Prolactin (PRL) ELISA Kit Instructions

  Prolactin (PRL) ELISA Kit Instructions

( Germany DRG : EIA1291)

1 , the principle of experiment

   DRG 's prolactinase-free assay is a solid-phase enzyme-free adsorption assay ( ELISA ) based on the sandwich method principle . The microassay well on the reaction plate is coated with a monoclonal antibody that binds to a unique antigenic site on the prolactin molecule. A patient sample containing endogenous prolactin was incubated with the enzyme conjugate in a coating well, which is an anti-prolactin antiserum linked to horseradish peroxidase. After incubation, the unbound enzyme conjugate was washed away with washings. The total amount of horseradish peroxidase bound is positively correlated with the concentration of prolactin in the sample. After the addition of the substrate solution, the apparent color intensity is proportional to the concentration of prolactin in the patient sample.

2 , kit components

1)        A microplate, 1 piece (96 wells ) , was coated with a monoclonal antibody against prolactin.

2)        Enzyme conjugate, 11ml , horseradish peroxidase-labeled prolactin antiserum.  

3)        Substrate solution, 11ml (TMB)

4)        Standard series, 6 (freeze-dried powder), concentration: 0 ; 5 ; 20 ; 50 ; 100 ; 200ng/ml

Conversion factor ( 1ng/ml=21.1mUI/mL )

5)        Stop Solution, 0.5M of H2SO4, 6ml

3 , the experiment requires equipment (but the kit does not provide)

1)        Microplate reader (450 ± 10nm) .

2)        Pipette

3)        Absorbent paper

4)        Standard water tank

5)        Distilled water

6)        Timer

4 , reagent storage :

   Unopened reagent at 2 - 8 ℃ stored, may remain active within the validity period. Do not use the expired reagent, enzyme conjugate, a substrate solution, zero standard and standards must be 2 - stored at 8 ℃.

   Microplates must be 2 - stored at 8 ℃. Once the bag is opened, it must be carefully sealed. If the package of the coated board is opened, its immunological activity is stable for 2 months, provided that it is sealed in a bag containing desiccant.

5 , sample collection

1)        Whole blood was collected by venipuncture, and the blood was coagulated. The serum was separated by a centrifuge at room temperature to avoid hemolysis. Note: This kit can only be used with samples that do not use any additives.

2)        The sample must be covered and stored for 48 hours at 2 - 8 °C prior to the experiment . If the sample requires a longer storage time, the sample should be stored frozen at -20 °C. Thawed samples must be turned upside down several times before testing.

Note: The sample used in this kit should not contain any additives.

6 , reagent preparation

Reference standard: 1.0 ml of double distilled water was added to the lyophilized standard .

Note: The prepared standard can be stored stably at 2-8 °C for 2 months. If you want to store it for a longer period of time, it should be stored at -20 °C.

7 , general considerations

1)        Before the start of the experiment, all reagents and samples were equilibrated to room temperature, a mixed reagent avoid foaming.

2)        Once the experiment begins , all steps should be carried out in its entirety.

3)        A new sampling tip is used for sampling each sample.

4)        Absorbance is related to incubation time and temperature. All reagents should be prepared after the start of the experiment to ensure consistent dosing time.

5)        The kit maximum absorbance (OD) between 1.200-2.000 (room temperature 22 ℃, the color within 10 minutes). If the maximum OD value is higher than the upper limit of your microplate reader or below 1.000 , the corresponding incubation time of the color reaction is reduced or prolonged. In general, the strength of the enzyme-free reaction is linear with time and temperature, which allows the operator to properly adjust the physical and chemical environment.

8 , experimental steps

1)        Place the required number of slats on the pallet.

2)        The luteinizing hormone family of standards (0; 5; 20; 50 ; 100; 200ng / ml), and quality control serum samples were added 25 μ l to the respective wells, each sample had the new sampling tip .

3)        Add 100 μ l of anti-prolactin enzyme conjugate to each well.

4)        Mixing for 10 seconds , it is important to mix thoroughly in this step.

5)        Incubate at room temperature (22 °C ) for 30 minutes.

6)        Discard the reaction solution in the well.

7)        The plate was washed 5 times with distilled water .

8)        Dry the plate on absorbent paper.

9)        By the original loading sequence, each well was added 100 μ l substrate solution.

10)    Incubate for 10 minutes at room temperature (22 °C ) .

11)    By sequential addition of substrate solution, 50 μ l per well of stop solution to each well.

12)    The absorbance was read at a wavelength of 450 ± 10 nm in 10 minutes .

9 , the result of calculation

1)        Calculate the average absorbance value for each standard, control, and patient sample.

2)        Using the absorbance value as the ordinate ( y ) and the concentration value as the abscissa ( x ), the corresponding concentration value ( mIU/ml ) of each standard is plotted to make a standard curve.

3)        The corresponding concentration was determined on the standard curve using the average absorbance value of each sample. Other inductive methods can also be used for calculation based on their actual experience or the function of the computer.

4)        If the concentration of the sample is higher than the concentration of the highest concentration of the standard or the concentration > 200 ng / ml , dilution is required, and any diluted sample must be taken into account in the calculation of the corresponding dilution factor.

10 , reference value

We strongly recommend that each experimental city establish its own normal and outliers. The following results are based on a certain number of blood samples from the normal population:

Sensitivity : The minimum detectable prolactin concentration is 0.35ng/ml

This translation is for reference only, please refer to the original for details.

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