Human FPR1 ELISA Kit

(1) The sample should be clear and transparent, and the suspended matter should be removed by centrifugation. Hemolysis of the sample will affect the results, so hemolyzed samples should not be used.
(2) If the sample is to be tested within 1 week after collection, it can be stored at 4°C. If it cannot be tested in time, please divide it into aliquots according to the amount used once and freeze it at -20°C (for testing within 1 month) or -80°C (for testing within 3-6 months) to avoid repeated freezing and thawing. Please bring the sample to room temperature before the experiment.

II. Sample dilution principles:

If your test sample needs to be diluted, the general dilution principles are as follows:

1. Dilute 50 times: one-step dilution. Take 5 μL of sample and add 245 μL of standard and sample diluent for a 50-fold dilution;

2. Dilute 100 times: one-step dilution. 3. 1000-fold dilution: Two-step dilution. Add 5 μL of sample to 95 μL of standard and sample diluent for a 20-fold dilution. Then, add 5 μL of the 20-fold diluted sample to 245 μL of standard and sample diluent for a 50-fold dilution, for a total of 1000-fold dilution. 4. 100,000-fold dilution: Three-step dilution. Add 5 μL of sample to 195 μL of Standard & Sample Diluent for a 40-fold dilution. Then, add 5 μL of the 40-fold diluted sample to 245 μL of Standard & Sample Diluent for a 50-fold dilution. Finally, add 5 μL of the 2,000-fold diluted sample to 245 μL of Standard & Sample Diluent for a 50-fold dilution, for a total dilution of 100,000-fold. 5. For each dilution step, use at least 3 μL of liquid, and the dilution factor should not exceed 100. Excessively small sample volumes can easily lead to greater errors during mixing. Ensure thorough mixing at each dilution step to avoid foaming.

6. If the dilution factor is very high, you can dilute with PBS first, and then use the standard and sample diluent provided in the kit as the final step.

III. Sample Dilution Recommendations

1. Normal, fresh serum/plasma samples are recommended for testing (Original solution).

2. Due to individual differences, the recommended dilution factor is for reference only. For actual testing, please estimate the sample concentration range in advance and determine the dilution factor of the sample to be tested through preliminary experiments.

IV. Pre-Test Preparation

1. Please remove the kit from the refrigerator 30 minutes in advance and equilibrate to room temperature.

2. Dilute the 25× concentrated wash buffer to 1× working solution with double-distilled water. Return the unused portion to 4°C. 3. Standards: Add 1.0 mL of Universal Standard & Sample Diluent to the lyophilized standard. Tighten the cap and let stand for 10 minutes to fully dissolve. Then gently mix (concentration: 10 ng/mL). Then, serially dilute the standard to 10 ng/mL, 5 ng/mL, 2.5 ng/mL, 1.25 ng/mL, 0.63 ng/mL, 0.32 ng/mL, and 0.16 ng/mL. Use 0 ng/mL as a blank well. Prepare the required amount of standard and set aside. It is recommended to add the prepared standard to the sample within 15 minutes; it is not recommended to leave it for an extended period. 4. Biotinylated Antibody Working Solution: Before the experiment, calculate the required amount of biotinylated antibody working solution (based on 100 μL/well, the actual amount should be 100-200 μL more). 15 minutes before use, dilute the concentrated biotinylated antibody (1:100) with biotinylated antibody diluent to the working concentration and use it on the same day. The dilution principle is to add 1 μL of concentrated biotinylated antibody to 99 μL of biotinylated antibody diluent and mix thoroughly with a pipette.

5. Enzyme Conjugate Working Solution: Before the experiment, calculate the required volume for the experiment (based on 100 μL/well; add 100-200 μL more). 15 minutes before use, dilute the concentrated HRP enzyme conjugate (1:100) with enzyme conjugate diluent to the working concentration for use that day. The dilution principle is to add 1 μL of concentrated enzyme conjugate to 99 μL of enzyme conjugate diluent and mix thoroughly with a pipette.

6. TMB Substrate - Use a pipette to aspirate the required volume of solution. Do not pour any remaining solution back into the reagent bottle.

V. Preparation before the experiment

1. Before use, equilibrate all materials and prepared reagents to room temperature. Before use, mix all reagents thoroughly, taking care not to produce any foam.

2. The user should calculate the number of samples that may be used in the entire experiment. Please reserve enough samples in advance.

3. Please estimate the concentration before measurement. If these values are not within the range of the standard curve, the user must determine the optimal sample dilution for their specific experiment.

VI. Operational Procedure

1. Before starting the experiment, all reagents should be equilibrated to room temperature and all reagents should be prepared in advance. When diluting reagents or samples, they must be mixed thoroughly and try to avoid foaming during mixing. If the sample concentration is too high, dilute it with sample diluent so that the sample is within the detection range of the kit. 2. Add 100 μL of the standard or sample to be tested (if the sample needs to be diluted, refer to the sample dilution guidelines for dilution methods). Be careful not to create bubbles. Add the sample to the bottom of the ELISA plate well, avoiding contact with the well walls. Gently shake to mix. Cover the plate or seal with film and incubate at 37°C for 80 minutes. To ensure the validity of the experimental results, use a fresh standard solution for each experiment. 3. Discard the liquid in the wells, spin dry, and wash the plate three times. Wash each well with 200 μL of wash buffer, soaking for 1-2 minutes. Spin off the liquid in the plate (or wash with a microplate washer). After the final wash, pat the plate dry on absorbent paper. 4. Add 100 μL of biotin antibody working solution to each well (can be prepared 15 minutes in advance). Cover the plate with film and incubate at 37°C for 50 minutes. 5. Discard the liquid in the wells and wash the plate three times. Wash each well with 200 μL of wash buffer, soak for 1-2 minutes, and discard the liquid in the plate (or use a plate washer). After the final wash, pat the plate dry on absorbent paper. 6. Add 100 μL of enzyme conjugate working solution to each well (can be prepared 15 minutes in advance) and incubate at 37°C for 50 minutes. 7. Discard the liquid in the wells and wash the plate five times. Wash each well with 200 μL of wash buffer, soak for 1-2 minutes, and discard the liquid in the plate (or use a plate washer). After the final wash, pat the plate dry on absorbent paper. 8. Add 90 μL of TMB chromogenic substrate solution to each well and incubate at 37°C in the dark for 20 minutes (shorten or extend the time as appropriate depending on the actual color development, but do not exceed 30 minutes). 9. Add 50 μL of stop solution to each well to terminate the reaction (the blue color will immediately turn yellow). The stop solution should be added in the same order as the developer. To ensure accurate experimental results, add the stop solution as soon as possible after the substrate reaction time expires. 10. Immediately measure the optical density (OD) of each well using a microplate reader at a wavelength of 450 nm. The instrument should be preheated and the assay program set before use. VII. Calculation of Results 1. Subtract the OD value of the blank well from the OD value of each standard and sample. If replicate wells are used, the average value should be used for calculation.

2. For ease of calculation, although concentration is the independent variable and OD value is the dependent variable, we still use the OD value of the standard as the horizontal axis (X-axis) and the concentration of the standard as the vertical axis (Y-axis) when drawing the graph. At the same time, for the intuitiveness of the experimental results, the figure provides the original data rather than the logarithmic value. Due to different experimental operating conditions (such as operators, pipetting techniques, plate washing techniques and temperature conditions, etc.), the OD value of the standard curve will vary. The standard curve provided is for reference only, and the experimenter needs to establish a standard curve based on his or her own experiment. The OD value of the used sample can be used to calculate the sample concentration on the standard curve, and then multiplied by the dilution factor to obtain the actual concentration of the sample. It is recommended to use professional curve drawing software, such as Curve Expert.

Sample Type

Recovery Range

Average Recovery

Serum (n=5)

87-95%

91%

EDTA Plasma (n=5)

93-107%

100%

heparin plasma(n=5)

87-99%

93%

Sample type

1:2

1:4

1:8

1:16

Serum (n=5)

79-91%

89-103%

82-95%

86-99%

EDTA Plasma (n=5)

87-96%

91-101%

87-98%

89-103%

heparin plasma(n=5)

87-98%

92-105%

95-107%

82-96%

Chinese name

96T

Storage conditions

ELISA plate (removable)

12 strips x 8 wells

4°C/-20°C

Lyophilized Standards

2

4°C/-20°C

Standards & Sample Diluent

20 mL

4°C/-20°C

Concentrated Biotinylated Antibody (100×)

120 L

4°C/-20°C

Biotinylated Antibody Dilution Buffer

12 mL

4°C/-20°C

Concentrated HRP Enzyme Conjugate (100 times;)

120 mL

4°C/-20°C

Enzyme Conjugate Dilution Buffer

12 mL

4°C/-20°C

Concentrated Wash Buffer (25×)

20 mL

4°C/-20°C

Chromogenic Substrate Solution (TMB)

10 mL

4°C/-20°C (protect from light)

Reaction stop solution

6 mL

4°C/-20°C

Sealing film

2

Normal temperature