TTR ELISA Kit

Catalog No. ABIN612801

Product Details TTR ELISA Kit

Target: TTR (Transthyretin (TTR))

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 0.122-31.25 ng/mL

Minimum Detection Limit: 0.122 ng/mL

Application: ELISA

Purpose: The AssayMax Human Prealbumin ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for detection of human prealbumin in plasma, serum, urine, saliva, milk, CSF, and cell culture samples. This assay employs a quantitative sandwich enzyme immunoassay technique that measures prealbumin in approximately 4 hours. A polyclonal antibody specific for prealbumin has been pre-coated onto a 96-well microplate with removable strips. Prealbumin in standards and samples is sandwiched by the immobilized antibody and biotinylated polyclonal antibody specific for prealbumin, which is recognized by a streptavidin-peroxidase conjugate. All unbound material is washed away and a peroxidase enzyme substrate is added. The color development is stopped and the intensity of the color is measured.

Brand: AssayMax™

Sample Type: Cell Culture Cells, Cerebrospinal Fluid, Milk, Plasma, Saliva, Serum, Urine

Analytical Method: Quantitative

Components: Human Prealbumin Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against human prealbumin. Sealing Tapes: Each kit contains 3 precut, pressure sensitive sealing tapes, which can be cut to fit the format of the individual assay. Human Prealbumin Standard: Human prealbumin in a buffered protein base (46.875 ng, lyophilized, 2 vials). Biotinylated Human Prealbumin Antibody (40x): A 40-fold concentrated biotinylated polyclonal antibody against prealbumin (150 l). MIX Diluent Concentrate (10x): A 10-fold concentrated buffered protein base (30 ml). Wash Buffer Concentrate (20x): A 20-fold concentrated buffered surfactant (30 ml, 2 bottles). Streptavidin-Peroxidase Conjugate (SP Conjugate): A 100-fold concentrate (80 l). Chromogen Substrate: A ready-to-use stabilized peroxidase chromogen substrate tetramethylbenzidine (8 ml). Stop Solution: A 0.5 N hydrochloric acid to stop the chromogen substrate reaction (12 ml).

Material not included: Microplate reader capable of measuring absorbance at 405 nm. Pipettes (1-20 µL, 20-200 µL, and multiple channel). Deionized or distilled reagent grade water. Incubator (37 °C)

Application Details

Sample Volume: 50 μL

Assay Time: 3 h

Plate: Pre-coated

Protocol:

• Step 1. Add 50 μL of Standard or Sample per well. Incubate 2 hours.
• Step 2. Wash, then add 50 μL of Biotinylated Antibody per well. Incubate 1 hour.
• Step 3. Wash, then add 50 μL of SP Conjugate per well. Incubate 30 minutes.
• Step 4. Wash, then add 50 μL of Chromogen Substrate per well. Incubate 30 minutes.
• Step 5. Add 50 μL of Stop Solution per well. Read at 450 nm immediately.

Reagent Preparation:

Freshly dilute all reagents and bring all reagents to room temperature before use. MIX Diluent Concentrate (10x): If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. Dilute the MIX Diluent Concentrate 10-fold with reagent grade water. Store for up to 30 days at 2-8 °C. Human Prealbumin Standard: Reconstitute the 46.875 ng of Human Prealbumin Standard with 1.5 mL of MIX Diluent to generate a 31.25 ng/mL standard stock solution. Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions. Prepare duplicate or triplicate standard points by further diluting the standard stock solution (31.25 ng/mL) 4-fold with MIX Diluent to produce 7.813, 1.953, 0.488, and 0.122 ng/mL solutions. MIX Diluent serves as the zero standard (0 ng/mL). Any remaining stock solution should be frozen at -20 °C and used within 2 days. Avoid repeated freeze-thaw cycles. Standard Point Dilution Prealbumin (ng/mL) P1 1 part Standard (31.25 ng/mL) 31.25 P2 1 part P1 + 3 parts MIX Diluent 7.813 P3 1 part P2 + 3 parts MIX Diluent 1.953 P4 1 part P3 + 3 parts MIX Diluent 0.488 P5 1 part P4 + 3 parts MIX Diluent 0.122 P6 MIX Diluent 0.0 5 Biotinylated Human Prealbumin Antibody (40x): Spin down the antibody briefly and dilute the desired amount of the antibody 40-fold with MIX Diluent. The undiluted antibody should be stored at -20 °C. Wash Buffer Concentrate (20x): If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. Dilute the Wash Buffer Concentrate 20-fold with reagent grade water. SP Conjugate (100x): Spin down the SP conjugate briefly and dilute the desired amount of the conjugate 100-fold with MIX Diluent. The undiluted conjugate should be stored at -20 °C.

Sample Collection: Plasma: Collect plasma using one-tenth volume of 0.1 M sodium citrate as an anticoagulant. Centrifuge samples at 3000 x g for 10 minutes. An 80000-fold sample dilution is suggested into MIX Diluent or within the range of 40000x to 160000x, however, user should determine optimal dilution factor depending on application needs. The undiluted samples can be stored at -20 °C or below for up to 3 months. Avoid repeated freeze-thaw cycles (EDTA or Heparin can also be used as an anticoagulant). Serum: Samples should be collected into a serum separator tube. After clot formation, centrifuge samples at 3000 x g for 10 minutes, and remove serum. An 80000-fold sample dilution is suggested into MIX Diluent or within the range of 40000x to 160000x, however, user should determine optimal dilution factor depending on application needs. The undiluted samples can be stored at -20 °C or below for up to 3 months. Avoid repeated freeze-thaw cycles. Urine: Collect urine using sample pot. Centrifuge samples at 800 x g for 10 minutes. Store samples at -20 °C or below for up to 3 months. Avoid repeated freeze-thaw cycles. Saliva: Collect saliva using sample tube. Centrifuge samples at 800 x g for 10 minutes. A 100-fold sample dilution is suggested into MIX Diluent, however, user should determine optimal dilution factor depending on application needs. The undiluted samples can be stored at -20 °C or below for up to 3 months. Avoid repeated freeze-thaw cycles. Milk: Collect milk using sample tube. Centrifuge samples at 800 x g for 10 minutes. A 1000-fold sample dilution is suggested into MIX Diluent, however, user should determine optimal dilution factor depending on application needs. The undiluted samples can be stored at -20 °C or below for up to 3 months. Avoid repeated freeze-thaw cycles. CSF: Collect cerebrospinal fluid (CSF) using sample pot. Centrifuge samples at 3000 x g for 10 minutes. A 4000-fold sample dilution is suggested into MIX Diluent, however, user should determine optimal dilution factor depending on application needs. The undiluted samples can be stored at -80 °C for up to 3 months. Avoid repeated freeze-thaw cycles. Cell Culture Supernatants: Centrifuge cell culture media at 3000 x g for 10 minutes to remove debris and collect supernatants. A 10-fold sample dilution is suggested into MIX Diluent, however, user should determine optimal dilution factor depending on application needs. The undiluted samples can be stored at -20 °C or below for up to 3 months. Avoid repeated freeze-thaw cycles. Refer to Sample Dilution Guidelines for further instruction. 4 Guidelines for Dilutions of 100-fold or Greater (for reference only, please follow the insert for specific dilution suggested) 100x 10000x A) 4 μL sample: 396 μL buffer (100x) = 100-fold dilution Assuming the needed volume is less than or equal to 400 μL. A) 4 μL sample : 396 μL buffer (100x) B) 4 μL of A : 396 μL buffer (100x) = 10000-fold dilution Assuming the needed volume is less than or equal to 400 μL. 1000x 100000x A) 4 μL sample : 396 μL buffer (100x) B) 24 μL of A : 216 μL buffer (10x) = 1000-fold dilution Assuming the needed volume is less than or equal to 240 μL. A) 4 μL sample : 396 μL buffer (100x) B) 4 μL of A : 396 μL buffer (100x) C) 24 μL of B : 216 μL buffer (10x) = 100000-fold dilution Assuming the needed volume is less than or equal to 240 μL.

Assay Procedure:

Prepare all reagents, standard solutions, and samples as instructed. Bring all reagents to room temperature before use. The assay is performed at room temperature (20-25 °C). Remove excess microplate strips from the plate frame and return them immediately to the foil pouch with desiccants inside. Reseal the pouch securely to minimize exposure to water vapor and store in a vacuum desiccator. Add 50 l of Human Prealbumin Standard or sample per well. Gently tap plate to thoroughly coat the wells. Break any bubbles that may have formed. Cover wells with a sealing tape and incubate for 2 hours. Start the timer after the last addition. Wash five times with 200 l of Wash Buffer manually. Invert the plate each time and decant the contents, hit 4-5 times on absorbent material to completely remove the liquid. If using a machine, wash six times with 300 l of Wash Buffer and then invert the plate, decanting the contents, hit 4-5 times on absorbent material to completely remove the liquid. Add 50 l of Biotinylated Human Prealbumin Antibody to each well. Gently tap plate to thoroughly coat the wells. Break any bubbles that may have formed. Cover wells with a sealing tape and incubate for 1 hour. Wash the microplate as described above. Add 50 l of Streptavidin-Peroxidase Conjugate per well. Gently tap plate to thoroughly coat the wells. Break any bubbles that may have formed. Cover wells with a sealing tape and incubate for 30 minutes. Turn on the microplate reader and set up the program in advance. Wash the microplate as described above. Add 50 l of Chromogen Substrate per well. Gently tap plate to thoroughly coat the wells. Break any bubbles that may have formed. Incubate for 30 minutes or till the optimal blue color density develops. Add 50 l of Stop Solution to each well. The color will change from blue to yellow. Gently tap plate to ensure thorough mixing. Break any bubbles that may have formed. 6 Read the absorbance on a microplate reader at a wavelength of 450 nm immediately. If wavelength correction is available, subtract readings at 570 nm from those at 450 nm to correct optical imperfections. Otherwise, read the plate at 450 nm only. Please note that some unstable black particles may be generated at high concentration points after stopping the reaction for about 10 minutes, which will reduce the readings.

Calculation of Results:

• Calculate the mean value of the duplicate or triplicate readings for each standard and sample.
• To generate a standard curve, plot the graph using the standard concentrations on the x-axis and the corresponding mean 450 nm absorbance (OD) on the y-axis. The best-fit line can be determined by regression analysis using log-log or four-parameter logistic curve-fit.
• Determine the unknown sample concentration from the standard curve and multiply the value by the dilution factor.

Assay Precision: Intra-assay and inter-assay coefficients of variation were 4.9 % and 7.5% respectively.

Restrictions: For Research Use only

Handling

Handling Advice: This product is for Research Use Only and is not intended for use in diagnostic procedures. Prepare all reagents (diluent buffer, wash buffer, standard, biotinylated antibody, and SP conjugate) as instructed, prior to running the assay. Prepare all samples prior to running the assay. The dilution factors for the samples are suggested in this insert. However, the user should determine the optimal dilution factor. Spin down the SP conjugate vial and the biotinylated antibody vial before opening and using contents. The Stop Solution is an acidic solution. The kit should not be used beyond the expiration date. 2

Storage: 4 °C/-20 °C

Storage Comment: Upon arrival, immediately store components of the kit at recommended temperatures up to the expiration date. Store SP Conjugate and Biotinylated Antibody at -20°C. Store Microplate, Diluent Concentrate (10x), Wash Buffer, Stop Solution, and Chromogen Substrate at 2-8°C. Unused microplate wells may be returned to the foil pouch with the desiccant packs and resealed. May be stored for up to 30 days in a vacuum desiccator. Diluent (1x) may be stored for up to 30 days at 2-8°C. Store Standard at 2-8°C before reconstituting with Diluent and at -20°C after reconstituting with Diluent.

References for TTR ELISA Kit (ABIN612801)

Pasella, Pinna, Deiana, Baralla, Dore, Mannu, Canu, Sotgiu, Zinellu, Mangoni, Sotgia, Carru, Deiana: "Plasma concentrations of transthyretin in older Sardinians including centenarians." in: Aging clinical and experimental research, (2015) (PubMed).

Murakami, Sango, Watabe, Niimi, Takaku, Li, Yamamura, Sunada: "Schwann cells contribute to neurodegeneration in transthyretin amyloidosis." in: Journal of neurochemistry, Vol. 134, Issue 1, pp. 66-74, (2015) (PubMed).

Target Details for TTR

Target: TTR (Transthyretin (TTR))

Alternative Name: Prealbumin (Transthyretin, TTR) (TTR Products)

Synonyms: zgc:103583 ELISA Kit, TTR ELISA Kit, LOC100226992 ELISA Kit, CTS ELISA Kit, CTS1 ELISA Kit, HsT2651 ELISA Kit, PALB ELISA Kit, TBPA ELISA Kit, AA408768 ELISA Kit, AI787086 ELISA Kit, D17860 ELISA Kit, prealbumin ELISA Kit, Lr1 ELISA Kit, Tbpa ELISA Kit, Prealbumin ELISA Kit, xTTR ELISA Kit, transthyretin (prealbumin, amyloidosis type I) ELISA Kit, transthyretin ELISA Kit, Transthyretin ELISA Kit, transthyretin (prealbumin, amyloidosis type 1) ELISA Kit, transthyretin-like ELISA Kit, transthyretin L homeolog ELISA Kit, ttr ELISA Kit, TTR ELISA Kit, vpb38 ELISA Kit, MRAD2831_RS38185 ELISA Kit, Caci_4246 ELISA Kit, LOC100226992 ELISA Kit, Ttr ELISA Kit, ttr.L ELISA Kit

Background: Prealbumin (Transthyretin, TTR, ATTR, TBPA) is a hepatic secretory protein thought to be important in the evaluation of nutritional deficiency and nutrition support (1). Prealbumin plays important physiological roles as a transporter of thyroxine and retinol-binding protein (2).

Gene ID: 7276

UniProt: P02766

Pathways: Hormone Transport