Cryptosporidium Diagnostic Assays: Microscopy.

Development of a lateral flow immunochromatographic assay for rapid detection of Mycoplasma pneumoniae-specific IgM in human serum specimens

Rapid detection of lactic acid bacteria by lateral flow immunochromatographic assay

Brucellosis, a prevalent zoonotic disease, induces substantial economic losses in livestock due to reproductive impairments and high mortality rates; it also presents considerable public health concerns worldwide. The current work highlights the potential application of the developed lateral flow immunochromatographic assay (LFIA) in the rapid and accurate detection of Brucella spp., underscoring its significance in disease management and control. The study investigated the purity of the extracted B. abortus smooth lipopolysaccharides-O (S-LPS-O) using high-performance liquid chromatography (HPLC). Moreover, it delved into the development and assessment of a novel diagnostic kit for Brucella using a recombinant protein A-lateral flow assay in comparison to the RBPT, SAT, MRT, I-ELISA as screening tests, also to C-ELISA, CFT as confirmatory tests. HPLC analysis indicated a distinctive absence of impurities in the extracted S-LPS-O, contrasting with the three peaks observed in the commercial LPS chromatogram. Sensitivity, specificity, and accuracy evaluations were performed, with the LFIA demonstrating promising results, showcasing its potential as a reliable diagnostic tool. Statistical analysis of the LFIA, RBPT, SAT, MRT, ELISA, CFT, and PCR results revealed notable sensitivity, specificity, and accuracy rates, affirming the diagnostic efficacy of the LFIA. The novel layout of the LFIA and the purity of extracted S-LPS-O showcased significant progress in the diagnostic capabilities of the developed LFIA. Moreover, the currently developed LFIA introduces a rapid and reliable diagnostic tool, particularly in resource-limited settings, owing to its ease of use and minimal equipment requirements with its competitively obtained results.

The development of Lateral Flow Immunochromatography Assay can be divided into two levels; standardizing membrane characteristics and optimizing molecular level immunoassay reaction between analyte and detector molecules. In the preliminary phase the reaction specificity of capture and detector antibodies with the analyte has to be checked with other techniques like ELISA. Molarity and pH of conjugation buffer have prime importance in the immunoreaction among analyte and antibodies. Epitope mapping of the capture and detector antibodies is also recommended to confirm the specificity of the assay. Standardization of membrane characteristics directly relates to the sensitivity of the assay through its porosity, hydrophobicity, protein holding/releasing capacity and wicking rate. Under optimised condition a perfect Lateral Flow Immunochromatography Assay should have high on-rate (target binding efficiency), low off-rate (target releasing efficiency) and low Cross-reactivity. In this manuscript, we share our experience, especially on developmental strategies and troubleshooting, that we have experienced during Lateral Flow Immunochromatography Assay kit development.

Nipah virus (NiV) and Hendra virus (HeV), which both belong to the genus henipavirus, are zoonotic pathogens that cause severe systemic, neurological, and/or respiratory disease in humans and a variety of mammals. Therefore, monitoring viral prevalence in natural reservoirs and rapidly diagnosing cases of henipavirus infection are critical to limiting the spread of these viruses. Current laboratory methods for detecting NiV and HeV include virus isolation, reverse transcription quantitative real-time PCR (RT-qPCR), and antigen detection via an enzyme-linked immunosorbent assay (ELISA), all of which require highly trained personnel and specialized equipment. Here, we describe the development of a point-of-care customized immunochromatographic lateral flow (ILF) assay that uses recombinant human ephrin B2 as a capture ligand on the test line and a NiV-specific monoclonal antibody (mAb) on the conjugate pad to detect NiV and HeV. The ILF assay detects NiV and HeV with a diagnostic specificity of 94.4% and has no cross-reactivity with other viruses. This rapid test may be suitable for field testing and in countries with limited laboratory resources.

Article Diagnostic performance of a SARS-CoV-2 IgG/IgM lateral flow immunochromatography assay in symptomatic patients presenting to the emergency department was published on September 1, 2020 in the journal Clinical Chemistry and Laboratory Medicine (CCLM) (volume 58, issue 9).

Evaluating the clinical performance of available methods to detect antibodies against Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has become a primordial issue in clinical laboratories. The aim of this study was to evaluate the clinical performance of two methods for SARS-CoV-2 antibodies detection, an automated Chemiluminescent Immunoassay (CLIA) and an immunochromatographic Lateral-Flow Assay (LFA) in patients with positive reverse transcription polymerase chain reaction (RT-PCR). Performance for CLIA method was Positive Agreement (PA) 56.6% and Negative Agreement (NA) 96,6% for IgM and PA 85.8%/NA 90,2% for IgG. Performance for LFA method was PA 56.2% and NA 100% for IgM and PA 95.5% and NA 100 % for IgG. LFA general agreement IgG was better than CLIA. In both methods, significant differences in Kappa index are observed when IgG and IgM are compared. When evaluating the data from a clinical perspective, we found that both method performance for IgM detection may not meet the expected requirements for their clinical utility and could lead to an inappropriate medical decision. The findings of this study show that both immunoassay methods might be reliable for assessing immunological response in COVID-19 patients. Our results also confirm that IgG measurement could be helpful, especially for epidemiological studies in our population. These results provide evidence to justify epidemiological studies in our population.

BackgroundGluten, a food allergen, is available in foods derived from wheat, rye and barley. It damages the small intestine and causes celiac disease. Herein, we designed a rapid immunochromatographic lateral flow test assay for detecting the gluten contents of raw materials. In this rapid test, the presence of gluten was screened through the capturing of gliadin (a toxic component of gluten) by two identical gliadin monoclonal antibodies. One of the antibodies was immobilized on the membrane in the test zone as a capture reagent. The other antibody was labeled with gold nanoparticles (AuNPs) as a detector reagent.ResultsGold nanoparticles with a size of about 20 nm were synthesized and conjugated to the gliadin monoclonal antibodies. The detection limit of the experimental assay was 20 ppm and positive results were visualized after 15 min using only 40 μL of the extracted sample for each test. Analysis of different flour samples identified the best sensitivity and specificity of the lateral flow test strip (LFTS).ConclusionThe experimental LFTS is an easy-to-use and rapid method for the screening of gluten level in raw materials. The LFTS may be employed to ensure the safety of foods.

In this study, a lateral flow immunochromatographic assay (LFIA) system for the detection of immunoglobulin M (IgM) antibodies, related to TORCH [(T)oxoplasmosis, (O)ther agents, (R)ubella (also known as German Measles), (C)ytomegalovirus, and (H)erpes simplex virus infections], based on gold magnetic nanoparticles, was established. Following modification with poly(methacrylic acid), the gold magnetic nanoparticles conjugated with an anti‑human IgM antibody (μ‑chain specific) to construct a probe. A lateral flow assay device was constructed based on these conjugates. IgM antibodies to four types of pathogens, notably toxoplasmosis, rubella virus, cytomegalovirus and herpes simplex virus type 2, were detected using this device. Compared with commercial colloidal gold‑based LFIA strips, our method exhibited higher sensitivity. No interference with triglycerides, hemoglobin and bilirubin occurred, and no cross‑reactivity was noted among the four pathogens. The gold magnetic nanoparticle‑LFIA strips were used to assess 41 seropositive and 121 seronegative serum samples. The sensitivity was 100% (162/162) and the specificity was 100% (162/162). This method cannot only be used for the detection of TORCH IgM-specific antibodies, but it can potentially be developed for use in the diagnosis of other acute or recently identified autoimmune diseases.

Survey of actual literature and scientific reports was made. The most substantial analytical and diagnostical methods were identified and described. Principles and limitations of the methods are described, and actual papers are cited in this review. Analytical and diagnostical methods like Polymerase Chain Reaction (PCR), Loop-mediated isothermal Amplification (LAMP), Lateral Flow Immunochromatography Assay (LFIA), Enzyme-Linked Immunosorbent Assay (ELISA), biosensors and ChemiLuminescence ImmunoAssay (CLIA) are discussed for assay of viral particles, antigens and specific host antibodies in blood, serum, plasma, nasopharyngeal swab and other samples in order to diagnose COVID-19. The Coronavirus Disease 2019 (COVID-19) is an emerging disease that has spread over the world since the end of year 2019. The global epidemic pointed at the necessity to introduce sensitive methods for instrumental diagnosis of COVID-19 and distinguishing it from the other viral diseases. (Tab. 2, Ref. 96).

Advantages of fluorescent microspheres compared with colloidal gold as a label in immunochromatographic lateral flow assays

Lateral flow immunochromatographic assay (LFIA) for tumor marker detection: technical evolution, clinical translation challenges, and future perspectives.

Lateral flow assay for rapid differentiation of Mycobacterium tuberculosis complex and 97 species of mycobacteria other than tuberculosis grown in Löwenstein-Jensen and TK-SLC medium

The current study aimed at characterizing the dynamics of SARS‐CoV‐2 nucleocapsid (N) antigenemia in a cohort of critically ill adult COVID‐19 patients and assessing its potential association with plasma levels of biomarkers of clinical severity and mortality. Seventy‐three consecutive critically ill COVID‐19 patients (median age, 65 years) were recruited. Serial plasma (n = 340) specimens were collected. A lateral flow immunochromatography assay and reverse‐transcription polymerase chain reaction (RT‐PCR) were used for SARS‐CoV‐2 N protein detection and RNA quantitation and in plasma, respectively. Serum levels of inflammatory and tissue‐damage biomarkers in paired specimens were measured. SARS‐CoV‐RNA N‐antigenemia and viral RNAemia were documented in 40.1% and 35.6% of patients, respectively at a median of 9 days since symptoms onset. The level of agreement between the qualitative results returned by the N‐antigenemia assay and plasma RT‐PCR was moderate (k = 0.57; p < 0.0001). A trend towards higher SARS‐CoV‐2 RNA loads was seen in plasma specimens testing positive for N‐antigenemia assay than in those yielding negative results (p = 0.083). SARS‐CoV‐2 RNA load in tracheal aspirates was significantly higher (p < 0.001) in the presence of concomitant N‐antigenemia than in its absence. Significantly higher serum levels of ferritin, lactose dehydrogenase, C‐reactive protein, and D‐dimer were quantified in paired plasma SARS‐CoV‐2 N‐positive specimens than in those testing negative. Occurrence of SARS‐CoV‐2 N‐antigenemia was not associated with increased mortality in univariate logistic regression analysis (odds ratio, 1.29; 95% confidence interval, 0.49‐3.34; p = 0.59). In conclusion, SARS‐CoV‐2 N‐antigenemia detection is relatively common in ICU patients and appears to associate with increased serum levels of inflammation and tissue‐damage markers. Whether this virological parameter may behave as a biomarker of poor clinical outcome awaits further investigations.

During the COVID-19 pandemic, scientists have developed many diagnostic techniques, such as biosensors, to detect whether a patient is infected by SARS-CoV-2. This could help the governments to plan an effective method to decrease the rate at which the illness is spreading across the globe. These diagnostic techniques are developed after the research of this foreign viral pathogen, SARS-CoV-2, by investigating the potential analytes that could be used and the various ways that the virus could infect a person after exposure. The three diagnostic techniques will be discussed in this paper, including lateral flow immunochromatography assay (LFIA), CRISPR/Cas12, and quantum dots (QDs)-based biosensing system. In addition, this research will also systematically discuss the advantages and disadvantages of these three monitoring and analysis methods. Although these three diagnostic techniques have very high selectivity and sensitivity, establishing a reliable result, there are pathogens that could result in similar symptoms of respiratory pneumonia just like SARS-CoV-2. The diagnostic techniques explained in this paper cant diagnose more than one pathogen or disease, therefore, it is essential to further develop a multiplexing assay to produce a more detailed result.