CN109521203B

CN109521203B - Application of soluble VSIG4 as biomarker in preparation of hematophagous lymphohistiocytosis diagnostic kit

Info

Publication number: CN109521203B
Application number: CN201811422840.1A
Authority: CN
Inventors: 宋世平; 刘兵; 袁顺宗
Original assignee: Fifth Medical Center of PLA General Hospital
Current assignee: Fifth Medical Center of PLA General Hospital
Priority date: 2018-11-27
Filing date: 2018-11-27
Publication date: 2022-02-01
Anticipated expiration: 2038-11-27
Also published as: CN109521203A

Abstract

The invention discloses a diagnostic kit for preparing human hemophagocytic lymphohistiocytosis by using human serum soluble VSIG4 as a biomarker. The serological detection result shows that the concentration of the soluble VSIG4 in the serum of a patient with tumor combined hemophagocytic lymphohistiocytosis is obviously higher than that of a normal population and the serum of a patient without the hemophagocytic lymphohistiocytosis, and the soluble VSIG4 has higher sensitivity, specificity and larger work curve area (> 0.94) of a subject when being used for diagnosing the hemophagocytic lymphohistiocytosis, so the soluble VSIG4 can be regarded as a new marker for diagnosing the hemophagocytic lymphoblastic lymphocytosis.

Description

Application of soluble VSIG4 as biomarker in preparation of hematophagous lymphohistiocytosis diagnostic kit

Technical Field

The invention belongs to an in vitro diagnosis marker, and relates to application of soluble VSIG4 as a biomarker in preparation of a hematophagous cytolymphocythemia diagnosis kit.

Background

Lymphohistiocytosis Haemophaga (HLH), also known as haemophagic syndrome (HPS), is considered a reactive proliferative histiocytosis of the macrophagy system. HLH is divided into two major classes, primary and secondary, with primary HLH resulting in activation of mononuclear macrophages mainly due to the production and release disorders of cytotoxic particles; secondary HLH occurs mainly in patients with infection or tumor, because cytotoxic T cells or NK cells are defective in function to cause antigen clearance, the mcmb system receives continuous antigen to stimulate excessive activation and proliferation, activates cascade effect of cytokines, generates cytokine storm, causes macrophages to activate and infiltrate phagocytic blood cells in large quantity and destroy tissues, and further causes multiple organ failure. HLH is mainly manifested by fever, splenomegaly, pancytopenia, high triglyceride, low fibrinogen, high serum ferritin, high serum soluble interleukin-2 receptor alpha (sCD25), and reduced activity of NK cells in peripheral blood, and hemophagocytosis can be found in bone marrow smear, spleen or lymph node biopsy. Since secondary HLH lacks standard treatment regimens and has rapid disease progression and extremely high mortality, efficient early diagnosis is important for HLH treatment.

Eight diagnostic criteria for secondary HLH are proposed in the HLH-2004 diagnostic criteria: 1) generating heat; 2) splenomegaly; 3) at least two lines of whole blood cells are reduced; 4) high triglycerides or low fibrinogen; 5) high serum ferritin; 6) increased serum soluble interleukin-2 receptor alpha (sCD 25); 7) a decrease in peripheral blood NK cell activity; 8) hematophagous cell events were found in bone marrow smears, spleen or lymph node biopsies. HLH can be diagnosed when five of eight standards are met, but in clinical practice, detection methods for the eight standards are not developed by every medical center. Therefore, the discovery of a new specific marker has very important significance for the diagnosis and treatment of HLH.

The early stage of the subject group finds that the soluble VSIG4 is abnormally and highly expressed in the serum of HLH patients, is closely related to the prognosis and outcome of the patients, and further considers that the soluble VSIG4 is possibly valuable for HLH diagnosis.

Disclosure of Invention

The invention aims to provide application of soluble VSIG4 as a biomarker in preparing an HLH diagnostic kit. In order to realize the purpose, the invention discloses the following technical scheme: use of soluble VSIG4 as a biomarker in the preparation of a HLH diagnostic kit.

The diagnosis kit comprises but is not limited to a serum sample HLH diagnosis kit, a VSIG4 concentration detection kit, a CRIg concentration detection kit, a Z39Ig concentration detection kit and a macrophage activation diagnosis kit.

The invention has the advantages that: according to the soluble VSIG4, the expression of the soluble VSIG4 in HLH patients is obviously higher than that of normal people as shown by a serological detection result, so that the HLH patients can be accurately identified. The diagnostic research analysis shows that the expression level of the soluble VSIG4 has higher sensitivity and specificity and larger area under the curve (> 0.94) when being used for diagnosing the HLH, has stable result and high diagnostic value, and particularly has good specificity on the HLH diagnosis of lymphoma/leukemia combination.

Drawings

Figure 1 expression measurements of soluble VSIG4 in the serum of normal population, breast cancer, lung cancer, gastric cancer, tumor patients without HLH incorporation and tumor patients with HLH incorporation showed that soluble VSIG4 expression was significantly higher in the serum of patients with HLH incorporation than in the other groups (× P < 0.0001).

FIG. 2 ROC curve of serum soluble VSIG4 for diagnosing lymphoma combined with HLH, area under the curve being 0.94.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1: ELISA detection of soluble VSIG4 in human peripheral blood serum

In order to detect soluble VSIG4 in human serum, the following ELISA detection method was performed, which specifically comprises the steps of:

1. coating: using carbonate buffer solution with pH9.0 as diluent, rabbit or mouse or goat anti-human VSIG4 extracellular domain antibody was diluted to 1. mu.g/ml, added to 96-well flat-bottom enzyme plate in polystyrene at 100. mu.l/well, sealed with preservative film, and placed in refrigerator at 4 ℃ overnight. The next day, the coating solution was discarded and the plate was washed 3 times with 300. mu.l of PBST buffer for 3 minutes each.

2. And (3) sealing: add 200. mu.l of 2% BSA-PBS buffer to each well, block for 1h at room temperature, and wash the plate 3 times for 3 minutes with 300. mu.l of PBST buffer.

3. Sample adding: serum (diluted 1: 10 in PBST buffer) or recombinant human VSIG4 protein diluted in a gradient was added to suspected HLH patients at 100. mu.l/well and incubated at 37 ℃ for 1h, with 2 parallel wells per sample. After incubation, antigen was discarded and the plates were washed 3min x 3 times with 350 μ l PBST buffer.

4. Adding enzyme labels to detect the antibody: mu.l/well of horseradish peroxidase-labeled rabbit anti-human VSIG4 antibody diluted with PBST buffer at 1. mu.g/ml was added as a detection antibody and incubated at room temperature for 1 h. The plate was then discarded and washed 3 times with 300. mu.l of PBST buffer for 3 minutes each.

5. Color development: the chromogenic solution A and chromogenic solution B (TMB substrate solution Set, Yinqiao Co.) were mixed in equal volumes at 15 minutes in advance, and were protected from light. Then, 200. mu.l/well of the buffer solution was added to the microplate, protected from light, and incubated for 20 minutes.

6. And (4) terminating: after color stabilization, 50. mu.l of 2M H was added per well₂The reaction was stopped with SO4 solution and gently mixed until color development was uniform.

7. Reading value: the microplate was placed in the microplate reader within 20 minutes and the absorbance at 450nm was read.

8. And (4) processing a result: the readings of 2 negative control wells were first determined and averaged to give a background reading. For each serum sample or standard, the inter-well variation (inter-well variation-difference between two-well OD/sum of two-well OD) is calculated, and if the variation (CV) < 15%, the sample is a valid sample, otherwise, the sample is discarded. The mean of two wells for a valid sample was taken as the OD value for that sample, and the background reading was subtracted as the valid OD value. A standard curve was then plotted using a gradient of diluted recombinant VSIG4 standards to calculate the protein concentration of soluble VSIG4 in each serum. Note: the materials used in the above experimental methods, except for special indications, are analytical reagents.

Example 2: clinical detection application of human serum soluble VSIG4 indirect double-sandwich ELISA detection method

Serum soluble VSIG4 concentrations were determined for lymphoma patients diagnosed with HLH (mean age 58, maximum age 84, minimum age 26) and lymphoma patients not containing HLH (mean age 46, maximum age 83, minimum age 10) using the above method of example 1. The results of the experiment and the standard curve show a linear correlation coefficient of 0.9995 between serum soluble VSIG4 concentration and lymphoma combined HLH, indicating that this concentration gradient falls substantially within the linear range of the detection reaction. The experimental result shows that the average concentration of serum soluble VSIG4 of patients without HLH lymphoma is 676.5pg/ml, the detection interval is 0-3258 pg/ml, the average concentration of serum soluble VSIG4 of patients with HLH lymphoma is 5569pg/ml, and the detection interval is 1037-13017 pg/ml. The difference of the two is significant through a one-way ANOVA test (p is less than 0.0001). Meanwhile, the average concentration of serum soluble VSIG4 of the healthy control group is 3.5pg/ml, and the detection interval is 0-173 pg/ml.

Example 2: parallel detection of HLH using control methods

When the serum soluble CD25 concentration of all ELISA samples of the foregoing example 2 was measured using an electrochemiluminescence method (a method conventionally used clinically), and a receiver curve (ROC curve) for HLH detection of soluble CD25 and soluble VSIG4 was plotted (fig. 2), it was found that the area under the curve of soluble CD25 was 0.92 and the area under the curve of soluble VSIG4 was 0.94, and the diagnostic values of these two proteins were substantially the same (DeLong nonparametric test, p ═ 0.6125). At this time, the threshold of serum soluble CD25 for best diagnostic effect was 8033U/ml, sensitivity was 95.00%, and specificity was 84.09%. The soluble VSIG4 threshold was 2195pg/ml with 95% sensitivity and 86.36% specificity.

The results show that the established ELISA detection method for serum soluble VSIG4 has the characteristics of good repeatability and high sensitivity. The marker is detected by serum soluble VSIG4 measurement of 120 healthy adults and HLH patients, and is high in detection sensitivity, so that the marker can be a potential early serum screening marker. Moreover, it is essentially the same as the diagnostic efficacy of soluble CD25, specifically, HLH can be detected independently using soluble VSIG4 alone, which has similar diagnostic value to the soluble CD25 currently used clinically.

Example 3: HLH detection kit for detecting soluble VSIG4

The invention also provides an HLH detection kit, which contains recombinant human soluble VSIG4 as a protein standard, an anti-human soluble VSIG4 antibody (coating antibody: working concentration 2 mu g/ml, dilution buffer 50mM carbonate buffer, pH 9.0; detection antibody: working concentration 1 mu g/ml, dilution buffer 150mM PBST buffer, pH 7.4), and other detection reagents in example 1.

64 clinical trials have been completed using the kit of the present invention using the method of example 1, with a positive predictive value of 76%, a negative predictive value of 97.44%, a sensitivity of 95%, and a specificity of 86.36%. According to the literature search, no report of the same work exists at home and abroad at present.

Claims

1. The application of soluble VSIG4 protein in serum as a biomarker in preparing a diagnosis kit for hemophagocytic lymphohistiocytosis is disclosed, the diagnosis kit is a VSIG4 concentration detection kit, the expression of VSIG4 in an HLH patient is obviously higher than that of a normal population, and the hemophagocytic lymphohistiocytosis is lymphomatoid lymphohistiocytosis combined with lymphoma.