HOLO Human Interleukin-12p70 (IL-12p70) Detection Kit 

Interleukin 12 (IL-12), also known as Natural Killer Cell Stimulatory Factor (NKSF), is a 70-75 kDa heterodimeric glycoprotein that belongs to the IL-12 family of heterodimeric cytokines. IL-12 consists of disulfide-linked subunits, IL-12A (p35) and IL-12B (p40). It is produced mainly by monocytes, macrophages, B-lymphocytes, and dendritic cells. Functionally, IL-12 enhances cytotoxic activity and induces the production of interferongamma (IFN-γ) production in NK cells, T cells and dendritic epidermal T cells. IFN-γ plays a pivotal role in Th1-type immune responses, and is essential for antiviral, antitumor responses, and the maintenance of immune tolerance.

MCE HOLO Human Interleukin-12p70 (IL-12p70) Detection Kit is a homogeneous luminescence-based assay used for the quantitative detection of human IL-12p70 concentrations in biological samples such as buffer solutions, cell culture supernatants, or serum.

The detection principle is as follows: Incubate the IL-12p70-specific antibody 1 (F molecule-labeled), IL-12p70-specific antibody 2 (biotin-labeled), acceptor beads (F molecule cross-linked), and the sample to form a sandwich immune complex (antibody 1 - sample - antibody 2). The immune complex is then incubated with donor beads (streptavidin cross-linked) to form a chemiluminescent complex, ensuring that the distance between the two beads is less than 200 nm. When the distance between the two beads is less than 200 nm, the donor beads is excited by light (680 nm), producing singlet oxygen which diffuses to the acceptor beads, inducing energy transfer. The acceptor beads absorb this energy and emit scattering light at 615 nm. The light signal is collected by a photosensitive detector, and the concentration of IL-12p70 in the sample is calculated through mathematical fitting. In the absence of human IL-12p70 in the sample, no immune complex is formed, or the distance between the two beads exceeds 200 nm, exceeding the transfer distance for singlet oxygen, and the acceptor beads do not emit any light.

Features of MCE HOLO Human Interleukin-12p70 (IL-12p70) Detection Kit

1. Easy to Use: The experiment is simple and fast, requiring no washing or separation steps, and is compatible with automated instruments.

2. Wide Detection Range: The dynamic detection range is 0 - 300,000 pg/mL, and the linear detection range is 1.99 - 30,000 pg/mL.

3. High Sensitivity: Small sample volume, with high sensitivity and strong specificity, and capable high-throughput detection.

4. Compatibility: Suitable for natural samples such as cell culture supernatants, buffer solutions, and serum.

4°C, 1 year

Keep away from light. Do not freeze.

1. It is recommended to aliquot the reconstituted standard and store at -20°C. Avoid repeated freeze-thaw cycles.

2. This product is for R&D use only, not for drug, household, or other uses.

3. For your safety and health, please wear a lab coat and disposable gloves to operate.

If the IL-12p70 concentration of the sample exceeds the detection range of the kit, it is recommended to dilute the sample appropriately before testing.

1. Cell Culture Supernatant: Centrifuge the cell culture supernatant at 1,000 × g for 10 min and collect the supernatant.

Note: The cell culture medium should contain at least 1% FBS or 0.1% BSA.

2. Serum: Collect the serum sample following standard procedures.

Note: For serum samples, it is recommended to use a negative serum for reconstitution or dilution of the standard, where the IL-12p70 content in the negative serum should be lower than the detection blank limit (blank limit concentration is provided in the appendix).

Dissolve the IL-12p70 standard (lyophilized powder) in 50 μL of deionized water to prepare a stock solution of 0.9 μg/mL. Then, dilute the stock solution with dilution buffer to prepare a series of gradient concentrations.

Choose one of the two following protocols according to the type of assay.

Protocol 1 (For IL-12p70 concentration detection in most common culture systems such as buffer solutions, DMEM, and fetal bovine serum)

1. Dual Antibody Premix Preparation: 10 μL A + 10 μL B + 10 μL C, mix well for use.

Note: It is recommended to use the dual antibody premix within 1 h after preparation.

2. Immune Complex Preparation: Add 10 μL of standard/sample to the above antibody premix, and incubate at 37°C for 15 min or at room temperature for 60 min.

Note: During incubation, it is recommended to cover the microplate or seal it to prevent evaporation of liquid.

3. Luminescent Complex Preparation: In a green light or dark environment, add 60 μL of D to the above immune complex mixture, and incubate at 37°C for 10 min or at room temperature for 30 min.

Note: a. The temperatures of both incubation should be consistent.

b. Reagent D is light-sensitive. It is recommended to protect from light (light intensity < 100 LUX) or perform the addition and incubation in a green light environment.

c. During incubation, it is recommended to cover the microplate or seal it to prevent evaporation of liquid.

4. Detection: Place the luminescent complex into a homogeneous chemiluminescence instrument or a multifunctional microplate reader with an Alpha module for reading the RLU.

5. Standard Curve Plotting: Plot the standard curve with IL-12p70 concentration (pg/mL) on the x-axis and RLU on the y-axis.

Note: It is recommended to generate a new standard curve for each assay, and perform 2-3 replicate wells for each standard concentration.

6. Calculation: Use the standard curve to calculate the IL-12p70 concentration in the sample.

Protocol 2 (For IL-12p70 concentration detection in RPMI culture system)

1. Dual Antibody Premix Preparation: In a green or dark environment, 10 μL A + 10 μL B + 20 μL D, mix thoroughly and incubate at room temperature for 30 min.

Note: a. Reagent C is light-sensitive. It is recommended to protect from light (light intensity < 100 LUX) or perform the addition and incubation in a green light environment.

b. During incubation, it is recommended to cover the microplate or seal it to prevent evaporation of liquid.

2. Immune Complex Preparation: In a green or dark environment, add 10 μL of standard/sample to the premix above, and incubate at 37°C for 25 min.

During incubation, it is recommended to cover the microplate or seal it to prevent evaporation of liquid.

3. Luminescent Complex Preparation: In a green or dark environment, In a green or dark environment, add 10 μL of C to the premix above, and incubate at 37°C for 10 min.

Note: During incubation, it is recommended to cover the microplate or seal it to prevent evaporation of liquid.

4. Detection: Place the luminescent complex into a homogeneous chemiluminescence instrument or a multifunctional microplate reader with an Alpha module for reading the RLU.

5. Standard Curve Plotting: Plot the standard curve with IL-12p70 concentration (pg/mL) on the x-axis and RLU on the y-axis.

Note: It is recommended to generate a new standard curve for each assay, and perform 2-3 replicate wells for each standard concentration.

6. Calculation: Use the standard curve to calculate the IL-12p70 concentration in the sample.

1. It is recommended to aliquot the reconstituted standard and store at -20°C. Avoid repeated freeze-thaw cycles.

2. This product is for R&D use only, not for drug, household, or other uses.

3. For your safety and health, please wear a lab coat and disposable gloves to operate.