simple mechanistic system of differential equations. Derivation includes expressions for the location of the peak concentration of bound reporter as well as that maximum concentration of target biomolecule at which immunoassay interference drives concentration of bound reporter to zero. This parsimonious, mechanistic, and generalized model may prove foundational to analytic study of immunoassay interference. More comprehensive models for specific classes of immunoassays have and can be developed.

Laboratory diagnosis of SARS-CoV-2 - A review of current methods.

At present the whole world is facing pandemic of the Coronavirus disease (COVID-19); caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This disease has rapidly spreads across the world from its origin of Wuhan, China and affected millions people worldwide and make them to remain in their homes. The knowledge of available laboratory methods is essential for early and correct diagnosis of COVID-19 to identify new cases as well as monitoring treatment of confirmed cases. In this review we aim to provide the updated information about selection of specimens and availability of various diagnostic methods and their utility with current findings for the laboratory diagnosis of SARS-CoV-2 infection. This will guide the healthcare professionals and government organizations to make strategy for establishing diagnostic facilities for SARS-CoV-2 infections.

A novel protein biochip screening serum anti-sperm antibody expression and natural pregnancy rate in a follow-up study in Chinese infertility.

Production of anti-sperm antibody (ASA) often suffers from autoimmune reaction against sperms in human infertility. The antibodies are measured in both blood and seminal plasma of males. Here, we reported a simple protein biochip methodology that takes advantage of a functionalized self-assembled monolayer modified by N-hydroxysuccinimide (NHS) and enables identification of anti-sperm antibody in Chinese male infertility. To validate this biochip platform, we immobilized purified sperm protein on the biochip surface and tested a variety of parameters in quality controls for the protein assay, respectively. Then, we analyzed serum samples from 368 patients with infertility and 116 healthy donors by means of this biochip simultaneously. We found that positive rate of serum ASA was 20.92% (77/368) in the cases and 1.72% (2/116) in the controls, respectively. Furthermore, we further corroborated the biochip assay in comparison with ELISA method. We found that both methods were compatible for the detection of serum ASA in the patients. In addition, a follow-up study for natural conception in ASA-positive and ASA-negative patients was conducted. The result showed a significant correlation between serum ASA expression and natural pregnancy rate 6.5% in ASA-positive patients while 18.9% in ASA-negative patients, indicating the potential roles of ASA in naturally reproductive processes.

Combination of Mechanical and Chemical Methods Improves Gene Delivery in Cell-based HIV Vaccines.

OBJECTIVE: Novel vaccination approaches are required to control human immunodeficiency virus (HIV) infections. The membrane proximal external region (MPER) of Env gp41 subunit and the V3/glycans of Env gp120 subunit were known as potential antigenic targets for anti-HIV-1 vaccines. In this study, we prepared the modified dendritic cells (DCs) and mesenchymal stem cells (MSCs) with HIV-1 MPER-V3 gene using mechanical and chemical approaches. METHODS: At first, MPER-V3 fusion DNA delivery was optimized in dendritic cells (DCs) and mesenchymal stem cells (MSCs) using three mechanical (i.e., uniaxial cyclic stretch, equiaxial cyclic stretch and shear stress bioreactors), and two chemical (i.e., TurboFect or Lipofectamine) methods. Next, the modified DCs and MSCs with MPER-V3 antigen were compared to induce immune responses in vivo. RESULTS: Our data showed that the combination of equiaxial cyclic stretch loading and lipofectamine twice with 48 h intervals increased the efficiency of transfection about 60.21 ± 1.05 % and 65.06 ± 0.09 % for MSCs and DCs, respectively. Moreover, DCs and MSCs transfected with MPER-V3 DNA in heterologous DC or MSC prime/ peptide boost immunizations induced high levels of IgG2a, IgG2b, IFN-γ and IL-10 directed toward Th1 responses as well as an increased level of Granzyme B. Indeed, the modified MSCs and DCs with MPER-V3 DNA could significantly enhance the MPER/V3-specific T-cell responses compared to MPER/V3 peptide immunization. CONCLUSIONS: These findings showed that the modified MSC-based immunization could elicit effective immune responses against HIV antigen similar to the modified DC-based immunization.

Development of an elisa for the diagnosis of reactive IgE antibodies anti-therapeutic horse sera.

Hypersensitive diseases that involve IgE reactivity are important concern of public, especially those encompassing the potential pathogenesis from the administration of horse serum-based therapeutics such as antivenoms. A method for the definitive diagnosis of reactive IgE is important for identifying allergic patients to control severe collateral effects during planned and emergency application of immunotherapies when the allergy source cannot be avoided for treatment. To date, no tests have been developed to accompany the wide range of antivenoms produced from horse sera. The aim of this was to develop a cost-effective ELISA of high sensitivity and specificity to detect circulating patient IgE that binds horse IgG3, the most prevalent antibody class in passive antibody therapies. Horse IgG3 was purified in a single step on jacalin-Sepharose and absorbed to standard ELISA plates as the capture molecule for reactive human IgE. The direct performance evaluation with allergenic and non-allergenic patient, together with competitive peptides assays, showed high sensitivity and specificity to detect human IgE that recognized horse IgG3. The analytical sensitivity and ED50 were calculated to be 0.01 µg mL-1 and 0.052 µg mL-1, respectively. The intra- and inter-assay coefficient of variation ranged from 3.3 to 11.1% and 4.0-8.0%, respectively. The horse IgG3-based ELISA assay can detect reactive allergenic IgE at picomolar concentrations. The coefficient of variation suggests that it can be easily standardized between laboratories, provide rapid and can be applied to population surveillance. Patient management during treatment for envenomation would be greatly improved by a robust and reliable diagnostic test for preexisting allergies to mitigate life-threating consequences of hypersensitivity.

DNA Amplification Techniques for the Detection of Toxoplasma gondii Tissue Cysts in Meat Producing Animals: A Narrative Review Article.

BACKGROUND: Toxoplasma gondii is an intracellular parasite, which infects one-third population of world. Humans and animals acquire infection by ingesting oocytes from feces of cats or by meat of other animals having cysts that may lead to congenital, ocular or cephalic toxoplasmosis. Either it is important to detect T. gondii from meat of food animals from retail shops or directly at slaughterhouses, which is meant for export. METHODS: The current research was done without time limitation using such terms as follows: "Toxoplasma gondii", "Meat", "Tissue cyst", "PCR", "LAMP", "Screening" and "Immunological assay" alone or in combination, in English language. The used electronic databases for searching included as follows: Pub-Med, Scopus, Google Scholar, Web of Science and Science Direct. The searches were limited to the published papers to English language. RESULTS: Sensitivity of different molecular techniques for diagnosis of Toxoplasma is real-time PCR > LAMP > conventional PCR. In addition to these DNA analysis tools, bioassay in mice and cats is considered as "gold standard" to detect T. gondii. CONCLUSION: This review article will help the readers for grasping advantages and limitations of different diagnostic tools for screening meat samples for T. gondii. This review also makes bibliography about the type of meat sample to be processed for diagnosis and different primers or sequences to be targeted for T. gondii by number of researches for its detection from meat or tissue sample using DNA amplification techniques.

Immunological-based assays for specific detection of shrimp viruses.

Among shrimp viral pathogens, white spot syndrome virus (WSSV) and yellow head virus (YHV) are the most lethal agents, causing serious problems for both the whiteleg shrimp, Penaeus (Litopenaeus) vannamei, and the black tiger shrimp, Penaeus (Penaeus) monodon. Another important virus that infects P. vannamei is infectious myonecrosis virus (IMNV), which induces the white discoloration of affected muscle. In the cases of taura syndrome virus and Penaeus stylirostris densovirus (PstDNV; formerly known as infectious hypodermal and hematopoietic necrosis virus), their impacts were greatly diminished after the introduction of tolerant stocks of P. vannamei. Less important viruses are Penaeus monodon densovirus (PmDNV; formerly called hepatopancreatic parvovirus), and Penaeus monodon nucleopolyhedrovirus (PemoNPV; previously called monodon baculovirus). For freshwater prawn, Macrobrachium rosenbergii nodavirus and extra small virus are considered important viral pathogens. Monoclonal antibodies (MAbs) specific to the shrimp viruses described above have been generated and used as an alternative tool in various immunoassays such as enzyme-linked immunosorbent assay, dot blotting, Western blotting and immunohistochemistry. Some of these MAbs were further developed into immunochromatographic strip tests for the detection of WSSV, YHV, IMNV and PemoNPV and into a dual strip test for the simultaneous detection of WSSV/YHV. The strip test has the advantages of speed, as the result can be obtained within 15 min, and simplicity, as laboratory equipment and specialized skills are not required. Therefore, strip tests can be used by shrimp farmers for the pond-side monitoring of viral infection.

Serum GP73 is complementary to AFP and GGT-II for the diagnosis of hepatocellular carcinoma.

Golgi protein 73 (GP73) is a resident Golgi type II transmembrane protein that has been reported to markedly increase in chronic liver disease, particularly in hepatocellular carcinoma (HCC). However, it remains unclear as to whether serum GP73 represents a reliable serum marker for the diagnosis of HCC. The aim of the present study was to evaluate the diagnostic value of serum GP73 in patients with HCC and to determine the diagnostic accuracy of measuring serum GP73 in combination with α-fetoprotein (AFP) and γ-glutamyl transferase isoenzyme II (GGT-II) in HCC. Serum GP73 was detected using a time-resolved fluorescence immunological assay (TRFIA) and enzyme-linked immunosorbent assay (ELISA) in 79 HCC cases, including 16 liver cirrhosis, 30 chronic hepatitis and 28 healthy individuals. The correlation between serum GP73 and tumor size and HCC grading was analyzed and the complementary diagnostic value of serum GP73, AFP and GGT-II was evaluated. TRFIA was established for the detection of serum GP73 and was sensitive and reproducible. The expression levels of serum GP73 were markedly higher in the patients with HCC when compared with those of the individuals with liver cirrhosis and chronic hepatitis or the healthy individuals. According to the receiver operating characteristic (ROC) curve, diagnostic sensitivity and specificity for HCC with a cut-off value of 78.1 ng/l were 73.4 and 79.0%, respectively. However, no correlation was identified among serum GP73 and tumor size or grading, and no correlations were identified among serum GP73, AFP and GGT-II. The diagnostic sensitivities for HCC, as detected by TRFIA of GP73, AFP and GGT-II, were 73.4, 55.6 and 68.4%, respectively, and the specificities were 80.0, 86.7 and 97.1%, respectively. The combined determination of these markers increased the diagnostic sensitivity to 96.3% for HCC. TRFIA functions as a sensitive and replicable assay for the detection of serum GP73. The levels of serum GP73 were significantly higher in the HCC group when compared with the individuals with benign liver diseases. Serum GP73 may serve as a potential independent diagnostic candidate for HCC and the combined determination of serum GP73, AFP and GGT-II may increase the diagnostic efficiency of HCC.