Advantages of stereolithographic 3D printing in the fabrication of the Affiblot device for dot-blot assays.

In stereolithographic (SLA) 3D printing, objects are constructed by exposing layers of photocurable resin to UV light. It is a highly user-friendly fabrication method that opens a possibility for technology sharing through CAD file online libraries. Here, we present a prototyping procedure of a microfluidics-enhanced dot-blot device (Affiblot) designed for simple and inexpensive screening of affinity molecule characteristics (antibodies, oligonucleotides, cell receptors, etc.). The incorporation of microfluidic features makes sample processing user-friendly, less time-consuming, and less laborious, all performed completely on-device, distinguishing it from other dot-blot devices. Initially, the Affiblot device was fabricated using CNC machining, which required significant investment in manual post-processing and resulted in low reproducibility. Utilization of SLA 3D printing reduced the amount of manual post-processing, which significantly streamlined the prototyping process. Moreover, it enabled the fabrication of previously impossible features, including internal fluidic channels. While 3D printing of sub-millimeter microchannels usually requires custom-built printers, we were able to fabricate microfluidic features on a readily available commercial printer. Open microchannels in the size range 200-300 µm could be fabricated with reliable repeatability and sealed with a replaceable foil. Economic aspects of device fabrication are also discussed.

Validation of dot blot immunoassay for measurement of complement opsonization of nanoparticles.

Complement plays a critical role in the immune response toward nanomaterials. The complement attack on a foreign surface results in the deposition of C3, assembly of C3 convertases, the release of anaphylatoxins C3a and C5a, and finally, the formation of membrane attack complex C5b-9. Various technologies can measure complement activation markers in the fluid phase, but measurements of surface C3 deposition are less common. Previously, we developed an ultracentrifugation-based dot blot immunoassay (DBI) to measure the deposition of C3 and other protein corona components on nanoparticles. Here, we validate the repeatability of the DBI and its correlation with pathway-specific and common fluid phase markers. Moreover, we discuss the advantages of DBI, such as cost-effectiveness and versatility, while addressing potential limitations. This study provides insights into complement activation at the nanosurface level, offering a valuable tool for nanomedicine researchers in the field.

Establishment and application of a reverse dot blot assay for 13 mutations of hearing-loss genes in primary hospitals in China.

Background: Hearing loss is a common sensorineural dysfunction with a high incidence in China. Although genetic factors are important causes of hearing loss, hearing-related gene detection has not been widely adopted in China. Objective: Establishing a rapid and efficient method to simultaneously detect hotspot hearing loss gene mutations. Methods: A reverse dot blot assay combined with a flow-through hybridization technique was developed for the simultaneous detection of 13 hotspot mutations of 4 hearing loss-related genes including GJB2, GJB3, SLC26A4, and the mitochondrial gene MT-RNR1. This method involved PCR amplification systems and a hybridization platform. Results: The technique can detect 13 hotspot mutations of 4 hearing loss-related genes. And a total of 213 blood samples were used to evaluate the availability of this method. Discussion: Our reverse dot blot assay was a simple, rapid, accurate, and cost-effective method to identify hotspot mutations of 4 hearing loss-related genes in a Chinese population.

In-process quality control in foot-and-mouth disease vaccine production by detection of viral non-structural proteins using chemiluminescence dot blot assay.

Foot-and-mouth disease (FMD) is a contagious viral disease of cloven-footed animals. Immunization with inactivated virus vaccine is effective to control the disease. Six-monthly vaccination regimen in endemic regions has proven to be effective. To enable the differentiation of infected animals from those vaccinated, non-structural proteins (NSPs) are excluded during vaccine production. While the antibodies to structural proteins (SPs) could be observed both in vaccinated and infected animals, NSP antibodies are detectable only in natural infection. Quality control assays that detect NSPs in vaccine antigen preparations, are thus vital in the FMD vaccine manufacturing process. In this study, we designed a chemiluminescence dot blot assay to detect the 3A and 3B NSPs of FMDV. It is sensitive enough to detect up to 20 ng of the NSP, and exhibited specificity as it does not react with the viral SPs. This cost-effective assay holds promise in quality control assessment in FMD vaccine manufacturing.

A dot-blot ELISA preliminary evaluation using PvMSP1-42 recombinant protein as antigen for serological diagnosis of Plasmodium vivax infection in Thailand.

Plasmodium vivax is the most prevalent cause of malaria in Thailand and is predominant in malarial endemic areas worldwide. P. vivax infection is characterized by low parasitemia, latent liver-stage parasites, or asymptomatic infections leading to underreported P. vivax cases. These are significant challenges for controlling and eliminating P. vivax from endemic countries. This study developed and evaluated a dot-blot enzyme-linked immunosorbent assay (ELISA) using PvMSP1-42 recombinant antigen for serological diagnosis based on the detection of antibodies against P. vivax. The optimal PvMSP1-42 concentration and dilutions of anti-human IgG horseradish peroxidase (HRP)-conjugated antiserum were tested on 88 serum samples from P. vivax, Plasmodium falciparum and bacterial infection, including healthy individuals. A cut-off titer of 1:800 produced optimal values for sensitivity and specificity of 90.9 and 98.2%, respectively, with an accuracy of 95.5%. The positive and negative predictive values were 96.8 and 94.7% respectively. The results from microscopic examination and dot-blot ELISA showed strong agreement with the 0.902 kappa index. Thus, the dot-blot ELISA using PvMSP1-42 antigen provided high sensitivity and specificity suitable for serodiagnosis of P. vivax infection. The test is a simple and quick diagnostic assay suitable for field testing as it does not require specific equipment or particular skills.

Comparative analysis of a novel N-butanol-prepared antigen vs thermo-resistant and sonicated antigens for human leptospirosis detection.

The diagnosis of human leptospirosis is mainly based on serological assays. Since the extraction by N-butanol has only been studied as an antigen for the diagnosis of cattle leptospirosis, this study aimed to investigate the feasibility of the N-butanol preparation for the diagnosis of human leptospirosis and compare it with sonicated and thermo-resistant antigens in IgM dot-blot test. Paired serum samples from 147 laboratory-confirmed leptospirosis cases were tested. The control group consisted of 148 serum samples from healthy individuals and nonleptospirosis cases. N-butanol antigens from serovar Copenhageni (ButC3) and serovar Patoc (ButP3) showed reactivity with antileptospiral antibodies from patients with confirmed leptospirosis. In the acute phase, sensitivities of IgM dot-blot assay with ButC3 and ButP3 antigens were 47.6% and 51.0%, respectively. In the convalescent phase, sensitivities were 95.9% (ButC3) and 93.2% (ButP3), and no significant differences were observed among the IgM dot-blot tests with other antigens. The specificity of the IgM dot-blot test with ButC3 antigen was good (92.6%), but with ButP3 (83.1%), it was significantly lower than with the other tests. The IgM dot-blot test described in this study is simple to perform and presents reliable visual results. Antigens prepared by N-butanol proved to be valuable diagnostic markers of leptospirosis.

Design of dot-blot hybridization assay for simultaneous detection of Campylobacter jejuni and Campylobacter coli: a preliminary study.

Objectives: Campylobacters are a major cause of gastroenteritis worldwide. These are fastidious in culture and false negative results are seen in many clinical laboratories. Among molecular methods, the dot-blot technique is widely used for a variety of purposes, especially diagnostics. So, the authors aimed to detect C. jejuni and C. coli simultaneously using a dot-blot assay. Methods: After evaluating the bioinformatics studies, a cadF-conserved fragment was selected for the design of primers and probe. DNAs from standard strains and a recombinant plasmid, prepared in this study, were used to assess the technique. The specificity of the method was also surveyed using DNAs from other enteric bacteria. The limit of detection was evaluated by recombinant plasmid and different concentrations of the designed probe. Results: A 95-bp fragment of cadF was selected, and in silico analysis studies showed that it is conserved between both species. Also, the non-specific annealing of the primers and probe with other bacteria was not seen theoretically. The technique with recombinant plasmid as well as DNAs of standard strains created black spots on the membrane, confirming that the probe was correctly synthesized. No non-specific reactions with other bacterial species were observed (specificity=100%). The limit of detection of the test was determined to be 50 µg/ml. Conclusions: This is the first study to simultaneously detect two important pathogens in the Campylobacter genus and was able to detect C. jejuni and C. coli with acceptable sensitivity and specificity.

Selection of collagen IV fragments forming the outer sphere of the native protein: Assessment of biological activity for regenerative medicine.

The aim of this research was to select the fragments that make up the outer layer of the collagen IV (COL4A6) protein and to assess their potential usefulness for regenerative medicine. It was expected that because protein-protein interactions take place via contact between external domains, the set of peptides forming the outer sphere of collagen IV will determine its interaction with other proteins. Cellulose-immobilized protein fragment libraries treated with polyclonal anti-collagen IV antibodies were used to select the peptides forming the outer sphere of collagen IV. In the first test, 33 peptides that strongly interacted with the polyclonal anti-collagen IV antibodies were selected from a library of non-overlapping fragments of collagen IV. The selected fragments of collagen IV (cleaved from the cellulose matrix) were tested for their cytotoxicity, their effects on cell viability and proliferation, and their impact on the formation of reactive oxygen species (ROS). The studies used RAW 264.7 mouse macrophage cells and Hs 680.Tr human fibroblasts. PrestoBlue, ToxiLight™, and ToxiLight 100% Lysis Control assays were conducted. The viability of fibroblasts cultured with the addition of increasing concentrations of the peptide mix did not show statistically significant differences from the control. Fragments 161-170, 221-230, 721-730, 1331-1340, 1521-1530, and 1661-1670 of COL4A6 were examined for cytotoxicity against BJ normal human foreskin fibroblasts. None of the collagen fragments were found to be cytotoxic. Further research is underway on the potential uses of collagen IV fragments in regenerative medicine.

Rapid Dot-Blot Immunoassay for Detecting Multiple Salmonella enterica Serotypes.

Salmonella, a major contributor to foodborne infections, typically causes self-limiting gastroenteritis. However, it is frequently invasive and disseminates across the intestinal epithelium, leading to deadly bacteremia. Although the genus is subdivided into >2,600 serotypes based on their antigenic determinants, only few serotypes are responsible for most human infections. In this study, a rapid dot-blot immunoassay was developed to diagnose multiple Salmonella enterica serotypes with high incidence rates in humans. The feasibility of 10 commercial antibodies (four polyclonal and six monoclonal antibodies) was tested using the 18 serotypes associated with 67.5% Salmonella infection cases in the United States of America (U.S.A) in 2016. Ab 3 (polyclonal; eight of 18 serotypes), Ab 8 (monoclonal; 13 of 18 serotypes), and Ab 9 (monoclonal; 10 of 18 serotypes) antibodies exhibited high detection rates in western blotting and combinations of two antibodies (Ab 3+8, Ab 3+9, and Ab 8+9) were applied to dot-blot assays. The combination of Ab 3+8 identified 15 of the tested 18 serotypes in 3 h, i.e., S. Enteritidis, S. Typhimurium, S. Javiana, S. I 4,[5],12:i:-, S. Infantis, S. Montevideo, S. Braenderup, S. Thompson, S. Saintpaul, S. Heidelberg, S. Oranienburg, S. Bareilly, S. Berta, S. Agona, and S. Anatum, which were responsible for 53.7% Salmonella infections in the U.S. in 2016. This cost-effective and rapid method can be utilized as an on-site colorimetric method for Salmonella detection.

Components Subcellular Localization: Cell Surface Exposure.

Surface-exposed proteins of Gram-negative bacteria are represented by integral outer membrane ß-barrel proteins and lipoproteins. There are no computational methods to predict surface-exposed lipoproteins, and therefore lipoprotein topology must be experimentally tested. This chapter describes several distinct but complementary methods for detection of surface-exposed proteins: cell surface protein labeling, accessibility to extracellular protease or antibodies, and SpyTag/SpyCatcher system.

Deciphering the human antibody response against Burkholderia pseudomallei during melioidosis using a comprehensive immunoproteome approach.

Introduction: The environmental bacterium Burkholderia pseudomallei causes the often fatal and massively underreported infectious disease melioidosis. Antigens inducing protective immunity in experimental models have recently been identified and serodiagnostic tools have been improved. However, further elucidation of the antigenic repertoire of B. pseudomallei during human infection for diagnostic and vaccine purposes is required. The adaptation of B. pseudomallei to very different habitats is reflected by a huge genome and a selective transcriptional response to a variety of conditions. We, therefore, hypothesized that exposure of B. pseudomallei to culture conditions mimicking habitats encountered in the human host might unravel novel antigens that are recognized by melioidosis patients. Methods and results: In this study, B. pseudomallei was exposed to various stress and growth conditions, including anaerobiosis, acid stress, oxidative stress, iron starvation and osmotic stress. Immunogenic proteins were identified by probing two-dimensional Western blots of B. pseudomallei intracellular and extracellular protein extracts with sera from melioidosis patients and controls and subsequent MALDI-TOF MS. Among B. pseudomallei specific immunogenic signals, 90 % (55/61) of extracellular immunogenic proteins were identified by acid, osmotic or oxidative stress. A total of 84 % (44/52) of intracellular antigens originated from the stationary growth phase, acidic, oxidative and anaerobic conditions. The majority of the extracellular and intracellular protein antigens were identified in only one of the various stress conditions. Sixty-three immunoreactive proteins and an additional 38 candidates from a literature screening were heterologously expressed and subjected to dot blot analysis using melioidosis sera and controls. Our experiments confirmed melioidosis-specific signals in 58 of our immunoproteome candidates. These include 15 antigens with average signal ratios (melioidosis:controls) greater than 10 and another 26 with average ratios greater than 5, including new promising serodiagnostic candidates with a very high signal-to-noise ratio. Conclusion: Our study shows that a comprehensive B. pseudomallei immunoproteomics approach, using conditions which are likely to be encountered during infection, can identify novel antibody targets previously unrecognized in human melioidosis.

Optimizing the Composition of the Substrate Enhances the Performance of Peroxidase-like Nanozymes in Colorimetric Assays: A Case Study of Prussian Blue and 3,3'-Diaminobenzidine.

One of the emerging trends in modern analytical and bioanalytical chemistry involves the substitution of enzyme labels (such as horseradish peroxidase) with nanozymes (nanoparticles possessing enzyme-like catalytic activity). Since enzymes and nanozymes typically operate through different catalytic mechanisms, it is expected that optimal reaction conditions will also differ. The optimization of substrates for nanozymes usually focuses on determining the ideal pH and temperature. However, in some cases, even this step is overlooked, and commercial substrate formulations designed for enzymes are utilized. This paper demonstrates that not only the pH but also the composition of the substrate buffer, including the buffer species and additives, significantly impact the analytical signal generated by nanozymes. The presence of enhancers such as imidazole in commercial substrates diminishes the catalytic activity of nanozymes, which is demonstrated herein through the use of 3,3'-diaminobenzidine (DAB) and Prussian Blue as a model chromogenic substrate and nanozyme. Conversely, a simple modification to the substrate buffer greatly enhances the performance of nanozymes. Specifically, in this paper, it is demonstrated that buffers such as citrate, MES, HEPES, and TRIS, containing 1.5-2 M NaCl or NH4Cl, substantially increase DAB oxidation by Prussian Blue and yield a higher signal compared to commercial DAB formulations. The central message of this paper is that the optimization of substrate composition should be an integral step in the development of nanozyme-based assays. Herein, a step-by-step optimization of the DAB substrate composition for Prussian Blue nanozymes is presented. The optimized substrate outperforms commercial formulations in terms of efficiency. The effectiveness of the optimized DAB substrate is affirmed through its application in several commonly used immunostaining techniques, including tissue staining, Western blotting assays of immunoglobulins, and dot blot assays of antibodies against SARS-CoV-2.

Epitope identification of a Lys63 linkage ubiquitin antibody by mass spectrometric epitope excision and extraction approaches.

Ubiquitin, a conserved protein in eukaryotic cells, exists as a monomer or polyubiquitin chains known as isopeptide-linked polymers. These chains are attached to a substrate or other ubiquitin molecules through a covalent bond between the α-amino group of lysine in ubiquitin and glycine in the C-terminal of the subsequent ubiquitin unit. The choice of the specific lysine residue in ubiquitin for forming ubiquitin-ubiquitin chains determines its biochemical and biological function. A detailed chemical structure-function evaluation of the respective polyubiquitin chain is required. Interestingly, specific lysine linkage polyubiquitin chains become covalently bonded to many pathological inclusions seen in serious human disease states which appear to be resistant to normal degradation, so the interaction between polyubiquitin chains and ubiquitin antibodies is very useful. For example, the neurofibrillary tangles of Alzheimer's disease and the Lewy bodies seen in Parkinson's disease are heavily ubiquitinated and can be readily visualized using specific ubiquitin antibodies. This study utilized synthetic ubiquitin building block peptides that contained various lysine residues (K6, K11, K33, K48, and K63) linked to a Gly-Gly dipeptide, with the aim of exploring the recognition specificity of the Lys63-polyubiquitin antibody. The interaction studies between different ubiquitin building blocks and the specific Lys63-ubiquitin (K63-Ub) antibody were performed by affinity-mass spectrometry (Affinity-MS) and immunoblotting which enables direct protein identification from biological material with unprecedented selectivity. Affinity-MS and dot blot data proved the specific binding of the K63-Ub antibody to the ubiquitin peptides containing Lys6 or Lys63 residues. In epitope excision for mass spectrometric epitope identification, the ubiquitin building block with Lys63 residue bound to the immobilized K63-Ub antibody was proteolytically cleaved using pronase. The resulting epitope and non-epitope fractions were subjected to matrix-assisted laser desorption/ionization-time of flight analysis, revealing that the epitope is located within the sequence ubiquitin(60-66). Epitope extraction-MS consistently confirmed these findings.

Evaluation of the Histoplasma capsulatum 100-kilodalton antigen dot blot for the rapid diagnosis of progressive histoplasmosis in HIV/AIDS patients.

Among people living with HIV (PLHIV), progressive disseminated histoplasmosis (PDH) represents an important cause of mortality. Since antigen detection allows a rapid diagnosis and the instauration of a specific treatment this study aimed to evaluate the analytical performance of the Hcp100 dot blot, an in-house assay that detects the Histoplasma capsulatum 100-kilodalton antigen in urine and compare it with 2 commercially available assays the Histoplasma Urine Antigen Lateral Flow Assay (MVD-LFA) (MiraVista® Diagnostics) and the Clarus Histoplasma Galactomannan EIA (Clarus HGM) (IMMY). Urine specimens from 23 PLHIV with PDH, 13 patients with other infectious diseases, and 20 healthy individuals were tested. The Hcp100 dot blot showed higher sensitivity (87.0%), specificity (97.0%) and accuracy (92.9%) than the MVD-LFA (73.9%, 78.8%, and 76.8%, respectively) and the Clarus HGM (78.3%, 90.9%, and 85.7%, respectively). The Hcp100 dot blot had high analytical performance and would be a valuable screening tool for diagnosing PDH among PLHIV.

Development of an instrument-free and low-cost ELISA dot-blot test to detect antibodies against SARS-CoV-2.

Most laboratory tests to detect the presence of anti-SARS-CoV-2 antibodies use enzyme-linked immunosorbent assays (ELISA) or chemiluminescence immunoassays (CLIA); however, equipment for these immunoassays is unavailable in many areas of low- and middle-income countries. Rapid lateral flow immunoassay (LFIA) tests are an equipment-free option, but their high price may make them less suitable for conducting seroprevalence surveys. Here, we describe a simple dual antigen ELISA dot-blot test to detect anti-SARS-CoV-2 IgG antibodies with high sensitivity (94-98%) and specificity (92-100%), compared to commercially available ELISA and CLIA options. Additionally, this ELISA dot-blot test can be completed in one hour using minimal laboratory equipment. Importantly, this immunoassay is significantly more affordable than most LFIA tests available on the global market. The dot-blot strips may be stored for up to 7 days under freezing conditions. This ELISA dot-blot test is a cost-effective option for conducting seroprevalence screenings in areas lacking ELISA or CLIA facilities, compared to LFIA tests.

A PCR-Reverse Dot Blot Hybridization Based Microfluidics Detection System for the Rapid Identification of 13 Fungal Pathogens Directly After Blood Cultures Over a Period of Time.

Introduction: It is time-consuming to identify fungal pathogens from positive blood cultures using the standard culture-based method. And delayed diagnosis of bloodstream infection leads to significantly increased mortality. Methods: We developed a PCR-reverse dot blot hybridization combined with microfluidic chip techniques to rapidly identify 13 fungal pathogens within 3-4 h using the sample of blood cultured over a period of time. Results: We performed clinical validation using 43 blood culture-positive samples with a sensitivity of 96.7%, a specificity of 100%, and a concordance rate of 97.7%. Samples with different culture durations were evaluated using our approach, showing a detection rate of 85.2% at 16 h and 96.3% at 24 h; the platform could reach a detection limit of 103cfu/mL for the Candida spp. and 103 copies/mL for Aspergillus spp. Discussion: The detection rate of the platform is much higher than the positive rates of concurrent blood cultures. This method bears substantial clinical application potential as it incorporates the microfluidic platform with low reagent consumption, automation, and low cost (about 10 dollars).

A microfluidic chip-based multiplex PCR-reverse dot blot hybridization technique for rapid detection of enteropathogenic bacteria.

Diarrhea caused by enteropathogenic bacteria is a major public health issue worldwide, especially in developing countries. In this study, a microfluidic chip-based multiplex polymerase chain reaction (PCR)-reverse dot blot hybridization technology for the rapid and simultaneous detection of 11 enteropathogenic bacteria was developed and the entire process was completed within 3-4 h. The specificity of this method was analyzed using 11 types of pure target bacterial colonies and another 7 types of pure bacterial colonies, and its sensitivity was evaluated with the serial 10-fold dilution of 11 types of pure target bacterial colonies. The detection limit of this method was as low as 103-102 CFU/mL, and it exhibited high specificity for enteropathogenic bacteria. A total of 60 clinical diarrheal fecal samples were detected using this method, the results of which were compared with those of the conventional reference method, which resulted in a positive coincident rate of 100% and a negative coincident rate of 93.75%. Based on the findings, it could be concluded that multiplex PCR-reverse dot blot hybridization based on the microfluidic chip is a rapid, economical, sensitive, specific, and high-throughput method for detecting enteropathogenic bacteria.

Detection of M2-Type Anti-Mitochondrial Autoantibodies against Specific Subunits in the Diagnosis of Primary Biliary Cholangitis in Patients with Discordant Results.

BACKGROUND: M2-type anti-mitochondrial autoantibodies are considered the hallmark of primary biliary cholangitis and are directed mainly against the E2 subunits of the 2-oxo acid dehydrogenase complex enzymes (PDC, BCOADC and OGDC). The aim of this study was to determine whether a Dot-blot that includes these E2 subunits separately could confirm the results of methods with non-separated subunits in patients with low positive or discordant results between techniques. METHODS: Sera of 24 patients with low positive or discordant results and of 10 patients with clear positive results by non-separated subunits methods were analyzed by Dot-blot with separated subunits. RESULTS: Autoantibodies against E2 subunits of PDC, BCOADC or OGDC were detected in all patients, except in one case from the low positive or discordant results group, by Dot-blot with separated subunits. CONCLUSIONS: It would be advisable to use methods that include the three E2 subunits, and a Dot-blot with separated subunits could confirm doubtful cases by non-separated assays.

Lateral flow immunoassay for rapid and sensitive detection of dsRNA contaminants in in vitro-transcribed mRNA products.

High purity is essential in mRNA-based therapeutic applications. A major contaminant of in vitro-transcribed (IVT) mRNA manufacturing is double-stranded RNA (dsRNA), which can induce severe anti-viral immune responses. Detection methods, such as agarose gel electrophoresis, ELISA, and dot-blot assay, are used to detect the existence of dsRNA in IVT mRNA products. However, these methods are either not sensitive enough or time-consuming. To overcome these challenges, we develop a rapid, sensitive, and easy-to-implement colloidal gold nanoparticle-based lateral flow strip assay (LFSA) with sandwich format for the detection of dsRNA from IVT process. dsRNA contaminant can be determined visually on the test strip or quantitatively with a portable optical detector. This method allows for a 15 min detection of N1-methyl-pseudouridine (m1Ψ)-containing dsRNA with a detection limit of 69.32 ng/mL. Furthermore, we establish the correlation between the LFSA test results and the immune response caused by dsRNA in mice. The LFSA platform allows the rapid, sensitive, and quantitative monitoring of purity in massive IVT mRNA products and aids for the prevention of immunogenicity by dsRNA impurities.

Evaluation of Flathead Grey Mullets (Mugil cephalus) Immunization and Long-Term Protection against Vibrio harveyi Infection Using Three Different Vaccine Preparations.

In recent years, flathead grey mullets (Mugil cephalus) cultured in Eilat (Israel) have been highly affected by Vibrio harveyi, showing neurological signs such as uncoordinated circular swimming followed by high mortality rates. Despite the advances in and different approaches to control vibriosis associated with Vibrio harveyi, including commercial vaccines, most of them have not succeeded in long-term protection. In this study, we evaluated the effectiveness, long-term protection, and antibody production of three vaccine preparations: heat-killed bacteria (HKB), membrane proteins denaturation (BME PROT), and internal proteins (INT PROT) developed specifically against Vibrio harveyi for grey mullets. Our results show that fish immunized with heat-killed bacteria emulsified with adjuvant presented the most effective and long-lasting protection against the bacterium, and a cross-protection against other bacteria from the harveyi clade. The effectiveness of each immunization treatment correlated with the levels of specific antibody production against Vibrio harveyi in the serum of the immunized fish.