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ELISA detection kit for mice-derived soluble CD74 proteins and detection method of ELISA detection kit

A detection kit and CD74 technology, which is applied in the fields of immunology and biology, can solve the problems of basic research obstacles, failure to detect mouse-derived soluble CD74 protein, etc., and achieve high sensitivity, easy access, and broad market prospects Effect

Inactive Publication Date: 2015-01-14
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Due to the species-specificity between mouse-derived CD74 and human-derived CD74, we failed to detect mouse-derived soluble CD74 protein using the human-derived soluble CD74 ELISA detection method (CN103207277A) in the early stage
Moreover, there is no report on the quantitative detection of mouse-derived soluble CD74 protein at home and abroad, which has caused certain obstacles to basic research. Therefore, it is necessary to develop a quantitative detection method for soluble CD74 in basic experimental biological samples. Lay the foundation for further research on the function of CD74 protein

Method used

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  • ELISA detection kit for mice-derived soluble CD74 proteins and detection method of ELISA detection kit
  • ELISA detection kit for mice-derived soluble CD74 proteins and detection method of ELISA detection kit
  • ELISA detection kit for mice-derived soluble CD74 proteins and detection method of ELISA detection kit

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Embodiment 1: experimental method

[0067] Preparation of detection ELISA plate: Dilute AF7478 to 0.25 μg / ml in coating buffer, add 100 μl to each well of the ELISA plate, seal the plate and incubate overnight at 4°C; use freshly prepared ELISA plate Plate eluent, 300 μl per well, wash 3-5 times with Elx50 (BioTek), an automatic plate washer, and pat dry on absorbent paper; add ELISA plate blocking solution, 200 μl per well, and incubate at room temperature for 2 hours; the same method as before Wash 3-5 times with ELISA microplate eluent, 300 μl per well, and pat dry; store at 4°C for later use.

[0068] For serum samples or BALF samples, dilute 1:2 with sample diluent, add samples at a volume of 100 μl / well, and incubate at room temperature for 2 hours; use ELISA plate elution solution as before, 300 μl per well, wash 3-5 Once, pat dry; dilute CD74 antibody MAB7478 to 1 μg / ml, 100 μl per well, incubate at room temperature for 2 hours; use ELISA plate eluent as before...

Embodiment 2

[0069] Example 2: condition optimization

[0070] 1. Optimization of coating antibody concentration:

[0071]Select different concentrations of coating antibody AF7478 (1 μg / ml, 0.5 μg / ml, 0.25 μg / ml, 0.125 μg / ml) to coat the ELISA microtiter plate, detect according to the operation steps in Example 1, add known Different concentrations of CD74 protein standards. According to the obtained OD value, select the one with the smallest OD value of the blank group and the closest linear relationship between the OD value (minus the OD value of the blank group) and the standard protein concentration as the optimal antibody coating concentration. The best antibody coating concentration of AF7478 is 0.25μg / ml.

[0072] The optimal concentration of the screening coating antibody AF7478 is detailed in Figure 6 .

[0073] 2. Optimization of blocking solution:

[0074] The blocking solution is PBS solution containing 1% bovine serum albumin + 1% sucrose, PBS solution containing 5% bov...

Embodiment 3

[0089] Example 3: ELISA kit detects recombinant mouse-derived CD74 protein and establishes a standard curve

[0090] Dilute the recombinant mouse-derived CD74 standard protein from 25ng / ml to 9 concentrations, 25ng / ml, 12.5ng / ml, 6.25ng / ml, 5.208ng / ml, 4.557ng / ml, 3.125ng / ml, 2.604 ng / ml, 0.913ng / ml, 0.651ng / ml, each sample set up 3 duplicate holes, 100μl / hole, repeat the detection 3 times according to the steps of Example 1, the results are similar, as figure 1 As shown, the reaction between the recombinant mouse CD74 protein and the antibody has a good concentration-dependent relationship, and there is an obvious linear correlation.

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Abstract

The invention provides an ELISA detection kit for mice-derived soluble CD74 proteins and a detection method of the ELISA detection kit, belonging to the technical field of immunology and biology. The kit can accurately detect the content of soluble CD74 proteins in mice body fluid, and a detection result is qualitatively analyzed by ELISA, so that the subjectivity of semiquantitative methods such as immunohistochemistry, immunofluorescence and western blot can be eliminated; moreover, the sensitivity is high, and the detected minimum content of the CD74 proteins can reach 781.25 pg / ml. No complicated instrument is needed in the detection, the ELISA detection kit and the detection method are easy to popularize and use in scientific research institutions and universities and medical institutions, basic research biological specimens can be detected on large scale, and a great amount of data and information related to the mice CD74 proteins can be rapidly acquired.

Description

technical field [0001] The invention belongs to the field of immunology and biotechnology, and in particular relates to an ELISA detection kit for mouse-derived soluble CD74 protein and a detection method thereof. Background technique [0002] CD74, the major histocompatibility complex (MHC-II-associated invariant chain, Ii), is a non-polymorphic type II transmembrane glycoprotein that can be divided into intramembrane segment, transmembrane segment, and extramembrane segment. CD74 mainly exists in cells, and as a molecular chaperone of MHC-II molecules, it promotes the processing and release of MHC-II molecules in the endoplasmic reticulum, transports them to endosomes, and prevents the binding of MHC-II molecules to endogenous polypeptides in the endoplasmic reticulum. In addition, about 2-5% of CD74 is expressed on the surface of cells, such as B cell lines, epithelial cells, monocytes, etc., and does not attach to MHC class II molecules, and its function has nothing to ...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/535
CPCG01N33/535G01N33/6893
Inventor 伍国胜孙瑜唐昊陈郑礼王星童唐洪泰
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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