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Introduction Chronic kidney disease (CKD) is a significant global health issue. As the prevalence of renal replacement therapy (RRT) in Thailand is increasing, early detection and management of CKD is the most important step to prevent CKD progression and the need for RRT. Current diagnostic tests for CKD are non‐specific and expensive. We aimed to develop and validate antibody‐based‐albumin point‐of‐care testing (POCT) to detect patients with impaired kidney function at early stage. Methods The prototype strip test was developed under the concept of competitive lateral flow immunochromatography assay, or strip test. Monoclonal antibodies (MAbs) to human serum albumin (HSA) were harvested from the hybridomas of spleen cells from immunized mice and mouse myeloma cells. Presence of MAbs was detected by enzyme‐linked immunosorbent assay (ELISA). Spot urine was obtained from patients with kidney disease, type I, or type II Diabetes Mellitus upon their visit at King Chulalongkorn Memorial Hospital during 2018–2019. All samples were analyzed for urine albumin with our POCT (CU microalbumin) and the other two commercial POCTs (Microalbu PHAN and MICRAL). The results were validated against standard method for urine microalbumin measurement. A urine microalbumin concentration of less than 20 ug/ml was defined as normal. The sensitivity, specificity, and predictive values were calculated in comparison with the standard laboratory method. Result A total of 100 adult patients were included. CU microalbumin had a sensitivity of 86%, a specificity of 94%, and a positive predictive value of 96%. Our POCT showed good correlation with the laboratory results. Conclusion CU microalbumin correlated well with the standard method for quantitative measurement of urine albumin. Therefore, it has the potential for early screening of CKD, especially in primary health care facilities in resource limited settings.

Clostridium acetobutylicum TISTR 1462 and Clostridium beijerinckii TISTR 1461 were chosen to optimize acetone–butanol–ethanol (ABE) fermentation by using glucose as a carbon source. The enhancement in its productivity by adding various concentrations of ammonium acetate was studied. Then, the variation of glucose/xylose ratios in the pre-grown medium was investigated. The results showed that both increased ammonium acetate in the production medium and D–xylose in the pre-grown medium could produce more ABE. With these conditions, using corncob hydrolysate as a substrate, 20.58 g/L ABE was produced from C. beijerinckii TISTR 1461 with 0.44 g/L/h and 0.45 of ABE productivity and yield, respectively.

Aflatoxin M1 (AFM1) is a toxic metabolite of the fungal product aflatoxin found in milk. For food safety concern, maximum residual limits of AFM1 in milk and dairy products have been differently enforced in many countries. A suitable detection method is required to screen a large number of product samples for the AFM1 contamination. In this study, monoclonal antibodies (MAbs) against AFM1 were generated using a conventional somatic cell fusion technique. After screening, five MAbs (AFM1-1, AFM1-3, AFM1-9, AFM1-11, and AFM1-17) were obtained that showed cross-reactivity with aflatoxin B1 (AFB1) and aflatoxin G1 (AFG1) but with no other tested compounds. An indirect competitive enzyme-linked immunosorbent assay (ELISA) using a partially purified MAb and antigen-coated plates yielded the best sensitivity with the 50% inhibition concentration (IC 50 ) and the limit of detection (LOD) values of 0.13 ng/mL and 0.04 ng/mL, respectively. This indirect competitive ELISA was used to quantify the amount of fortified AFM1 in raw milk. The precision and accuracy in terms of % coefficient of variation (CV) and % recovery of the detection was investigated for both intra- ( n = 6) and inter- ( n = 12) variation assays. The % CV was found in the range of 3.50–15.8% and 1.32–7.98%, respectively, while the % recovery was in the range of 92–104% and 100–103%, respectively. In addition, the indirect ELISA was also used to detect AFM1 fortified in processed milk samples. The % CV and % recovery values were in the ranges of 0.1–33.0% and 91–109%, respectively. Comparison analysis between the indirect ELISA and high performance liquid chromatography was also performed and showed a good correlation with the R 2 of 0.992 for the concentration of 0.2–5.0 ng/mL. These results indicated that the developed MAb and ELISA could be used for detection of AFM1 in milk samples.

Background Black tiger shrimp Penaeus monodon is one of the common causes of shellfish allergy that is increasing worldwide. One of the important problems in the management of shellfish allergy is the lack of accurate diagnostic assay because the biological and immunological properties of allergens in black tiger shrimp have not been well characterized. This study aims to detect proteins with the ability to bind and cross-link immunoglobulin E (IgE) from black tiger shrimp by enzyme-linked immunosorbent assay (ELISA), Western blot, and a humanized rat basophilic leukemia reporter cell line RS-ATL8. Methods Sera from shrimp allergic subjects were subjected to ELISA and Western blots using raw or cooked shrimp extract as antigens. Pooled sera were used to sensitize the RS-ATL8 reporter cell line and cells were activated by shrimp extract. Eluted protein extracts separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were tested on the RS-ATL8 cell line and subjected to mass spectrometry to identify potential candidate allergens. Results Allergic sera reacted stronger to raw shrimp extract than cooked shrimp extract (P=0.009). Western blot demonstrated that major IgE reactivity protein bands were at 32-39 kDa and 91-230 kDa in both raw and cooked shrimp extracts. The eluted protein bands at the molecular weight of 38 and 115 kDa from raw shrimp extract induced IgE cross-linking as assayed by the RS-ATL8 cell line. These protein bands were subjected to mass spectrometry for analysis. Ubiquitin-activating enzyme and crustacyanin were identified as potential candidate novel shrimp allergens. Conclusions The RS-ATL8 reporter cell line can be used to identify potential new shrimp allergens that can functionally cross-link IgE and induce mast cell degranulation.

Norfloxacin belongs to the group of fluoroquinolone antibiotics which has been approved for treatment in animals. However, its residues in animal products can pose adverse side effects to consumer. Therefore, detection of the residue in different food matrices must be concerned. In this study, a single chain variable fragment (scFv) that recognizes norfloxacin antibiotic was constructed. The cDNA was synthesized from total RNA of hybridoma cells against norfloxacin. Genes encoding VH and VL regions of monoclonal antibody against norfloxacin (Nor155) were amplified and size of VH and VL fragments was 402 bp and 363 bp, respectively. The scFv of Nor155 was constructed by an addition of (Gly4Ser)3 as a linker between VH and VL regions and subcloned into pPICZαA, an expression vector of Pichia pastoris. The sequence of scFv Nor155 (GenBank No. AJG06891.1) was confirmed by sequencing analysis. The complementarity determining regions (CDR) I, II, and III of VH and VL were specified by Kabat method. The obtained recombinant plasmid will be useful for production of scFv antibody against norfloxacin in P. pastoris and further engineer scFv antibody against fluoroquinolone antibiotics.

A novel monoclonal antibody (MAb) against oxytetracycline (OTC) was generated and characterized. The MAb was used in the development of an enzyme-linked immunosorbant assay (ELISA)-based detection system. An OTC-bovine serum albumin (BSA) conjugate was prepared and used in the immunization of mice. A conventional somatic cell fusion technique was used to generate MAb-secreting hybridomas denoted 2-4F, 7-3G, and 11-11A. An indirect competitive ELISA (icELISA) was applied to measure the sensitivity and specificity of each MAb in terms of its 50% inhibitory concentration (IC50) and percentage of cross-reactivity, respectively. MAb 2-4F exhibited the highest sensitivity, with an IC50 of 7.01 ng/ml. This MAb showed strong cross-reactivity to rolitetracycline, but no cross-reactivity to other unrelated antibiotics. When MAb 2-4F was used to detect OTC from shrimp samples, the recoveries were in the range of 82%–118% for an intra-assay and 96%–113% for an inter-assay. The coefficients of variation of the assays were 3.9%–13.9% and 5.5%–14.9%, respectively.

After 24 h of chemical permeabilization with 20% (v/v) methanol at 25 °C, the amount of daidzein released from soybean seeds is 15 to 20% of the amount (0.0423 ± 0.0045 mg/g seed dry wt) obtained by physical grinding. With this chemical permeabilization condition, 70% of the permeabilized seeds are still able to germinate. The release of daidzein is enhanced to 33% with the addition of XAD-4 to 20% (v/v) methanol without affecting seed viability. © Rapid Science Ltd. 1998

Enrofloxacin is a fluoroquinolone antibiotic approved for the treatment of infections in animals. Because of the side effects to consumers of animal products, the maximum residue limits (MRLs) of enrofloxacin in animal tissues for consumption are regulated. In this study, a monoclonal antibody (mAb) against enrofloxacin was prepared and characterized for the development of a direct competitive enzyme-linked immunosorbent assay (ELISA). The obtained mAb, Enro44, was highly specific for enrofloxacin and had a 50% inhibition concentration (IC(50)) of 1.99 ng/ml in a competitive ELISA, and the limit of detection (LOD) was 0.50 ng/ml. The cross-reactivity of the mAb with other quinolones and fluoroquinolones was lower than 0.01%. The subclass of the mAb Enro44 was identified as IgG1. The antigen (Ag)-captured direct competitive ELISA using the mAb Enro44 was tested on different spiked samples, including chicken muscle, cattle milk, and cattle urine, and the assay demonstrated recoveries of 82-112%, 80-125%, and 78-124%, respectively. Furthermore, the quantitation of enrofloxacin obtained from the ELISA and from high-performance liquid chromatography (HPLC) was in good agreement, with the linear regression coefficient between 0.933 and 1.056. The cDNAs encoding a heavy-chain Fd fragment (VH and CH1) and a light chain of the mAb Enro44 were cloned and sequenced. Taken together, the results obtained reveal a potential use of this mAb in an ELISA for the detection of enrofloxacin in food samples. The information of amino acid sequence of this mAb will be useful for further modification and production of the mAb in a bioreactor.

Indigofera tinctoria was permeabilized with 20% methanol (v/v) at 25 °C and released 8±2 μg indigo g−1 dry plant material (excluding roots). This is equivalent to 42±11% of the total indican within the cells. The plants began to recover after 2 weeks.