• Model: EF006163
  • 100 Units in Stock
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Packing  96Tests


Shorten Name  4-HNE

Target Name  4-Hydroxynonenal

Alternative Name

Human 4-HNE ELISA KIT ,Human 4-Hydroxynonenal ELISA KIT ,Human 4-hydroxy-2-nonenal ELISA KIT ,Human HNE ELISA KIT ,Human 4-Hydroxynon-2-enal ELISA KIT


Human 4-HNE ELISA KIT Detect Range 0.62-40ng/ml


Intended use 

Human 4-HNE ELISA KIT allows for the in vitro quantitative determination of 4-HNE concentrations in serum, plasma, tissue homogenates,cell culture supernates or other biological fluids.


Human 4-HNE ELISA KIT Content List

Reagent  Quantity

Assay plate      1      

Standard  2      

Sample Diluent         1 × 20mL 

Assay Diluent A        1 × 10mL 

Assay Diluent B        1 × 10mL 

Detection Reagent A       1 × 60μL  

Detection Reagent B       1 × 120μL         

Wash Buffer(25 x concentrate)        1 × 30mL 

Substrate          1 × 10mL 

Stop Solution    1 × 10mL 

Plate sealer      5      


Test principle 

Human 4-HNE ELISA KIT is based on the competitive binding enzyme immunoassay technique. The microtiter  plate provided in Human 4-HNE ELISA KIT has been pre-coated with an antibody specific to target antigen, During the reaction, target antigen in the sample or standard competes with a fixed amount of biotin-labeled target antigen for sites on a pre-coated antibody specific to target antigen. Excess conjugate and unbound sample or standard are washed from the plate. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB substrate solution is added to each  well.  The  enzyme-substrate  reaction is  terminated by the addition of a sulphuric acid solution and the color change is measured spectro- photometrically at  a  wavelength  of  450  nm ± 2 nm.  The  concentration of target  antigen  in  the  samples is then determined by comparing the O.D. of the samples to the standard curve.


Sample collection and storage     

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℃.

Plasma - Collect plasma 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℃.

Tissue homogenates - The preparation of tissue homogenates will vary depending upon tissue type. For this assay, tissue was rinsed with ice-cold 1×PBS to remove excess blood, homogenized in ice-cold 1×PBS and stored overnight at ≤-20℃。 In most cases, 10% homogenate (eg.1g of tissue in 10mL of ice-cold 1×PBS) is recommended. After two freeze-thaw cycles were

performed to break the cell membranes, the homogenates were centrifuged  for 5 minutes at 5000 x g. Remove the supernate and assay immediately or aliquot and store at   ≤-20℃.

Cell culture supernates and Other biological fluids - Remove  particulates

by centrifugation and assay immediately or aliquot and store samples at -20℃ or -80℃.

Fresh  samples are first choice. If not, avoid freeze-thaw of samples.



Reagent preparation     

Standard – Please refer to the Data Sheet inserting in the ELISA kit.

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

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


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. Avoid foaming. Keep appropriate numbers  of

strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20℃ until the kits expiry date. Prepare  all  reagents,  working  standards  and  samples  as  directed  in the previous sections. 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 dilutions for their particular experiments.

1.      Add 50μL of Standard, Blank, or Sample per  well.

2.      Immediately add 50 μL  of Detection A working solution to each well. Cover

with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C.

3.      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  orautowasher, and  let  it  sit  for 1~2 minutes. Complete removal of liquid at each step is essential for 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.

4.      Add 100μL of Detection Reagent B working solution to each well. Cover with a new Plate sealer. Incubate for 45 minutes at 37℃.

5.      Repeat the aspiration/wash process for five times as conducted in step 3.

6.      Add 90μL of Substrate Solution to each well. Cover with a new Plate sealer. Incubate within 15-30 minutes at 37°C. Protect from light.

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

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


Calculation of results     

Average the duplicate readings for each standard, control, and sample and sample, then subtract the average 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, construct 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 target antigen concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. It is recommended to use professional software to do this calculation, such as CurveExpert. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.


Important note      

1.      Please carefully reconstitute Standards or working Detection Reagent A and B according to the instruction, and avoid foaming and mix gently until the crystals have completely dissolved. The reconstituted Standards, Detection Reagent A and B can be used only once.

2.      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 have been added to the well strips, DO NOT let the strips DRY at any time during the assay.

3.      To avoid cross-contamination, change pipette tips between additions of each standard level, between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent.

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

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

6.      Kits from different batches may be a little different in detection range, sensitivity and color developing time. Please perform the experiment exactly according to printed instruction inside in the kit while electronic ones from our website is for reference only.

7.      Do not substitute reagents from one lot to another. Use only the reagents

in the same kit supplied by manufacturer.

8.      Even the same operator might get different results in two separate experiments. In order to get better reproducible results, the operation of every step in the assay should be controlled. Furthermore, a preliminary experiment before assay for each batch is recommended.

9.      Each ELISA kit has been strictly passed Q.C test. However, results from end users might be inconsistent with our in-house data due

to some unexpected transportation conditions or different lab equipments. Intra-assay variance among kits from different batches might arise from above factors, too.

10.   ELISA Kits from different manufacturers for the same item might produce different results, since we haven’t compared our products

with other manufacturers.

11.   Period of validity: six months.




The Stop Solution provided with Human 4-HNE ELISA KIT is an acid solution. Wear eye, hand, face, and clothing protection when using this material.


4-HNE Related Products

4-HNE Related ELISA KITs


4-HNE ELISA Kit| Mouse 4-Hydroxynonenal ELISA Kit

4-HNE ELISA Kit| Rat 4-Hydroxynonenal ELISA Kit

4-HNE Related antibodies

Anti-GSTA4 antibody


*Please noted that 4-HNE ELISA KIT|Human is only for RUO (Research Use Only), Not for the diagnostic Purpose!

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