Elisa kit for checking soluble PD-1 protein and checking method

An enzyme-linked immunoassay detection and kit technology, applied in the field of medical and biological detection, to achieve the effect of accurate detection and excellent linearity

Inactive Publication Date: 2009-01-07
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is still no ELISA kit for the detection of sPD-1 using these two monoclonal antibodies

Method used

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  • Elisa kit for checking soluble PD-1 protein and checking method
  • Elisa kit for checking soluble PD-1 protein and checking method
  • Elisa kit for checking soluble PD-1 protein and checking method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1, making an enzyme-linked detection kit for detecting soluble PD-1 protein

[0044] For convenience of description, first list the main reagents and instruments used in the examples:

[0045] Biotin: N-hydroxysuccinimide ester ((BNHS) Sigma)

[0046] Coating solution: volume ratio Tirs-HCL: H 2 O=1:9

[0047] Cleaning solution: a solution containing 1% Tween 20 by mass fraction prepared in PB (pH=7.4)

[0048] Blocking solution: PBS solution with mass fraction of 1% BSA (Sigma company)

[0049] Horseradish peroxidase: volume ratio Avidin-HRP (Sigma company): BSA=1: 16667

[0050] Chromogenic solution: volume ratio tetramethylbenzidine (TMB) (Sigma company): H 2 O=1:2

[0051] Stop solution: 2M sulfuric acid

[0052] Enzyme label assay plate (12×8 wells, Nunc company);

[0053] Enzyme label analyzer (Bio-Rad company)

[0054] An enzyme-linked detection kit for detecting soluble PD-1 protein, comprising:

[0055] 1 vial of PD-1Ig fusion protein standard...

Embodiment 2

[0108] Example 2: An example of the use of an enzyme-linked detection kit using soluble PD-1 protein, the flow chart can be found in figure 2

[0109] (1) Collect the data of normal people, patients with aplastic anemia (AA), patients with acute lymphoblastic leukemia (ALL), patients with acute myeloid leukemia (AML), patients with non-Kirosch lymphoma (NHL), patients with liver transplantation, and patients with hyperthyroidism. Serum, frozen at minus 20°C;

[0110] (2) Dilute the standard PD-1Ig (concentration 50ng / ml) to 7 gradient concentrations by 0.4 times with double distilled water. The standard curve in the kit is composed of 7 standard products with different concentrations. Concentrations are 50ng / ml, 20ng / ml, 8ng / ml, 3.2ng / ml, 1.28ng / ml, 0.512ng / ml, 0.2048ng / ml; the serum of the sample to be tested is also diluted to 7 gradient concentrations by 0.4 times;

[0111] (3) Antigen-antibody reaction: Add the standard substance and the gradient dilution solution of th...

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Abstract

The invention provides an enzyme-linked immune detection reagent kit for detecting soluble PD-1 protein and a detection method thereof. The reagent kit consists of a PD-1 fusion protein standard compounds, a monoclonal antibody coating plate, an enzyme labeled monoclonal antibody and an auxiliary reagent; wherein, the reagent kit applies two strains of mouse anti-human PD-1 hybridomas and monoclonal antibodies secreted by the hybridomas which are called 1F2 and 5F10 and that are developed independently by the inventor and establishes the enzyme-linked immune detection reagent kit for detecting the soluble PD-1 (sPD-1) protein and the detection method thereof according to different characteristics of recognition sites of the two single strains of monoclonal antibodies. The reagent kit can detect the sPD-1 protein accurately and sensitively. The method can be applied to the detection of scientific research as well as clinical autoimmune diseases and hematological diseases, and the like, and provides the evaluation for diagnosis, monitoring and prognosis of relevant clinical diseases.

Description

technical field [0001] The invention relates to the field of medical and biological detection, in particular to an enzyme-linked immunoassay kit for detecting soluble PD-1 protein, and a method for detecting soluble protein using the kit. Background technique [0002] PD-1 is a 50-55kD immunoglobulin superfamily type I transmembrane glycoprotein, which is mainly expressed in activated T and B cells and specific stages of bone marrow development (see: Ishida Y et al., The EMBOJournal, 11: 3887-3895 (1992); Agata Y et al., Int. Immunol, 8: 765-772 (1996)), the initial study showed that two tyrosine residues in the cytoplasmic region of PD-1 interact with downstream signaling molecules It can play a role in negative immune regulation (see: Latchman Y et al., Nat Immunol, 2(3): 261-268 (2001); Ravetch JV et al., Science, 290(5489): 84-89 (2000) .PD-1 can also promote the proliferation of T cells, which depends on the CD28 co-stimulatory signal (see: Rio ML et al., Eur J Immunol...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/543
Inventor 明志君张学光吴海竞陈永井
Owner SUZHOU UNIV
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