CN105823882B - ATP is used to evaluate the purposes and evaluation method of immunocompetence as biomarker - Google Patents

Abstract

The present invention relates to biological field, more particularly to ATP is used to evaluate the purposes and evaluation method of immunocompetence as biomarker.The present invention is not required to use the material of any isotope marks therefore overcomes the defect that prior art is unfavorable for safety operation；The cell of separation need not for a long time be cultivated and overcome prior art detection cycle shortcoming long detecting；Cell need to only crack can quick detection, overcome the cumbersome deficiency of prior art operation, and influenceed small by experiment condition.Present invention also offers the evaluation method of non-diseases diagnostic purpose immunocompetence, safety easy to operate, sensitiveness is good, and accuracy is high, can detect wide ranges, as a result can more accurately obtain the immune state of body.

Description

ATP is used to evaluate the purposes and evaluation method of immunocompetence as biomarker

Technical field

The present invention relates to biological field, more particularly to ATP as biomarker be used for evaluate immunocompetence purposes and Evaluation method.

Background technology

The material base that body undertakes immunologic function is immune system (immune system).Immune system is by immune group Knit and constituted with organ, immunocyte and immune molecule.Immune system can be divided into natural immune system (i.e. nospecific immunity system System) and acquired immune system (i.e. specific immune system).Acquired immune system may be generally divided into two major classes：Body fluid is exempted from Epidemic disease and cellular immunity.Humoral immunity relates generally to the antibody that can be combined with various exogenous antigens in human body, and they are that a class is big Molecule protein, also referred to as immunoglobulin, including IgG, IgA, IgM and IgD, the types of IgE five.Cell-mediated is immune anti- (cell-mediated immunity, CMI) is answered to relate generally to two major class cells：B cell and T cell, T cell can also be divided into Cd4 cell and cd8 cell etc.；Functionally briefly, B cell can produce various antibody, and T cell can inherently be killed Or phagocytosis enters internal various pathogen.

What " immunity degradation " referred to is exactly the reduction of internal antibody level or T, the reduction of B cell function so that human body is easy The various infection of generation.Fatigue, malnutrition, chronic disease (such as tuberculosis, hepatitis, malignant tumour, diabetes), AIDS, Medicine (such as chemotherapeutic or immunodepressant) can all cause immunity of organisms to decline.Clinically, detection immunity is main Index is that serum immune globulin (Ig) is quantitative and T cell subgroup is analyzed.If Ig amounts are reduced or B cell, T cell number drop Low or dysfunction (particularly CD4, cd8 cell) is all pointed out to there may be immunity degradation.

The body of hypoimmunity is easy to infected or cancer stricken；Immunity is extraordinary also to produce insalubrious knot Really, allergic reaction, autoimmune disease are such as triggered.Therefore, accurate detection immunity of organisms height has important reality meaning Justice.

The technology of detection body immunity mainly has at present：1. lymphopoiesis detection technique：Lymphocyte precursor There is no any effector function, but can breed when antigenic stimulus is run into, lymphopoiesis is assessment body immunity A kind of important method.The clinically conventional lymphopoietic method of detection has 3H-TdR incorporation methods, mtt assay and stream at present Formula cytometry.2.T cell secreting function determination techniques：The T cell of in vitro culture is through various mitogens or antigenic stimulus Afterwards, various cell factors are secreted, by detecting each cytokine content, BA or gene expression dose, to reflect that T is thin Born of the same parents' function.Conventional detection method has Elisa, Elispot, PCR/RT-PCR.3.T cell-mediated determination of cytotoxic activity skill Art：Cytotoxic T lymphocyte (CTLs) is a kind of effector cell that T cell is produced after antigenic stimulus in vivo or in vitro, Can Specific lytic target cell, this ability can reflect the anti-tumor capacity of body, therefore cytotoxicity assay can be anti- The immunocompetence of film projector body.Conventional method has chromium to discharge detection and LDH release detections.

But immunity detection method has following problem：1. the defect of lymphopoiesis detection technique：3H-TdR mixes Enter method it represent be cell in a certain quantitative culture liquid DNA synthesis total amount, rather than the reagent number of specific T-cells, Influenceed by condition of in vitro culture but also there is radionuclide contamination pollution problem；MTT methods are influenceed very big by experiment condition, Cultivating system needs strict control；Flow Cytometry analysis need certain appointed condition and required experimental period cycle compared with It is long.The defect of 2.T cell secreting function determination techniques：The time required to Elisa methods and Elispot methods operate cumbersome detection More long, the T cell of detection must be able to secrete targeted cytokines；There is the result of false positive and false negative in PCR/RT-PCR, no What is necessarily obtained is exactly purpose fragment, the strict otherwise easily pollution of operation, it is most important that the PCR inspections of routine do not measure latency Purpose fragment.The defect of 3.T cell-mediated determination of cytotoxic activity technology：Chromium has radioactivity, is unfavorable for safety operation and gives up Thing is disposed and is not quantitative analysis and operating procedure is more；LDH is the product that cell is metabolized in itself, it is understood that there may be spontaneous release shows As, and damage is also had after effector cell acts on target cell, LDH can be equally discharged, so as to influence determination of cytotoxic activity Accuracy.

Related products all in the market and detection means can not carry out effective dividing to the therapeutic effect of patient Analysis and assessment, and can not be applicable for patient HIV or the low person of immunocompetence.Therefore it provides a kind of immunity power Testing index has realistic meaning.

The content of the invention

In view of this, the present invention provides ATP is used to evaluate the purposes and evaluation method of immunocompetence as biomarker. Good with ATP evaluations immunocompetence sensitiveness, accuracy is high, can detect wide ranges, as a result can more accurately obtain the immune shape of body State.

In order to realize foregoing invention purpose, the present invention provides following technical scheme：

It is used to evaluate the purposes of immunocompetence as biomarker the invention provides ATP.

Atriphos (ATP) is played an important role in the vital movement of organism：The conjunction of serum immune globulin Into and the process of secretion or even function be required for the participation of ATP；Another aspect T cell function is killed or swallowed ATP being also required into internal various pathogen, energy is provided；Biography of the second messengers such as Ca2+, DAG for immune signal path The essential effect that plays is led and amplifies, but their synthesis secretion and transdermal delivery are required for ATP；In addition ATP is produced Can be secreted into extracellular after life, be combined with its acceptor, and then activate second messenger, carry out signal transduction.Therefore, the triphosphoric acid of cell The measure of adenosine (ATP) value has important clinical meaning for detection body immunity, and immunocompetence is made objectively Assessment.And then prevention can infect ahead of time, therapeutic intervention immunity disease is carried out early, and can detect the effect that treatment recovers.

In some specific embodiments of the invention, the standard of the evaluation is：The concentration ＜ 150ng/ml of ATP or Concentration >=the 500ng/ml of ATP is immunocompetence exception.

In other specific embodiments of the invention, the concentration ＜ 150ng/ml of the ATP are hypoimmunity.

In other specific embodiments of the invention, the concentration >=500ng/ml of the ATP is that immunity is extraordinary.

In other specific embodiments of the invention, the concentration ＜ 500ng/ml of 150ng/ml≤ATP are immunity Normally.

Present invention also offers a kind of evaluation method of non-diseases diagnostic purpose immunocompetence, comprise the following steps：

Step 1：Obtain the ATP in sample；

Step 2：Determine the concentration of the ATP；

Step 3：According to the concentration of the ATP, the power of adaptive immune ability.

In some specific embodiments of the invention, the standard of the evaluation method is the concentration ＜ 150ng/ml of ATP Or concentration >=the 500ng/ml of ATP is immunocompetence exception.

Specific to other specific embodiments of the invention, the concentration ＜ 150ng/ml of ATP are in the evaluation method Hypoimmunity.

Specific to other specific embodiments of the invention, the concentration >=500ng/ of ATP described in the evaluation method Ml is that immunity is extraordinary.

Specific to other specific embodiments of the invention, the concentration ＜ of 150ng/ml≤ATP in the evaluation method 500ng/ml is that immunity is normal.

Fluorescence method is the experimental technique of the most frequently used measure ATP, and principle is according to firefly luciferase (firefly Luciferase need ATP to provide energy when) oxyluciferin produces fluorescence to develop.When firefly luciferase and fluorescence When element is all excessive, the generation of fluorescence and the concentration of ATP are directly proportional in certain concentration range.Can thus examine with sensitivity The ATP concentration surveyed in solution.Concrete principle is as follows：

It is used to evaluate the purposes of immunocompetence as biomarker the invention provides ATP.The present invention is not required to taking office Therefore the material of what isotope marks overcomes the defect that prior art is unfavorable for safety operation；When the cell of separation does not need long Between cultivate and overcome prior art detection cycle shortcoming long detecting；Cell need to only crack can quick detection, overcome existing There is the deficiency that technical operation is cumbersome, and influenceed small by experiment condition.Present invention also offers non-diseases diagnostic purpose immunocompetence Evaluation method, safety easy to operate, sensitiveness is good, and accuracy is high, can detect wide ranges, as a result can more accurately obtain machine The immune state of body.This method can detect the ATP concentration of specific cells, it is to avoid the vacation sun or false that prior art is likely to result in Feminine gender, improves the accuracy of measure；This method is analyzed by the immune state to cell integral level, it is adaptable to HIV Patient or the low person of immunocompetence.

Brief description of the drawings

In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing The accompanying drawing to be used needed for having technology description is briefly described.

Fig. 1 shows sample statistics figure in embodiment 1；Wherein abscissa H shows normal person, and D shows immunocompromised person；Ordinate shows The concentration of ATP, unit is ng/mL；

Fig. 2 shows sample statistics figure in embodiment 1；Wherein circle shows the respective value of each sample；Abscissa H shows normal person, D Show immunocompromised person；Ordinate shows the concentration of ATP, and unit is ng/mL；

Fig. 3 shows the detectable scope of ATP and sensitivity in solution to be measured in embodiment 1；Wherein, ordinate is relatively glimmering Light unit；Abscissa for ATP concentration, unit for μM；It can be seen that, detection range is 0.01-10uM, and accuracy is high, can detect model Enclose wide；

Fig. 4 shows that embodiment 2 detects the testing result of sample；

Fig. 5 shows that embodiment 3 detects the testing result of sample；

Fig. 6 shows the testing result of comparative example.

Specific embodiment

It is used to evaluate the purposes and evaluation method of immunocompetence, this area as biomarker the invention discloses ATP Technical staff can use for reference present disclosure, be suitably modified technological parameter realization.In particular, all similar replacements Apparent to those skilled in the art with changing, they are considered as being included in the present invention.The method of the present invention And application is described by preferred embodiment, related personnel can substantially not depart from present invention, spirit and model Enclose interior be modified to method described herein and application or suitably change is realized and apply the technology of the present invention with combining.

The ATP that the present invention is provided is used for examination used in the purposes and evaluation method for evaluate immunocompetence as biomarker Agent can be bought by market.Wherein, ATP standard items (Guangzhou this good), ATP detects liquid (sigma companies), RPMI1640 (gibco Company), Ficoll (sigma companies), PHA (sigma companies), TRIS (sigma), EDTA (sigma).

With reference to embodiment, the present invention is expanded on further：

Embodiment 1

Experimental group：

Part I：(i) separate and obtain cell (separation of Ficoll methods)

1. a 15ml cones bottom pipe is taken, 6-9ml PBS or RPMI1640 is added, 3-6ml blood samples are added, mixed.

2. it is another to take a new 15ml cones bottom pipe, 3-6ml Ficoll separating liquids are added, carefully the blood sample of dilution is being added To Ficoll upper stratas, make layering clear.

3., 20 to 40min is centrifuged, Brake is tuned into 0 by≤1000 DEG C, 25 DEG C.

4. upper plasma is transferred in a new 1.8ml cryopreservation tube, -80 DEG C of preservations；Blood plasma to white is suctioned out to be layered On (i.e. mononuclear cell layer) at about 0.5-1cm.

5. PBMCs to a new 1.5ml centrifuge tube is carefully shifted；≤ 1000g, is centrifuged 4-10min by 20 DEG C -25 DEG C.Plus Enter PBS to cumulative volume be 1ml re-suspended cells.

6. change PBS and repeat this step washing 3 times.Trypan Blue 1-5min numbers cell quantity and survival rate.

(ii) paramagnetic particle method separates cell (by taking CD4 magnetic beads as an example)

1.EDTA or ACD heparin tubes gather venous blood.

2. 125ul whole bloods are taken, is diluted with 350ulPBS, add 25ulCD4beads.

3. room temperature mixes 10min, is placed in and 2min is adsorbed on magnetic sheet.

4. supernatant is abandoned, is washed three times with 300ulPBS.

Part II stimulates cell detection ATP values

7. the cell of acquisition is divided into 4 parts, cell concentration is 1-3 × 10⁵Per hole：It is set to blank (-), 5- 15ug/ml PHA, relevant disease specific antigen (such as tuberculosis ESAT6/CFP-10 5-15ug/ml) stimulates 16-24h, It is placed in 37 degree of 5%CO2 cultures.

8. overnight 20 DEG C -25 DEG C, 5-10min is centrifuged afterwards by cell≤800g, adds 200ul lysates fully to crack carefully Born of the same parents (may be selected：4 DEG C, 12000g is centrifuged, 5-10min takes supernatant).

9. it is immediately placed on to be measured on ice.

10. standard items are prepared：Stand-by reagent is dissolved on ice, ATP standard liquids is diluted to ATP lysates appropriate dense Degree gradient.Specific concentration need to be adjusted according to the concentration of ATP in sample.

11. plus 100ulATP detects liquid to 96 holes.Room temperature is placed 3-5 minutes, so that the ATP of background is all consumed Fall, so as to reduce background.

12. add 10-100ul samples or standard items, are mixed with rifle (micropipettor) rapidly, after being at least spaced 2 seconds, stand Determined with ELIASA 562nm.The stand-by period is 2 seconds before detection, and detection time is 10 seconds.

13. are analyzed according to ATP contents.

The third part immunocompetence

Samples sources：Guangzhou chest hospital.Wherein, to have determined as immunity extraordinary for sample 1-2, and sample 3-59 determines For immunity is normal, sample 60-71 is defined as hypoimmunity.ATP Concentration Testings the results are shown in Table 1.

The data statistics figure of above-mentioned sample is shown in Fig. 1, Fig. 2, wherein, H:Normal person, D：Have been determined as immunocompromised person.

As shown in Table 1, the concentration >=500ng/ml of concentration the ＜ 150ng/ml or ATP of ATP is immunocompetence exception；Its In, the concentration ＜ 150ng/ml of ATP are hypoimmunity；Concentration >=the 500ng/ml of ATP is that immunity is extraordinary；150ng/ml≤ The concentration ＜ 500ng/ml of ATP are that immunity is normal.

Knowable to the above analysis, can be with the power of adaptive immune ability by the concentration for detecting ATP.

ATP is detected：

1. standard items preparation is carried out on ice：Dissolving ATP standard stocks liquid (10umol/L), molten ATP standards on ice Liquid ATP lysates are diluted to 0,0.01,0.025,0.05,0.075,0.1,0.25,0.5,1.0,10.0umol/L concentration ladder Degree.ATP concentration of standard solution can be adjusted according to the concentration of ATP in sample.

2. add 10ul samples or standard items in 96 hole White-opalescent ELISA Plates on ice, be placed in ELIASA and select LU, end-point method, 562nm detects the fluorescent value of its background.

3. plus 100ulATP detects that liquid, in the 96 hole White-opalescent ELISA Plates containing sample or standard items, uses rapidly the volley of rifle fire Mix, select LU, end-point method, 562nm to determine (setting in advance) with ELIASA immediately.

4. the concentration of ATP in sample is calculated according to standard curve.

Testing result is shown in Fig. 3.From the figure 3, it may be seen that detectable scope is 0.01-10uM, and the cell or tissue of routine is cracked The concentration of ATP is only 0.1-1 μm of ol/L in liquid.Therefore, testing accuracy of the invention is high, and sensitivity is good, can detect scope wide.

Embodiment 2

Take 1 normal person and 7 TB patient whole bloods 6ml, Ficoll methods separate PBMC, with 200ul Lysis cell lysis, Detect the ATP concentration (ordinate) of sample, adaptive immune ability.

Method：

1. PBMC is separated

1. blood (room temperature is placed and is no more than 8 hours) is gathered with anticoagulant heparin pipe, separates front upper and lower reverse mixing 8 times, made Cell distribution is uniform.

2. a 15ml centrifuge tube is taken, 5ml RPMI1640 (without FBS) is added, is added with pasteur pipet isometric (RPMI1640：Blood is 1:1) blood sample, mixing 8 times of lightly turning upside down, reaches color homogeneous.

3. a new 15ml centrifuge tube is separately taken, 5ml Ficoll separating liquid (separating liquid and blood is added with electronic liquid-transfering gun Sample volume ratio is 1:2) centrifuge tube, is inclined into certain angle, the blood sample of dilution is added to Ficoll upper stratas with pasteur pipet is careful, Make layering clear.

4. it is 800g by centrifuge condition setting, 20 DEG C, acce, Brake is set to 0, and 30min is centrifuged.

5. blood plasma to white is discarded with pasteur pipet after being centrifuged to be layered on (i.e. mononuclearcell layer) at about 0.5-1cm i.e. Can, it is sure not agitation tunica albuginea layer.

6. tunica albuginea layer to a new 1.5ml centrifuge tube is carefully shifted with pasteur pipet again (sucking-off volume is 1.5ml)；

7. it is 5000rpm by centrifuge condition setting, 20 DEG C, 10min, liquid juxtaposition in evacuation centrifuge tube after centrifugation is centrifuged In on the blotting paper of sterilizing, the cell being attached on wall is only stayed, it is 1.5ml to cumulative volume to add RPMI (without FBS), gently blows and beats weight Outstanding cell.

8. it is 3000rpm by centrifuge condition setting, 20 DEG C, 10min, liquid juxtaposition in evacuation centrifuge tube after centrifugation is centrifuged In on the blotting paper of sterilizing, the cell being attached on wall is only stayed, be subsequently adding 1.5ml RPMI (without FBS), gently blown and beaten resuspended thin Born of the same parents.

9. centrifuge condition setting is 400g by third time, 20 DEG C, 10min, liquid in evacuation centrifuge tube after centrifugation is centrifuged It is placed on blotting paper, only stays the cell being attached on wall, adds 1ml RPMI (without FBS) culture medium gently to blow and beat re-suspended cell. 10ul is taken for Trypan Blue, survival rate is counted and calculate (concrete operations refer to cell count).

2.ATP is detected

1. the PBMC cell per wells 300,000 for separating counting as stated above are taken, are added in 1.5ml centrifuge tubes, 800g, 20 DEG C, 7min is centrifuged, is placed on blotting paper after liquid in evacuation centrifuge tube after centrifugation and inhaled, add 200ul lysates (Tris-EDTA：20mmol/L Tris+2mmol/LEDTA, pH7.75) in abundant cell lysis 5min on ice, it is subsequently placed in 1min is boiled in 100 degree of water-baths, is immediately placed on to be measured on ice.

2. standard items preparation is carried out on ice：Dissolving ATP standard stocks liquid (10umol/L), molten ATP standards on ice Liquid is diluted to 0,0.01,0.05,0.1,0.5,1.0,5.0,10.0umol/L concentration gradients with ATP lysates.ATP standard liquids Concentration can be adjusted according to the concentration of ATP in sample.

3. add 10ul samples or standard items in 96 hole White-opalescent ELISA Plates on ice, be placed in ELIASA and select LU, end-point method, 562nm detects the fluorescent value of its background.

4. plus 100ulATP detects that liquid, in the 96 hole White-opalescent ELISA Plates containing sample or standard items, uses rapidly the volley of rifle fire Mix, select LU, end-point method, 562nm to determine (setting in advance) with ELIASA immediately.

The concentration of ATP in sample is calculated according to standard curve.

As shown in Figure 4：TB patient immune functions are less than normal person, and patient 4,5,6 belongs to hypoimmunity.

It can be seen that, there is feasibility and accuracy come the power of adaptive immune ability by detecting the concentration of ATP.

Embodiment 3

Take 1 normal person and 6 TB (pulmonary tuberculosis) patient whole blood 6ml, Ficoll methods separate PBMC, use 200ul Lysis Cell lysis, detect the ATP concentration (ordinate) of sample, adaptive immune ability.Method is with embodiment 2.

As shown in Figure 5：TB patient immune functions are less than normal person, and patient 1,5 belongs to hypoimmunity, and patient 4 is by anti- Leave hospital after tuberculosis therapy, immunologic function recovers normal, belongs to immunity normal.

It can be seen that, there is feasibility and accuracy come the power of adaptive immune ability by detecting the concentration of ATP.

Comparative example

The isolated T cell of whole blood, 20h is stimulated with 5ug/mlPHA, detection cell factor (such as IFN-γ, IL-2 etc.) Level, to reflect the function of T cell.

Method：Take 1 normal person (normal person's sample 5 in embodiment 1) whole blood 6ml, Ficoll method and separate PBMC, use 5ug/ After mlPHA stimulates 20h, 1200g, 10min centrifuge cell, careful adherent absorption supernatant, ELISA detections.

ELISA steps：

A) it is coated with：100ul R1 (coated antibody) is added to arrive ELISA Plate bottom per hole.4 DEG C, 200rpm shaken over night.

B) wash：Add 200ul R2 (cleaning solution) per hole, be stored at room temperature 1min, outwell and pat dry, wash 3 times.

C) close：200ul R3 (confining liquid), room temperature, 200rpm shake closings 1h are added per hole.

D) wash：Add 200ul R2 (cleaning solution) per hole, be stored at room temperature 1min, outwell and pat dry, wash 3 times.

E) dry：Remaining unspent ELIAS strip air-dries 45min in fume hood, then seals, and 4 DEG C preserve one week left side It is right.

F) it is loaded：Sample, standard items dilute (50ul+50ul) with R3.100ul is in ELISA Plate for addition, room temperature, 200rpm Shake is incubated 2h.

G) wash：Add 200ul R2 (cleaning solution) per hole, be stored at room temperature 1min, outwell and pat dry, wash 5 times.

H) secondary antibody is incubated：100ul R4 are added per hole, room temperature, 200rpm shakes are incubated 1h.

I) wash：Add 200ul R2 (cleaning solution) per hole, be stored at room temperature 1min, outwell and pat dry, wash 5 times.

J) develop the color：Add 100ul bottom R5 (substrate nitrite ion), lucifuge colour developing 15min.

K) ELIASA setting：ABS end-point methods detect 450nm light absorption values, and 630nm is reference wavelength.

L) terminate：OD450 wavelength is surveyed in 50ul terminate liquids R6 (1M sulfuric acid), 5min, 630nm is reference wavelength.

Standard curve is as shown in Figure 6：After PHA stimulates 20h, T cell activation is can detect, but the method is experimental period long, But the IFN-γ content of the method detection is low (pik rank), and detection range is narrow, accuracy reduction, it is impossible to which reaction T is thin well Born of the same parents' immunocompetence, and experimental period is long, complex operation is cumbersome.

The results are shown in Table 2.

The testing result of table 2

Group	IFN-γ content (pg/mL)
		Do not stimulate	42.24
PHA stimulates	226.18

As seen from the results in Table 1, after PHA stimulates 20h, IFN-γ content significantly rises, and illustrates T cell activation.But IFN-γ Content is low (pik rank), it is impossible to embody immunocompetence well.

The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should It is considered as protection scope of the present invention.

Claims (1)

1. a kind of evaluation method of non-diseases diagnostic purpose immunocompetence, it is characterised in that separate PBMC cells, determines basis ATP, comprises the following steps：

（1）Blood is gathered with anticoagulant heparin pipe, front upper and lower reverse mixing 8 times is separated, makes cell distribution uniform；

（2）A 15ml centrifuge tube is taken, RPMI1640s of the 5ml without FBS is added, RPMI1640 and blood are added with pasteur pipet Volume ratio is 1:1 blood sample, mixing 8 times of lightly turning upside down, reaches color homogeneous；

（3）A new 15ml centrifuge tube separately is taken, 5ml Ficoll separating liquids are added with electronic liquid-transfering gun, make separating liquid and blood sample Volume ratio is 1:2, centrifuge tube is inclined into certain angle, the blood sample of dilution is added to Ficoll upper stratas with pasteur pipet is careful, make Layering is clear；

（4）It is 800g by centrifuge condition setting, 20 DEG C, acce, Brake is set to 0, and 30min is centrifuged；

（5）Blood plasma is discarded with pasteur pipet at about 0.5-1cm, be sure not agitation tunica albuginea layer to white layering after centrifugation；

（6）Tunica albuginea layer to a new 1.5ml centrifuge tube is carefully shifted with pasteur pipet again, sucking-off volume is 1.5ml；

（7）It is 5000rpm by centrifuge condition setting, 20 DEG C, 10min is centrifuged, liquid is placed in evacuation centrifuge tube after centrifugation On the blotting paper of sterilizing, the cell being attached on wall is only stayed, it is 1.5ml to cumulative volume to add the RPMI without FBS, gently blows and beats weight Outstanding cell；

（8）It is 3000rpm by centrifuge condition setting, 20 DEG C, 10min is centrifuged, liquid is placed in evacuation centrifuge tube after centrifugation On the blotting paper of sterilizing, the cell being attached on wall is only stayed, be subsequently adding RPMIs of the 1.5ml without FBS, gently blow and beat re-suspended cell；

（9）Centrifuge condition setting is 400g by third time, 20 DEG C, 10min, liquid juxtaposition in evacuation centrifuge tube after centrifugation is centrifuged In the cell being attached on wall on blotting paper, is only stayed, RPMI culture mediums of the 1ml without FBS is added gently to blow and beat re-suspended cell；Take 10ul is used for Trypan Blue, counts and calculate survival rate；

（10）The PBMC cell per wells 300,000 for separating counting as stated above are taken, is added in 1.5ml centrifuge tubes, 800g, 20 DEG C, 7min is centrifuged, it is placed on blotting paper after liquid in evacuation centrifuge tube after centrifugation and is inhaled, 200ul lysates are added, on ice Abundant cell lysis 5min, is subsequently placed in 100 degree of water-baths and boils 1min, is immediately placed on to be measured on ice；Wherein, lysate by 20 mmol/L Tris and 2 mmol/L EDTA are constituted, and pH is 7.75；

（11）Standard items preparation is carried out on ice：10umol/L ATP standard stock liquid is dissolved on ice, and ATP standard liquids are used ATP lysates are diluted to 0,0.01,0.05,0.1,0.5,1.0,5.0,10.0 umol/L concentration gradients；ATP standard liquids are dense Degree can be adjusted according to the concentration of ATP in sample；

（12）Add 10ul samples or standard items in 96 hole White-opalescent ELISA Plates on ice, be placed in ELIASA and select LU, end-point method, 562nm detects the fluorescent value of its background；

（13）It is mixed with the volley of rifle fire rapidly plus 100ulATP detects liquid in the 96 hole White-opalescent ELISA Plates containing sample or standard items It is even, select LU, end-point method, 562nm to determine with ELIASA immediately；

（14）It is analyzed according to ATP contents：Concentration >=the 500ng/ml of concentration the ＜ 150ng/ml or ATP of ATP is immunity energy Power exception；Wherein, the concentration ＜ 150ng/ml of ATP are hypoimmunity；Concentration >=the 500ng/ml of ATP is that immunity is extraordinary； The concentration ＜ 500ng/ml of 150ng/ml≤ATP are that immunity is normal.