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ELISA test kit of human-derived soluble CD74 protein and detection method thereof

A detection kit, CD74 technology, applied in the field of immunology and biology, can solve problems such as blanks, and achieve the effects of easy access, broad market prospects, and simple and convenient operation

Active Publication Date: 2013-07-17
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

According to the above theory, is the extracellular segment of CD74 separated from the cell membrane the soluble CD74 (soluble CD74, sCD74)? At present, the research on sCD74 is still blank at home and abroad

Method used

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  • ELISA test kit of human-derived soluble CD74 protein and detection method thereof
  • ELISA test kit of human-derived soluble CD74 protein and detection method thereof
  • ELISA test kit of human-derived soluble CD74 protein and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Preparation of detection ELISA plate: Dilute the anti-C-16 epitope human CD74 antibody to 250ng / ml in the coating buffer, add 100μl to each well of the ELISA plate, seal the plate and place it at 4°C Incubate overnight; shake off the liquid in each well, wash 3-5 times with freshly prepared ELISA plate eluent, 300 μl per well, and dry; add ELISA plate blocking solution, 200 μl per well, and incubate at room temperature 2 hours; Shake off the liquid in each well, add ELISA microplate eluent, 300 μl per well, wash 3-5 times, dry; put in 4 ° C, set aside.

[0053] Detection of serum samples and standard proteins: Serum samples were diluted 1:10 with sample diluent, added at a volume of 100 μl / well, and incubated at room temperature for 2 hours; standard proteins were diluted with sample diluent into different concentration gradients, and 100 μl / well Add the volume of the sample, and incubate at room temperature for 2 hours; shake off the liquid in each well, add ELISA plat...

Embodiment 2

[0054] Embodiment 2: optimization of conditions

[0055] 1. Optimization of coating antibody concentration:

[0056] Select different coating antibody (C-16) concentrations (5μg / ml, 2.5μg / ml, 1μg / ml, 0.5μg / ml, 0.25μg / ml, 0.125μg / ml) to coat the ELISA plate, according to The operation steps in Example 1 were used for detection, and known CD74 protein standards with different concentrations were added. According to the obtained OD value, select the blank group with the smallest OD value and the closest linear relationship between the OD value and the standard protein concentration as the optimal antibody coating concentration. The optimal antibody coating concentration of C-16 is 0.25μg / ml.

[0057] 2. Optimization of blocking solution:

[0058] The blocking solution is PBS solution containing 1% bovine serum albumin + 1% sucrose, PBS solution containing 5% bovine serum albumin + 1% sucrose, SuperBlock blocking solution; the blocking time is 1 hour at room temperature, 2 hour...

Embodiment 3

[0065] Example 3: Evaluation of the specificity and sensitivity of the ELISA kit for human-derived soluble CD74 protein

[0066] 1. Specificity test

[0067] The established ELISA method was used to detect the serum samples of 100 patients undergoing cardiac surgery, and the recombinant CD74 standard protein was used as a positive control. According to the obtained OD value, the serum samples were divided into positive samples with soluble CD74 protein (sCD74 + serum) and negative specimens without soluble CD74 protein (sCD74 - Serum), and further verified by Western blot method. The positive and negative samples were diluted 8 times, 16 times and 32 times respectively, and after SDS page electrophoresis, they were detected with a mouse anti-human CD74 antibody (MB741, #555538, BD Pharmingen) different from the ELISA method, such as figure 1 The positive specimens (6B and 6C) and the standard protein can detect CD74 protein, but the negative specimen (10B) cannot detect obvi...

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Abstract

The present invention belongs to the field of immunology and biotechnology, and provides an ELISA test kit of human-derived soluble CD74 protein and a detection method thereof. The kit of the present invention can accurately detect the content of human-derived soluble CD74 protein in body fluid or cell supernatant, and results are quantitatively analyzed by an enzyme-labeling instrument to exclude subjectivity of semi-quantitative methods such as immunohistochemistry, immunofluorescence, Western blot and the like; the kit has high sensitivity, the detected content of CD74 protein is as low as 800 pg / ml; according to the invention, no complicated instrument is necessary in detection; the kit is suitable for popularization and application in scientific research institutions and medical institutions, is suitable for large-scale detection of clinical samples, and can rapidly obtain massive data and information related to human-derived CD74 protein.

Description

technical field [0001] The invention belongs to the field of immunology and biotechnology, and in particular relates to an ELISA detection kit and a detection method for human-derived soluble CD74 protein. Background technique [0002] CD74 is the major histocompatibility complex class II molecule-associated invariant chain (major histocompatibility complex, MHC-II-associated invariant chain, Ii), also known as HLA-DRγ, and its gene is located on chromosome 5 long arm 3 in humans Band 1 to band 3 (5q31-q33), including 9 exons. CD74 is mainly expressed in antigen-presenting cells such as dendritic cells, monocyte-macrophages, B cells, and Langerhans cells. As an MHC-II molecular chaperone, the main function of CD74 is related to antigen presentation; as an II-type membrane protein, 2-5% of CD74 is expressed on the cell membrane, and its function has nothing to do with MHC-II molecules. Recent studies have found that CD74 is the receptor of macrophage migration inhibitory fa...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/535
Inventor 夏照帆孙瑜唐昊韩姝
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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