Determination of urinary Tamm-Horsfall protein by ELISA using a maleimide method for enzyme-antibody conjugation

Uto, Iwao; Ishimatsu, Takashi; Hirayama, Hideo; Ueda, Shoichi; Tsuruta, Junji; Kambara, Takeshi

doi:10.1016/0022-1759(91)90067-p

Cited by 20 publications

(7 citation statements)

References 18 publications

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“…al. reported that storage at -70 °C did not alter the concentration of Tamm-Horsfall protein (THP) compared to fresh urine, while storage -30°C resulted in a 3fold increase of THP in human urine [9]. Innanen found that one or two freeze-thaw cycles decreased the concentration of microalbumin; however, mixing after thawing restored microalbumin concentration to normal [10].…”

Section: Discussionmentioning

confidence: 99%

Collection, storage, preservation, and normalization of human urinary exosomes for biomarker discovery

Zhou¹,

Yuen²,

Pisitkun

et al. 2006

Kidney International

536

457

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Urinary exosomes containing apical membrane and intracellular fluid are normally secreted into the urine from all nephron segments, and may carry protein markers of renal dysfunction and structural injury. We studied methods for collection, storage, and preservation of urinary exosomal proteins. We collected urine from healthy volunteers, added protease inhibitors, and stored urine samples at 4, -20, and -80 degrees C for 1 week or 7 months. Samples were thawed with and without extensive vortexing, and three fractions were isolated: urinary sediment, supernatant, and exosome fraction. Protein concentration, electrophoresis patterns, and abundance of seven exosome-associated proteins were measured. Exosome-associated proteins were not detected in sediment or supernatant fractions. Protease inhibitors prevented degradation of exosome-associated proteins. Freezing at -20 degrees C caused a major loss in exosomes compared to fresh urine. In contrast, recovery after freezing at -80 degrees C was almost complete. Extensive vortexing after thawing markedly increased exosome recovery in urine frozen at -20 or -80 degrees C, even if frozen for 7 months. The recovery from first and second morning urine was similar. The abundance of cytosolic exosome-associated proteins did not decrease during long-term storage. We concluded: (1) protease inhibitors are essential for preservation; (2) storage at -80 degrees C with extensive vortexing after thawing maximizes the recovery of urinary exosomes; (3) the difference between first and second morning urine exosome-associated protein was small, suggesting minimal protein degradation in the urinary tract/bladder; (4) urinary exosomes remain intact during long-term storage. These urine collection, storage, and processing conditions may be useful for future biomarker discovery efforts.

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Section: Discussionmentioning

confidence: 99%

Collection, storage, preservation, and normalization of human urinary exosomes for biomarker discovery

Zhou¹,

Yuen²,

Pisitkun

et al. 2006

Kidney International

536

457

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“…The E. coli periplasmic extracts were electrophoresed on a 10% sodium dodecylsulfate-polyacrylamide gel (SDS-PAGE) for 20 min at 50 V, followed by 1 h at 150 V. The samples were then electrotransferred to a nitrocellulose membrane for 30 min at 15 V by a Semi-Dry Transfer Cell (Biorad) with a transfer buffer (12 …”

Section: Western Blot Assaymentioning

confidence: 99%

“…In most enzyme-labeling systems, enzymes are chemically coupled with biomolecules (known as detection molecules) such as antigens or antibodies and with DNA probes for application in immunoassays and gene assays, respectively [8]. Generally, chemical coupling involves the cross-linking of an enzyme and a detection molecule by using bifunctional reagents (e.g., glutaraldehyde and periodate) [9,10] or the introduction of free SH groups on to the surface of enzyme proteins and antigens or antibodies, which leads to the formation of bioconjugates through S-S bonding [11,12]. However, most chemical coupling procedures are random reactions that often produce many undesired conjugates through self-coupling and inappropriate coupling; these conjugates might block the active sites of conjugate partners, which may consequently influence the specificity and sensitivity of the assays [8,13,14].…”

Section: Introductionmentioning

confidence: 99%

Application of methyl parathion hydrolase (MPH) as a labeling enzyme

et al. 2008

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Methyl parathion hydrolase (MPH) is an enzyme that catalyzes the degradation of methyl parathion, generating a yellow product with specific absorption at 405 nm. The application of MPH as a new labeling enzyme was illustrated in this study. The key advantages of using MPH as a labeling enzyme are as follows: (1) unlike alkaline phosphatase (AP), horseradish peroxidase (HRP), and glucose oxidase (GOD), MPH is rarely found in animal cells, and it therefore produces less background noise; (2) its active form in solution is the monomer, with a molecular weight of 37 kDa; (3) its turnover number is 114.70±13.19 s −1 , which is sufficiently high to yield a significant signal for sensitive detection; and (4) its 3D structure is known and its C-terminal that is exposed to the surface can be easily subjected to the construction of genetic engineering monocloning antibody-enzyme fusion for enzyme-linked immunosorbent assay (ELISA). To demonstrate its utility, MPH was ligated to an single-chain variable fragment (scFv), known as A1E, against a white spot syndrome virus (WSSV) with the insertion of a [-(Gly-Ser) 5 -] linker peptide. The resulting fusion protein MPH-A1E possessed both the binding specificity of the scFv segment and the catalytic activity of the MPH segment. When MPH-A1E was used as an ELISA reagent, 25 ng purified WSSV was detected; this was similar to the detection sensitivity obtained using A1E scFv and the HRP/Anti-E Tag Conjugate protocol. The fusion protein also recognized the WSSV in 1 μL hemolymph from an infected shrimp and differentiated it from a healthy shrimp.

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“…In solution, THP tends to form a gel; this process is accelerated by various factors found in the urine, such as sodium and calcium ions, 14 albumin 15 and Bence Jones proteins, 16 whereupon THP aggregates in the urine (M r : 7 Â 10 7 Da) and is implicated in the formation of urinary casts. 17 There are significant differences in the urinary THP concentrations measured using ELISA between studies in the range of 2.1-19.3 lg/mL for males and 12.9-45.4 lg/mL for females, 18 to 4 AE 1.8 lg/mL, 19 to 55-77.9 lg/mL for males and 70.7-96 lg/mL for females. 20 These data suggest that the gelation and instability of THP interfere with ELISA.…”

Section: Introductionmentioning

confidence: 99%

Highly sensitive reversed-phase high-performance liquid chromatography assay for the detection of Tamm–Horsfall protein in human urine

et al. 2015

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Determination of urinary Tamm-Horsfall protein by ELISA using a maleimide method for enzyme-antibody conjugation

Cited by 20 publications

References 18 publications

Collection, storage, preservation, and normalization of human urinary exosomes for biomarker discovery

Collection, storage, preservation, and normalization of human urinary exosomes for biomarker discovery

Application of methyl parathion hydrolase (MPH) as a labeling enzyme

Highly sensitive reversed-phase high-performance liquid chromatography assay for the detection of Tamm–Horsfall protein in human urine

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