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Due to the recent pandemic caused by coronavirus disease 2019 (COVID-19), the lateral flow immunoassay used for its rapid antigen test is more popular than ever before. However, the history of the lateral flow immunoassay is about 60 years old, and its original purpose of use, such as a COVID-19 rapid antigen test or a pregnancy test, was the qualitative detection of a target analyte. Recently, the demand for quantitative analysis of lateral flow immunoassays is increasing in various fields. Lateral flow immunoassays for quantitative detection using various materials and sensor technologies are being introduced, and readers for analyzing them are being developed. Quantitative analysis readers are highly anticipated for their future development in line with technological advancements such as optical, magnetic field, photothermal, and electrochemical sensors and trends such as weight reduction, miniaturization, and cost reduction of systems. In addition, the sensing, processing, and communication functions of portable personal devices such as smartphones can be used as tools for the quantitative analysis of lateral flow immunoassays. As a result, lateral flow immunoassays can efficiently achieve the goal of rapid diagnosis by point-of-care testing. Readers used for the quantification of lateral flow immunoassays were classified according to the adopted sensor technology, and the research trends in each were reviewed in this paper. The development of a quantitative analysis system was often carried out in the assay aspect, so not only the readers but also the assay development cases were reviewed if necessary. In addition, systems for quantitative analysis of COVID-19, which have recently been gaining importance, were introduced as a separate section.

Bottom-up, end-user based feed, and food analysis through smartphone quantification of lateral flow assays (LFA) has the potential to cause a paradigm shift in testing capabilities. However, most developed devices do not test the presence of and implications of inter-phone variation. Much discussion remains regarding optimum color space for smartphone colorimetric analyses and, an in-depth comparison of color space performance is missing. Moreover, a light-shielding box is often used to avoid variations caused by background illumination while the use of such a bulky add-on may be avoidable through image background correction. Here, quantification performance of individual channels of RGB, HSV, and LAB color space and ΔRGB was determined for color and color intensity variation using pH strips, filter paper with dropped nanoparticles, and colored solutions. LAB and HSV color space channels never outperformed the best RGB channels in any test. Background correction avoided measurement variation if no direct sunlight was used and functioned more efficiently outside a light-shielding box (prediction errors < 5%/35% for color/color intensity change). The system was validated using various phones for quantification of major allergens (i.e., gluten in buffer, bovine milk in goat milk and goat cheese), and, pH in soil extracts with commercial pH strips and LFA. Inter-phone variation was significant for LFA quantification but low using pH strips (prediction errors < 10% for all six phones compared). Thus, assays based on color change hold the strongest promise for end-user adapted smartphone diagnostics.

ObjectiveThe development of an enzymatic assay for the specific quantification of the C1-oxidation product, i.e. gluconic acid of cellulose active lytic polysaccharide monooxygenases (LPMOs).ResultsIn combination with a β-glucosidase, the spectrophotometrical assay can reliably quantify the specific C1- oxidation product of LPMOs acting on cellulose. It is applicable for a pure cellulose model substrate as well as lignocellulosic biomass. The enzymatic assay compares well with the quantification performed by HPAEC-PAD. In addition, we show that simple boiling is not sufficient to inactivate LPMOs and we suggest to apply a metal chelator in addition to boiling or to drastically increase pH for proper inactivation.ConclusionsWe conclude that the versatility of this simple enzymatic assay makes it useful in a wide range of experiments in basic and applied LPMO research and without the need for expensive instrumentation, e.g. HPAEC-PAD.

Background After hematopoietic stem cell transplantation (HSCT) T- and B-cell reconstitution from primary lymphoid organs are a prerequisite for an effective early lymphocyte reconstitution and a long-term survival for adult patients suffering from acute leukemia. Here, we asked whether quantification of T cell receptor excision circle, (TREC) and kappa-deleting recombination excision circle (KREC) before and within six month after allogeneic HSCT could be used to measure the thymic and bone marrow outputs in such patients. Methods We used a duplex real time PCR assay to quantify the absolute copy counts of TREC and KREC, and correlated the data with absolute cell counts of CD3 + CD4 + T-cell and CD19 + B-cell subsets determined by flow cytometry, respectively. Results By comparing two recently proposed naïve T cell subsets, CD31 + naive and CD31 - naive T cells, we found a better correlation for the CD31 + subset with TREC level post alloHSCT, in line with the assumption that it contained T cells recently derived from the thymus, indicating that TREC levels reflected real thymic de novo production. Transitional as well as naïve B cells highly correlated with KREC levels, which suggested an association of KREC levels with ongoing bone marrow B cell output. CD45RO + memory T cells and CD27 + memory B cells were significantly less correlated with TREC and KREC recovery, respectively. Conclusion We conclude that simultaneous TREC/ KREC quantification is as a suitable and practicable method to monitor thymic and bone marrow output post alloHSCT in adult patients diagnosed with acute leukemia.

Erythropoietin (EPO) quantification during cell line selection and bioreactor cultivation has traditionally been performed with ELISA or HPLC. As these techniques suffer from several drawbacks, we developed a novel EPO quantification assay. A camelid single-domain antibody fragment directed against human EPO was evaluated as a capturing antibody in a label-free biolayer interferometry-based quantification assay. Human recombinant EPO can be specifically detected in Chinese hamster ovary cell supernatants in a sensitive and pH-dependent manner. This method enables rapid and robust quantification of EPO in a high-throughput setting.

In Thailand, Hb H (α0(-thal/α(+)-thal) disease is highly prevalent. We designed 3 primer sets (A, B and C) to detect -α (3.7) and -α (4.2) deletion types of α(+)-thal by quantitative (q)PCR. The A and C primer sets were used to amplify DNA sequences at the 3' terminal regions of HBA2 and HBA1 gene, respectively, and the B primer set was used to amplify an upstream DNA sequence at the 5' flanking region of HBA1 gene. The relative quantities of the PCR products (based on threshold cycle (CT) values) of the 3 primer sets were calculated according to the equation R = 2-ΔΔCT, and these values were used to distinguish between -α (3.7) and -α (4.2) deletion mutations. The type of α(+)-thal mutations was determined by calculating the difference between R (C-A) and R (C-B), yielding a value either of 0.5 or 1.0, which indicates the copy number of the target DNA compared with normal diploid control. Measured values that are close to 0.5 indicate there is a single allele of the target DNA. This method was applied to 250 DNA samples recruited for this study, and the R (C-A) and R (C-B) value determined for 185 cases of non α-thal was 1.03 ± 0.04 and 0.95 ± 0.08, respectively, for 41 cases of -α (3.7) α-thal trait 0.49 ± 0.04 and 0.45 ± 0.04, respectively, and for 2 cases of -α (4.2) α(+)-thal trait 0.5 ± 0.1 and 1.01 ± 0.06, respectively. The allele frequency of -α (3.7) and -α (4.2) mutation was 0.092 and 0.004, respectively. These results were in con- cordance with those obtained by conventional gap-PCR. The method described here is simple, accurate and feasible for screening of α(+)-thal carriers and should provide valuable information for genetic counselling of patients at risk of having a child with Hb H disease.

Objectives The purpose of this study was to develop a quantification assay to measure airborne concentrations of obeche wood allergen at workplaces. Methods Specific polyclonal antibodies to obeche wood were produced in rabbit and used to develop an inhibition enzyme immunoassay (EIA). Inhalable dust samples from three wood-processing companies were taken with a stationary sampling device (Gravicon VC25). The loaded dust filters were extracted under standardized conditions and measured with the assay. In addition, the antigen and allergen contents of obeche wood from different sources (Cameroon, N= 5; Ghana, N= 4) were analyzed from immunoblots, detected with rabbit immunoglobulin (Ig) G and human Ig E. Results Polyclonal antibodies specific for obeche wood allergens, without cross-reactivity to other woods, were used to establish the inhibition enzyme immunoassay. The assay is able to quantify allergen concentrations from 30 to 300 ng/ml. With inhibition enzyme immunoassay, exposure to airborne obeche wood allergen can be monitored in wood-processing companies. Inhalable dust samples from workplaces contained an average allergen concentration of 15 μg/g dust. Significantly lower protein and allergen contents were measured for obeche wood from Cameroon (ayous) in one company. IgE immunoblots indicated that the lower antigen and allergen contents of the ayous wood may be the result of its lacking the major obeche wood allergen, Trip s 1. Conclusions The data showed that the total dust concentration in workplaces containing obeche wood dust did not correspond to the assessed allergen concentration. To estimate exposure limits regarding sensitization risk, it is necessary to measure the allergen content directly.

This chapter contains sections titled: Introduction Development of a One‐Step Assay for the Semiquantification of Anti‐Human Interferon Beta‐1 Neutralizing Antibodies Development of a One‐Step Assay for the Semiquantification of Neutralizing Antibodies against Tumor Necrosis Factor Alpha Antagonists Discussion Acknowledgment References

The process of building new blood vessels (angiogenesis) and controlling the propagation of blood vessels (anti‐angiogenesis) are fundamental to human health, as they play key roles in wound healing and tissue growth. More than 500 million people may stand to benefit from anti‐ or pro‐angiogenic treatments in the coming decades [National Cancer Institute (USA), Cancer Bulltetin, volume 3, no. 9, 2006]. The use of animal models to assay angiogenesis is crucial to the search for therapeutic agents that inhibit angiogenesis in the clinical setting. Examples of persons that would benefit from these therapies are cancer patients, as cancer growth and spread is angiogenesis‐dependent, and patients with aberrant angiogenesis in the eye, which may lead to blindness or defective sight. Recently, anti‐angiogenesis therapies have been introduced successfully in the clinic, representing a turning point in tumor therapy and the treatment of macular degeneration and heralding a new era for the treatment of several commonly occurring angiogenesis‐related diseases. On the other hand, pro‐angiogenic therapies that promote compensatory angiogenesis in hypoxic tissues, such as those subjected to ischemia in myocardial or cerebral hypoxia due to occluding lesions in the coronary or cerebral arteries, respectively, and in cases of poor wound healing, are also being developed. In this review, the current major and newly introduced preclinical angiogenesis assays are described and discussed in terms of their specific advantages and disadvantages from the biological, technical, economical and ethical perspectives. These assays include the corneal micropocket, chick chorioallantoic membrane, rodent mesentery, subcutaneous (s.c.) sponge/matrix/alginate microbead, s.c. Matrigel plug, s.c. disc, and s.c. directed in vivo angiogenesis assays, as well as, the zebrafish system and several additional assays. A note on quantitative techniques for assessing angiogenesis in patients is also included. The currently utilized preclinical assays are not equivalent in terms of efficacy or relevance to human disease. Some of these assays have significance for screening, while others are used primarily in studies of dosage‐effects, molecular structure activities, and the combined effects of two or more agents on angiogenesis. When invited to write this review, I was asked to describe in some detail the rodent mesenteric‐window angiogenesis assay, which has not received extensive coverage in previous reviews.