Introduction

Chemokine (CC motif) ligand 8 / monocyte chemokine 2 (CCL8 / MCP-2) consists of 97 amino acids and belongs to the CC subfamily. CCL8 / MCP-2 consists of monocytes, fibroblasts and Epithelial cells are produced,

For CD4 + T cells, CD8 + T cells and NK cells have chemotactic activity. Although the biological activity of CCL8 / MCP-2 in mice is still unknown, but recent reports have shown that plasma CCL8 / MCP-2 concentration increased It is closely related to the incidence of graft-versus-host disease (GVHD), which is a complication of bone marrow transplantation, and studies have shown that CCL8 / MCP-2 is closely related to the immune response in mice.

Experimental principle

This kit is a sandwich enzyme-linked immunosorbent assay kit that uses two highly specific antibodies. TMB is used as a developer, and the color intensity of the final solution is proportional to the amount of CCL8 / MCP-2 in the mouse sample

examination range

78.13 ~ 5,000 pg / mL

expected usage

For research only, not for diagnosis

This kit can be used for quantitative detection of CCL8 / MCP- in mouse serum, EDTA plasma and heparin plasma

Kit components

Coated plate, coated with anti-mouse CCL8 / MCP-2 (81) rabbit IgG 96 well

Labeled antibody,

30X concentrated, HRP labeled anti-mouse CCL8 / MCP-2 rabbit IgG monoclonal antibody, Fab affinity purified 0.4ml x 1

Standard, recombinant mouse CCL8 / MCP-2 0.5mlx 2

EIA buffer, 1% BSA, PBS containing 0.05% Tween 20 30mlx 1

Labeled antibody dilution, 1% BSA, 0.05% Tween 20 in PBS 12mlx 1

Developer, TMB, 15mlx 1

Stop solution, 1N sulfuric acid, 12mlx 1

Wash buffer concentrate, 40X concentrated, 0.005% Tween 20 phosphate buffer 50mlx 1

Materials required for the experiment but not provided in the kit

Microplate reader (450nm) pipette and pipette tip

Graduated cylinder and beaker with deionized water

Refrigerator (4 ℃) Coordinate paper (log / log)

Dilute test tube with absorbent paper standard

Incubator (37 ± 1 ℃) for washing bottles

Disposable test tube

Reagent preparation

Preparation of washing buffer: The washing solution of the kit is 40 times concentrated. Before use, the washing concentrated solution is first equilibrated to room temperature, mixed well, and then 50ml of concentrated solution and 1950ml of deionized water are mixed and mixed. The prepared diluted washing solution should be kept in the refrigerator and used up within 2 weeks

Preparation of diluted labeled antibody: Dilute the labeled antibody with labeled antibody diluent 30 times, that is.

Example: If only one plate and 8 wells are tested, 800ul of labeled antibody is needed (30ul of concentrated labeled antibody + 870ul of labeled antibody diluent, mix well, add 100ul per well when used)

This step is completed before the experiment

The remaining labeled antibody concentration should be packed in a sealed bottle and stored at 4 ℃

Standard preparation: Pipette 0.5ml of deionized water into the standard bottle and mix well to obtain mouse CCL8 / MCP-2 standard with a concentration of 10,000 pg / mL

Standard dilution: Prepare 8 test tubes for standard dilution, add 230ul EIA buffer to each tube

The concentration of each tube is as follows

Tube 1 5,000 pg / mL

Tube 2 2,500 pg / mL

Tube 3 1,250 pg / mL

Tube 4 625 pg / mL

Tube 5 312.5 pg / mL

Tube 6 156.25 pg / mL

Tube 7 78.13 pg / mL

Tube 8 0ng / ml (test sample blank)

Pipette 230ul of the standard product with a concentration of 10,000 pg / mL into No. 1 tube and mix it, then draw 230ul from No. 1 tube into No. 2 tube and serially dilute them in succession. The concentration is 0 pg / mL

Sample dilution: If the sample needs to be diluted, please dilute with EIA buffer. Generally, the serum and plasma samples of normal mice are recommended to be approximately 200-500 times. However, the dilution rate should be optimized according to the specific conditions of each laboratory. , Because the mouse CCL8 / MCP-2 varies with the individual's immune status and breed.

Experimental procedure

Before use, all samples should be equilibrated to room temperature for about 30 minutes, and then mixed thoroughly to ensure that there is no change in reagent quality. The standard curve and sample detection are performed simultaneously

Reagent sample standard sample blank reagent blank

Test sample 100ul Dilution standard (1-7 tubes) 100ul EIA buffer (8th tube) 100ul EIA buffer 100ul

Cover plate, incubate at 37 ℃ for 60min

Wash the plate 7 times

Enzyme labeled antibody 100ul 100ul 100ul-

Cover plate, incubate at 4 ℃ for 30min

Wash plate 9 times

Developer 100ul 100ul 100ul 100ul

Incubate at room temperature in the dark for 30min

Stopping fluid 100ul 100ul 100ul 100ul

Read OD value at 450nm within 30min after adding stop solution

Set reagent blank control wells, and add 100ul EIA buffer to the corresponding microwells.

Determine the sample blank well, sample well and standard well, then add 100ul of sample blank (tube 8), test sample and diluted standard (tube 1-7) to the corresponding microwells.

Cover plate, incubate at 37 ℃ for 60min

Wash the plate vigorously with a wash bottle, then add washing solution to the microwells and let stand for 15-30 seconds, then sprinkle the washing solution. Repeat 7 times, finally pat dry on absorbent paper

If you use an automatic plate washer to wash the plate, first wash the plate 4 times automatically, and then wash the plate 3 times manually as described above

Add 100ul of diluted labeled antibody to each well, except the reagent blank control well

Cover plate, incubate at 4 ℃ for 60min

Follow step 4) Wash the plate 9 times

Take the required amount of developer into the test tube, then add 100ul of developer into each microwell.

To avoid contamination, do not pour the remaining reagent into the original reagent bottle.

Incubate at room temperature in the dark for 30 minutes. After adding the developer, the color of the solution will become blue

Add 100ul of stop solution to each well, tap the edge of the plate to mix, then the color of the solution will turn yellow

Wipe off the dirt or water droplets on the bottom of the microplate and make sure that no bubbles are generated in the reaction solution. Read the reading at 450nm of the microplate reader within 30min after the stop solution

pay attention

Test samples should be tested or frozen immediately after collection. The frozen samples should avoid repeated freezing and thawing. Before testing, they should be completely dissolved and mixed at low temperature.

The test sample is diluted with EIA buffer

Test samples and standards are recommended for double detection

The test sample should be in the neutral pH range, the contamination of the organic solvent may affect the test results

Only wash the plate with the washing solution provided by the kit. Insufficient or excessive washing will cause the test to fail

Pat dry microplate on absorbent paper, can not wipe

The TMB substrate solution should be stored in a dark place because it is sensitive to light and avoid contact with metals

It must be read in the microplate reader within 30min after adding the stop solution

Result calculation

Before drawing the curve, all data should be subtracted from the blank value of the test sample, including the standard and unknown samples. Draw the standard curve on the log-logarithmic graph paper with the OD value and concentration of the standard, and the concentration of the unknown sample can be directly read from the standard curve

The above typical standard curve is only for demonstration and can not be used to obtain test data. A standard curve should be established for each test

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

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