HPL human placental prolactin ELISA kit
US DRG imported, article number: EIA1283
US DRG China exclusive agent "Shenzhen Kerunda Biological Engineering Co., Ltd." dedicated to serve you
1. Description of the preface
DRG's HPL (Human Placental Prolactin) ELISA kit can be used for the quantitative detection of HPL in human serum. This kit is for in vitro diagnostic use only.
The physiological function of HPL has not been determined so far, but it is largely a regulator of fetal placental and physiological changes during pregnancy, which indicates that maternal serum HPL levels can display the function of the placenta.
Human placental prolactin (HPL) levels are reduced in cases of (fetal) intrauterine death, infant labor pain, and fetal asphyxia during production. This correlation is particularly pronounced if low levels of HPL are repeated, suggesting that long-term uterus in this state can damage the fetus. If the pregnancy is normal, the level of maternal HPL is generally not (in the case of). In a single pregnancy, an elevated pregnancy rate means the best pregnancy outcome. However, high levels of HPL may also indicate important pathological causes of certain specific diseases in infants, such as congenital obesity, neonatal hemolysis, and fetal edema in diabetic infants.
2 , the principle of experiment
DRG's HPL enzyme-free assay is a solid-phase enzyme-linked immunosorbent assay (ELISA) based on the sandwich principle. The microassay well on the reaction plate is coated with a monoclonal antibody that binds to a unique antigenic site on the HPL molecule. A patient sample containing endogenous HPL is incubated with the enzyme conjugate in a coated well, which is an anti-HPL antiserum linked to horseradish peroxidase. After incubation, the unbound enzyme conjugate was washed away with washings. The total amount of horseradish peroxidase binding is positively correlated with the concentration of HPL in the sample. After the addition of the substrate solution, the apparent color intensity is proportional to the concentration of HPL in the patient sample.
3 , reagents
1) Microwell reaction plate, detachable, 12 x 8 (96 well), coated with monoclonal antibody against HPL.
2) Standard (standard 1-4), 4, 0.5 ml, ready for use. Concentration: 1.25; 5.0; 10.0; 20 mg/L, conversion factor: 1 mg/L = 1 mIU/L. Contains 0.3% Proclin preservative.
3) Zero standard (also used as sample diluent), 1 branch, 90ml, stand-by, 0mg/L. Contains 0.3% Proclin preservative.
4) Enzyme conjugate, 1 branch, 11 ml, anti-HPL antibody linked to horseradish peroxidase. Contains 0.3% Proclin preservative.
5) substrate solution, 1 branch, 11ml, stand-by, TMB (tetramethylbenzidine)
6) Stop solution, 1 branch, 6 ml, containing 0.5 M H2SO4. Please do not touch the stop solution to avoid irritating the skin and burning.
4 , storage conditions Â
Unopened reagents are stored at 2-8 ° C and remain active during the pot life. Do not use expired reagents. Enzyme conjugates, substrate solutions, standards and zero standards must be stored at 2-8 °C.
The microwell reaction plate must be stored at 2-8 °C. Once the bag is opened, it must be carefully sealed. After unpacking, the immunological activity of the coated microwell reaction plate can be stabilized for about 6 weeks, but must be sealed in a bag containing desiccant.
5 , the equipment required for the experiment (but not provided by the kit)
1) Microplate reader (450±10nm) (such as DRG's microplate reader)
2) Precision sampling gun with disposable 10μl, 50μl, 100μl and 1000μl nozzles
3) Absorbent paper.
4) distilled water
5) Test tube (12 x 75 mm) for sample dilution.
6 , sample
Serum will be used in this experiment. Do not use samples that are easily hemolyzed, jaundice or lipemia. Note: Samples containing sodium azide are not available for this experiment.
1) Sample collection: serum, venous puncture to collect whole blood, to be coagulated, and serum was separated by a centrifuge at room temperature. Do not centrifuge before incomplete coagulation. Patients who have received anticoagulant therapy may require longer clotting times.
2) Storage of samples: The samples must be covered and stored for up to 5 days at 2-8 °C prior to the experiment. Samples require longer storage times and must be cryopreserved at -20 °C prior to the experiment. The melted sample must be turned upside down several times before testing.
3) Dilution of the sample: The sample was diluted 1:100 with a zero standard before the start of the experiment. First add 1ml of zero standard to each sample tube, then add 10μL of sample to the corresponding tube and mix all the tubes on the vortex mixer for 10 seconds (avoid bubbles)
4) In the first experiment, the HPL concentration in the sample higher than the highest standard must be further diluted with zero standard. This dilution factor should be taken into account when calculating the sample concentration.
7 , general considerations
1) All reagents and samples must be equilibrated to room temperature before use. All reagents must be shaken but not foamed.
2) Once the test starts, all the steps must be carried out without interruption.
3) In order to avoid cross-screening, new sampling tips must be used for sampling each standard, control, and sample.
4) The absorbance value is determined by the time and temperature of incubation. Before the experiment begins, it is recommended that all the medicines be prepared to unscrew the lid.
Fix the microplate strips to be tested on the plate holder, etc. These preparations will ensure that the time required for each dosing step is the same, without interruption.
5) According to the general rule, the intensity of the enzyme-free reaction is linearly proportional to time and temperature.
8 , experimental steps
8.1 Operation Precautions
1) Manual loading: It is recommended to do no more than 32 test holes per experiment. Alternatively, it is recommended to add all standards, samples and controls within three minutes.
2) Automatic sample loading: 96 test holes can be made for each experiment. Alternatively, it is recommended to add all standards, samples and controls within three minutes.
3) All standards, samples and controls must be doubled at the same time to ensure that all test conditions are the same. There must be a standard curve for each test.
8.2 Specific experimental steps
1) Secure the required number of slats to the pallet.
2) Add 10 μL of the standard, control, and diluted samples to the corresponding microwells with a new sampling tip.
3) Add 100 μL of enzyme conjugate to each well.
4) Mix well for 10 seconds, it is important to mix thoroughly in this step.
5) Incubate for 30 minutes at room temperature.
6) Quickly discard the reactants in the wells, wash the plate 5 times with 400 μL of distilled water per well, and pat dry on absorbent paper. Note: The correct operation of the washing step will significantly affect the sensitivity and accuracy of the experiment.
7) Add 100 μl of substrate to each well.
8) Incubate for 10 minutes at room temperature.
9) Add 50 μl of stop solution to each well to stop the enzyme reaction.
10) Within 10 minutes after the addition of the stop solution, the OD value was measured at 450 ± 10 nm using a microplate reader.
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), plot the corresponding concentration values ​​for the absorbance values ​​of each standard to make a standard curve.
3) Determine the corresponding concentration on the standard curve using the average absorbance value of each sample.
4) Automatic calculation method: The result on the IFU is automatically calculated using the four-parameter curve. Other inductive methods may yield slightly different results.
5) The concentration of the sample can be read directly from the standard curve. Further dilution of the HPL concentration in the sample above the highest standard must be used. This dilution factor should be taken into account when calculating the sample concentration.
10 , expected value
It is strongly recommended that each laboratory establish normal and abnormal values ​​for its own laboratory. In one study, we used the DRG ELISA of DRG to obtain the following results:
Gestational week | Value (mg/L) |
10-12 | 0.05-1.00 |
12-14 | 0.10-1.7 |
14-16 | 0.3-2.8 |
16-18 | 0.5-3.5 |
18-20 | 0.9-4.0 |
20-22 | 1.1-5.0 |
22-24 | 1.3-5.8 |
24-26 | 1.6-6.7 |
26-28 | 2.0-7.7 |
28-30 | 2.7-8.5 |
30-32 | 3.2-9.5 |
32-34 | 3.7-10.1 |
34-36 | 4.0-10.7 |
36-38 | 4.3-11.2 |
38-40 | 4.4-11.7 |
40-42 | 4.3-11.6 |
Product performance characteristics
1. Specificity
The antigen used for detection is equivalent to HPL
Human chorionic gonadotropin 2000IU/ L could not be detected
Prolactin 200μg/L could not be detected
Alpha-fetoprotein 300KIU/L is not detected
Human growth hormone 100μg/L could not be detected
2. Sensitivity
The minimum concentration of HPL measured by this method was 0.043 mg/L.
3. Accuracy
a. Batch internal error accuracy
This accuracy was determined by repeated measurements of three different controls in one experiment. The differences within the batch are listed below:
Serum sample | 1 | 2 | 3 |
repeat times | 18 | 18 | 18 |
HPL average (mg/L) | 0.66 | 2.34 | 6.24 |
Standard deviation | 0.04 | 0.13 | 0.42 |
Difference coefficient (%) | 6.06 | 5.55 | 6.73 |
b. Inter-assay experiment accuracy
This accuracy is determined by repeated measurements of three different controls in several experiments. The differences between batches are listed below:
Serum sample | 1 | 2 | 3 |
repeat times | 39 | twenty four | twenty four |
HPL average (mg/L) | 0.68 | 2.52 | 6.87 |
Standard deviation | 0.06 | 0.18 | 0.39 |
Difference coefficient (%) | 8.82 | 7.14 | 5.67 |
4. Repeatability and linearity
Repeatability
Different patient serum samples of known HPL levels were mixed and double-tested. The average repetition rate was 101.17%.
Expected concentration value (mg/L) measured concentration value (mg/L) repetition rate (%)
b. Linear
In a linear study, two patient samples were serially diluted with zero standards. The average repetition rate was 99.7%.
Patient sample number Dilution Expected concentration value (mg/L) Measured concentration value (mg/L) Repeat rate (%)
5. Hook's effect
In this experiment, the Hook's effect was not produced when the HPL concentration reached 700 mg/L.
l quality control
Good experimental operation requires a calibration curve to be used for each control. A batch of statistically significant quantities of control were assayed to establish an average and normal human concentration range to ensure proper experimental procedures. Controls containing azide should not be used.
l Product restrictions
1) Have a good understanding of the instructions in the package and have good experimental operation when doing the experiment, so that reliable and repeatable results can be obtained.
2) Results obtained using this kit should only be used as a help for other diagnostic procedures and provide useful information to physicians.
3) The rinsing step is critical. Inadequate flushing results in low accuracy and high luminosity readings.
This translation is for reference only, please refer to the original for details.
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