人转甲状腺素蛋白(TTR)ELISA Kit

 

This immunoassay kit allows for the in vitro quantitative determination of human transthyretin, TTR concentrati*** in cell culture supernates, serum, pla***a and other biological fluids.

Transthyretin (TTR) is a serum and cerebrospinal fluid carrier of the thyroid hormone thyroxine (T4).TTR was originally called prealbumin because it ran faster than albumins on electrophoresis gels.

It functi*** in concert with two other thyroid hormone binding proteins: thyroxine-binding globulin (TBG) and albumins. TTR also acts as a carrier of retinol (vitamin A) through an association with retinol binding protein (RBP).

Numerous other ***all molecules are known to bind in the thyroxine binding sites, inTTRding many natural products (such as resveratrol), drugs (diflunisal, flufenamic acid), and toxins PCB. Since TTR binds promiscuously to many aromatic compounds, and generally does not bind T4 in serum, there is speculation that TTR's "true function" is to generally sweep up toxic and foreign compounds in the blood stream.

TTR is a 55 kDa homotetramer with a dimer of dimers configuration that is synthesized in the liver, choroid plexus and retinal pigment epithelium. Each monomer is a 127 residue polypeptide rich in beta sheet structure. Association of two monomers forms an extended beta sandwich. Further association of another identical set of monomers produces the homotetrameric structure. The two thyroxine binding sites per tetramer sit at the interface between the latter set of dimers.

 

The microtiter plate provided in this kit has been pre-coated with an antibody specific to TTR. Standa*** or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for TTR and ***idin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB substrate solution is added to each well. Only those wells that contain TTR, biotin-conjugated antibody and enzyme-conjugated ***idin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a w***elength of 450 nm &plu***n; 2 nm. The concentration of TTR in the samples is then determined by comparing the O.D. of the samples to the standard curve.

 

Reagent                                              Quantity

Assay plate                                                                    1 × 20ml

Standard                                             2

Sample Diluent                                                                    1 × 20ml

Assay Diluent A                                                                   1 × 10ml

Assay Diluent B                                                                  1 × 10ml

Detection Reagent A                                                           1 × 120μl

Detection Reagent B                                                         1 × 120μl

Wash Buffer(25 x concentrate)                          1 × 30ml

Substrate                                                                         1 × 10ml

Stop Solution                                                                             1 × 10ml

Plate sealer for 96 wells                                                      5

Instruction                                                                  1

Luminometer.

Pipettes and pipette tips.

EP tube

Deionized or distilled water.

Serum - Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at approximately 1000 × g. Remove serum and assay immediately or aliquot and store samples at -20℃ or -80℃.

Pla***a - Collect pla***a using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 × g at 2 - 8℃ within 30 minutes of collection. Store samples at -20℃ or -80℃. ***oid repeated freeze-thaw cycles.

Cell culture supernates and other biological fluids - Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20℃ or -80℃. ***oid repeated freeze-thaw cycles.

Note: Serum, pla***a, and cell culture supernatant samples to be used within 7 days may be stored at 2-8 ℃, otherwise samples must stored at -20℃ (≤ 1 months) or -80℃ (≤ 2 months) to ***oid loss of bioactivity and contamination. ***oid freeze-thaw cycles. When performing the assay slowly bring samples to room temperature.

DO NOT USE HEAT-TREATED SPECIMENS.

Serum/pla***a samples require a 20 fold dilution. A suggested 20-fold dilution is 50uLsample + 950uL Sample Diluent.

 

1.  The kit should not be used beyond the expiration date on the kit label.

2.    Do not mix or substitute reagents with those from other lots or sources.

3.    If samples generate values higher than the highest standard, further dilute the samples with the Assay Diluent and repeat the assay. Any variation in standard diluent, operator, pipetting technique, washing technique, incubation time or temperature, and kit age can cause variation in binding.

4.    This assay is designed to eliminate interference by soluble receptors, ligands, binding proteins, and other factors present in biological samples. Until all factors h***e been tested in the Quantikine Immunoassay, the possibility of interference cannot be exTTRded.

 

Bring all reagents to room temperature before use.

Wash Buffer - If crystals h***e formed in the concentrate, warm to room temperature and mix gently until the crystals h***e completely dissolved. Dilute 30 mL of Wash Buffer Concentrate into deionized or distilled water to prepare 750 mL of Wash Buffer.

Standard - Rec***titute the Standard with 1.0 mL of Sample Diluent. This rec***titution produces a stock solution of 30 ng/ml. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making serial diluti*** (Making serial dilution in the wells directly is not permitted). The undiluted standard serves as the high standard (30 ng/ml). The Sample Diluent serves as the zero standard (0 ng/ml).

 

Detection Reagent A and B - Dilute to the working concentration using Assay Diluent A and B (1:100),respectively.

 

Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37℃ directly.). All the reagents should be mixed thoroughly by gently swirling before pipetting. ***oid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at 4℃ until the kits expiry date. Prepare all reagents, working standa*** and samples as directed in the previous secti***. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample diluti*** for their particular experiments.

 

1. Add 100 μl of Standard, Blank, or Sample per well. Cover with the Plate sealer. Incubate for 2 hours at 37℃.

2. Remove the liquid of each well, don’t wash.

3. Add 100 μl ofDetection Reagent A working solution to each well. Cover with the Plate sealer. Incubate for 1 hour at 37℃. Detection Reagent A working solution may appear cloudy. Warm to room temperature and mix gently until solution appears uniform.

4. Aspirate each well and wash, repeating the process three times for a total of three washes. Wash by filling each well with Wash Buffer (approximately 400 μl) using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.

5. Add 100 μl of Detection Reagent B working solution to each well. Cover with a new Plate sealer. Incubate for 1 hours at 37℃.

6. Repeat the aspiration/wash as in step 4.

7. Add 90 μl of Substrate Solution to each well. Cover with a new Plate sealer. Incubate within 30 minutes at 37℃. Protect from light.

8. Add 50 μl of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.

9. Determine the optical density of each well at once, using a microplate reader set to 450 nm.

 

1. Absorbance is a function of the incubation time. Therefore, prior to starting the assay it is recommended that all reagents should be freshly prepared prior to use and all required strip-wells are secured in the microtiter frame. This will ensure equal elapsed time for each pipetting step, without interruption.

2. Please carefully rec***titute Standa*** or working Detection Reagent A and B according to the instruction, and ***oid foaming and mix gently until the crystals h***e completely dissolved. The rec***tituted Standa*** can be used only once. This assay requires pipetting of ***all volumes. To minimize imprecision caused by pipetting, ensure that pipettors are calibrated. It is recommended to suck more than 10μl for once pipetting.

3.    To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary. Do not allow wells to sit uncovered for extended periods between incubation steps. Once reagents h***e been added to the well strips, DO NOT let the strips DRY at any time during the assay.

4.    For each step in the procedure, total dispensing time for addition of reagents to the assay plate should not exceed 10 minutes.

5.    To ***oid cross-contamination, change pipette tips between additi*** of each standard level, between sample additi***, and between reagent additi***. Also, use separate reservoirs for each reagent.

6.    The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings.

7.    Duplication of all standa*** and specimens, although not required, is recommended.

8.    Substrate Solution is easily contaminated. Please protect it from light.

 

This assay recognizes recombinant and natural human TTR. No significant cross-reactivity or interference was observed.

 

The minimum detectable dose of human TTR is typically less than 0.24 ng/mL.

The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero.

 

0.47 -30 ng/mL. The standard curve concentrati*** used for the ELISA’s were 30 ng/mL, 15 ng/mL, 7.5 ng/mL, 3.75 ng/mL, 1.88 ng/mL,0.94 ng/mL, 0.47 ng/mL.

 

***erage the duplicate readings for each standard, control, and sample and subtract the ***erage zero standard optical density. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, c***truct a standard curve by plotting the mean absorbance for each standard on the x-axis against the concentration on the y-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the TTR concentrati*** versus the log of the O.D. and the best fit line can be determined by regression analysis. It is recommended to use some related software to do this calculation, such as curve expert 13.0. This procedure will produce an adequate but less precise fit of the data. If samples h***e been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.

1.   Unopened test kits should be stored referring to the package label for frequent use, and stored at -20℃ for long time storage. The unused strips should be kept in a sealed bag and stored at 2-8℃ in their pouch with the desiccant provided to minimize exposure to damp air. The test kit may be used throughout the expiration date of the kit (six months from the date of manufacture). Opened test kits will remain stable until the expiring date shown, provided it is stored as prescribed above.

2.   There may be some foggy substance in the wells when the plate is opened at the first time. It will not h***e any effect on the final assay results.

3.   Do not remove microtiter plate from the storage bag until needed.

4.   A microtiter plate reader with a bandwidth of 10nm or less and an optical density range of 0-3 OD or greater at 450nm w***elength is acceptable for use in absorbance measurement.

5.   Use fresh disposable pipette tips for each transfer to ***oid contamination.

6.   Do not substitute reagents from one kit lot to another. Use only the reagents supplied by manufacturer.

7.   Valid period: six months.

 

The Stop Solution suggested for use with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material.