CN104965077A - Pepsinogen I enzymatic chemiluminescence detection kit - Google Patents

Abstract

The invention discloses a pepsinogen I enzymatic chemiluminescence detection kit, and belongs to the technical field of immunodetection assay. The kit is composed of an enzyme labeling solution, a pepsinogen I standard substance, an anti-pepsinogen I monoclonal antibody coated immune magnetic bead, a sample diluting solution, a chemiluminescence substrate solution and a washing solution. The principle of the kit is characterized in that a pepsinogen I antibody is connected to the surface of the magnetic bead as a solid phase reagent, and a solid phase-antibody-antigen-enzyme labeled antibody sandwich immune compound is formed after pepsinogen I in a sample is captured and an enzyme-labeled anti-pepsinogen I monoclonal antibody reagent is added. The kit prepared by combining a chemiluminescence technology with an immune magnetic bead technology has the advantages of high sensitivity, good specificity, wide linear range, good stability, and meeting of clinic demands of stomach function detection.

Description

A kind of pepsinogen Cgene enzyme-catalyzed chemical luminescence detection kit

Technical field

The invention belongs to technical field of immune assay, particularly relate to a kind of pepsinogen Cgene enzyme-catalyzed chemical luminescence detection kit.

Background technology

This test is for diagnosing severe atrophy body of stomach gastritis, and prompting early carcinoma of stomach risk.Serum or blood plasma PGI (P-PGI, S-PGI) detection method detect a kind of reliable method of severe atrophy body of stomach gastritis, and its Sensitivity and Specificity is respectively 92% and 90%.

Propepsin (Pesinogen, PG) is pepsic inactive precursor in gastric juice, by chief cell and the mucilage cell secretion of body of stomach.Major part is secreted into gastral cavity, can be detected in blood on a small quantity.The quantity of PGI level and body of stomach mucous membrane chief cell has reliable correlativity.Serum PG level reflects the morphology and function of different parts stomach lining: PG I is the index detecting oxyntic gland cell function, and gastric acid secretion increases PG I rising, and secretion reduces or stomach lining body of gland atrophy PG I reduces; The correlativity comparatively large (relative to antral gastric mucosa) of PG II and non-cancer lesion at the bottom of stomach, its raise with fundus gland shrink tube, gastric metaplasia or Pseudopyloric gland metaplasia, special-shaped increment are relevant: PG I/II ratio Progressive symmetric erythrokeratodermia reduces and is in progress relevant to Mucosal atrophy.Therefore, simultaneous determination PG I and PG II ratio can play the effect of fundus gland mucous membrane " serum biopsy survey ".

Chemiluminescence rise in eighties of last century be the eighties continue Enzyme-multiplied immune technique and the emerging technology that grows up after putting immune technology, due to its high sensitivity, high specific, while method is easy, quick, mark bond is stablized, without features such as emitting isotope damage and pollutions, obtaining develop rapidly in recent years.Chemiluminescent principle is the excited state intermediate of the instability generated in chemical reaction, when it gets back to ground state, and release photon.In immune detection is analyzed, use chemiluminescence, sensing range can be made to reach 6 orders of magnitude, and sensitivity is very high, add that label is stablized, the term of validity is long, makes it receive increasing concern and application.Magnetic particle separation enzyme-linked immunoassay technology is a kind of is solid phase carrier of separating with magnetic particle, immune magnetic particle isolation technics is combined with Enzyme-multiplied immune technique and a kind of Novel immune detection method of setting up.Conventional ELISA method, antigen, the association reaction of antibody carries out on solid phase (elisa plate reacting hole) surface, and Magneto separate enzyme-linked immune detection method, the association reaction of antigen, antibody also carries out under the condition of approximate liquid phase, and thus reaction fast, thoroughly.There is the advantage that automaticity is high, highly sensitive, the detection used time is few compared with traditional E LISA.But detect PG I kit in the market mainly based on traditional E LISA method.

Summary of the invention

The technical issues that need to address of the present invention are to provide a kind of pepsinogen Cgene enzyme-catalyzed chemical luminescence detection kit, make to achieve robotization, high specific and sensitivity to human pepsinogen I detection.

A kind of pepsinogen Cgene enzyme-catalyzed chemical luminescence detection kit, it is characterized in that, this kit comprises enzyme mark liquid, pepsinogen Cgene standard items, wraps by the immunomagnetic beads of pepsinogen Cgene monoclonal antibody, Sample dilution, Chemoluminescent substrate, cleansing solution.

Described a kind of pepsinogen Cgene enzyme-catalyzed chemical luminescence detection kit, it is characterized in that, pepsinogen Cgene monoclonal antibody is coated on magnetic bead microsphere surface, through catching pepsinogen Cgene in sample and after adding enzyme mark pepsinogen Cgene monoclonal antibody reagent, forming solid phase-antibody-antigene-enzyme labelled antibody sandwich immunoassay compound.

Described a kind of pepsinogen Cgene enzyme-catalyzed chemical luminescence detection kit, is characterized in that, described bag is the magnetic microsphere containing being marked with former I monoclonal antibody of antipepsin by the immunomagnetic beads of pepsinogen Cgene monoclonal antibody;

In described enzyme mark liquid, the enzyme of enzyme labelled antibody is horseradish peroxidase;

Described cleansing solution is the damping fluid containing Tween-20;

Described chemical luminous substrate solution is enzyme-catalyzed chemical luminescence substrate solution.

Let alone the kit described in, it is characterized in that, described bag is obtained as follows by the immunomagnetic beads of pepsinogen Cgene monoclonal antibody:

(1) preparation of first wash buffer:

Take the MES hygrate of 0.1952g in the pure water of 100ml, adjustment pH to 5.0;

Add 50 μ l Tween-20s, be made into 0.01mol/L MES damping fluid, adjustment pH to 5.0;

(2) preparation of coupling buffer:

Take 0.38138g Na ₂b ₄o ₇10H ₂o is dissolved in 40ml pure water, obtains the borax soln of 0.05mol/L;

Take 0.1237g H ₃bO ₃be dissolved in 10ml pure water, obtain the BAS of 0.2mol/L;

Get the Na of 18ml 0.05mol/L ₂b ₄o ₇the H of solution and 2ml 0.2mol/L ₃bO ₃solution mixes, and is settled to 80ml and obtains 0.05mol/L borate buffer;

(3) preparation of whole washing lotion:

Take 2.78g Na ₂hPO ₄be dissolved in 100ml pure water and obtain 0.2mol/L Na ₂hPO ₄solution;

Take 1.2g NaH ₂pO ₄be dissolved in 50ml pure water and obtain 0.2mol/L NaH ₂pO ₄solution;

Get 81ml 0.2mol/LNa ₂hPO ₄solution and 19ml 0.2mol/L NaH ₂pO ₄solution mixes, and obtains 0.2mol/L PB buffer solution and obtains 0.2mol/LPB buffer solution;

Get 50ml 0.2mol/L PB damping fluid, 2 times are diluted to 100ml, add 0.9gNaCl, and the BSA of 0.5g0.5%-1%, 0.02-0.03% biological preservative, 0.05% polysorbas20, fully dissolve mixing;

(4) preparation of confining liquid:

Get 100ml Tris damping fluid, the pH of solution is adjusted between 8.5-9.0;

Add 2%BSA wherein;

(5) preparation of EDC solution and NHS solution:

Take 1-(3-the dimethylamino-propyl)-3-ethyl carbodiimide of 25mg/ml, be dissolved in the first wash buffer of 1ml, be mixed with the EDC solution of 25mg/ml;

The N-hydroxy-succinamide taking 25mg/ml is dissolved in first wash buffer, is configured to 25mg/ml NHS solution;

(6) magnetic bead bag is by process:

A, previous cleaning: get 100 μ l magnetic beads, be diluted to the magnetic bead concentration of 10mg/ml, therefrom draw 100 μ l; Magneto separate frame is separated, removes supernatant; Wash three times, Magneto separate with first washing lotion 250 μ l, remove supernatant;

B, activation: get the magnetic bead that previous cleaning completes and add 150 μ lMES, then add the NHS solution of 50 μ l EDC and 50 μ l, fully mix.The lower 25 DEG C of activation 30min of room temperature, have activated Magneto separate and have removed supernatant, with the resuspended washing of 250 μ l coupling buffer three times, divided supernatant of leaving away;

C, dilute anti-G-17 monoclonal antibody:

Anti-G-17 monoclonal antibody taken out from-20 DEG C of refrigerators, after it melts, take out 20 μ l, be diluted to 400 μ l with coupling buffer 1:20, the antibody concentration after dilution is 0.0397mg/ml.

D, coupling:

Add the magnetic bead after the resuspended activation of anti-G-17 monoclonal antibody of 400 μ l coupling buffer dilutions, fully mix, its magnetic bead bag is 1mg magnetic bead by ratio: 0.0159mg antibody;

37 DEG C of coupling 2h, 37 DEG C of shaking tables rock constant temperature blending;

E, close:

Magnetic bead coupling terminated is separated on Magneto separate frame, removes supernatant;

Add 400 μ l confining liquids, 1h closed by 37 DEG C of shaking tables;

Close after terminating and remove supernatant;

F, wash preservation eventually:

With the magnetic bead that the whole wash liquid of 400 μ l has been closed, in triplicate, final constant volume is to 500 μ l.

Principle of work of the present invention is a kind of detection method that double antibody sandwich method chemiluminescence combines with immunomagnetic bead technique.In sample, add quantitative bag marked anti-PG I monoclonal antibody by the immunomagnetic beads of pepsinogen Cgene monoclonal antibody and HRP.37 DEG C hatch after, wrap by the immunomagnetic beads of pepsinogen Cgene monoclonal antibody and HRP mark anti-PG I monoclonal antibody respectively in sample the different epi-positions of PG I molecule be combined, form magnetic bead-antibody-antigen-antibody compound.Direct precipitation in externally-applied magnetic field, namely separable.Abandoning supernatant, the compound of washing and precipitating, then adds enzyme-catalyzed chemical luminescence substrate.Substrate is catalytic pyrolysis under the effect of enzyme, form unstable excited state intermediate, just have issued photon when excited state intermediate gets back to ground state, form luminescence-producing reaction, the luminous intensity of light-emitting appearance detection reaction can be used, PG I content in sample can be calculated according to typical curve.In sensing range, luminous intensity is directly proportional to PG I concentration in sample.

The invention is characterized in: the method detecting pepsinogen Cgene in serum employs following reagent:

The present invention use enzyme mark liquid for HRP marks anti-PG I monoclonal antibody

Pepsinogen Cgene standard items of the present invention are the BSA protein solutions containing a certain amount of PG I antigen.

Sample dilution contained by the present invention is the PBS solution containing 1%BSA.

Chemoluminescent substrate of the present invention is enzyme-catalyzed chemical luminescence substrate solution.

Cleansing solution of the present invention is the damping fluid containing Tween-20 and ProCline-300.

Pepsinogen Cgene enzyme-catalyzed chemical luminescence detection kit method of operating of the present invention is as follows:

One, magnetic bead bag is buffered liquid preparation:

1. the preparation of first wash buffer:

1.1 take the MES hygrate of 0.1952g in the pure water of 100ml, adjustment pH to 5.0.

1.2 add 50 μ l Tween-20s, are made into 0.01mol/L MES damping fluid, adjustment pH to 5.0.

2. the preparation of coupling buffer:

2.1 take 0.38138g Na ₂b ₄o ₇10H ₂o is dissolved in 40ml pure water, obtains the borax soln of 0.05mol/L.

2.2 take 0.1237g H ₃bO ₃be dissolved in 10ml pure water, obtain the BAS of 0.2mol/L.

2.3 Na getting 18ml 0.05mol/L ₂b ₄o ₇the H of solution and 2ml 0.2mol/L ₃bO ₃solution mixes, and is settled to 80ml and obtains 0.05mol/L borate buffer.

3. the preparation of whole washing lotion:

3.1 take 2.78g Na ₂hPO ₄be dissolved in 100ml pure water and obtain 0.2mol/L Na ₂hPO ₄solution.

3.2 take 1.2g NaH ₂pO ₄be dissolved in 50ml pure water and obtain 0.2mol/L NaH ₂pO ₄solution.

3.3 get 81ml 0.2mol/LNa ₂hPO ₄solution and 19ml 0.2mol/L NaH ₂pO ₄solution mixes, and obtains 0.2mol/L PB buffer solution and obtains 0.2mol/LPB buffer solution.

3.3 get 50ml 0.2mol/L PB damping fluid, and 2 times are diluted to 100ml, add 0.9gNaCl, and 0.5gBSA (0.5%-1% adjustment) 0.02-0.03% biological preservative, 0.05% Tween-20, fully dissolve mixing.

4. the preparation of confining liquid:

4.1 get 100ml Tris damping fluid, during the pH of solution is adjusted to 8.5-9.0.

4.2 add 2%BSA to it.

The preparation of 5.EDC solution and NHS solution:

5.1 1-(3-the dimethylamino-propyl)-3-ethyl carbodiimide taking 25mg/ml, are dissolved in the first wash buffer of 1ml, are mixed with the EDC solution of 25mg/ml.

5.2 N-hydroxy-succinamides taking 25mg/ml are dissolved in first wash buffer, are configured to 25mg/mlNHS solution.

Two, magnetic bead bag is by process

1. previous cleaning:

1.1 get 100 μ l magnetic beads, are diluted to the magnetic bead concentration of 10mg/ml, therefrom draw 100 μ l.

1.2 Magneto separate framves are separated, remove supernatant.

1.3 wash three times, Magneto separate with first washing lotion 250 μ L, remove supernatant.

2. activate:

2.1 get the magnetic bead that previous cleaning completes adds 150 μ LMES, then adds the NHS solution of 50 μ L EDC and 50 μ L, fully mixes.The lower 25 DEG C of activation 30min of room temperature.

2.2 have activated Magneto separate removes supernatant, with the resuspended washing of 250 μ L coupling buffer three times, divides supernatant of leaving away.

3. dilute anti-PG I monoclonal antibody:

Anti-PG I monoclonal antibody is taken out from-20 DEG C of refrigerators, after it melts, takes out 40 μ L, be diluted to 400 μ L with coupling buffer 1:10.

4. coupling:

4.1 add the complete magnetic bead of the resuspended activation of anti-PG I monoclonal antibody of 0.077mg/ml concentration of 400 μ L coupling buffers dilutions, fully mix.Magnetic bead bag is that 1mg magnetic bead bag is by 0.0308mg antibody by ratio.

4.237 DEG C of coupling 2h, 37 DEG C of shaking tables rock constant temperature blending.

5. close:

5.1 magnetic beads coupling terminated are separated on Magneto separate frame, remove supernatant.

5.2 add 400 μ L confining liquids, and 1h closed by 37 DEG C of shaking tables.

Supernatant is removed after 5.3 closed end.

6. wash preservation eventually:

6.1 magnetic beads closed with the whole wash liquid of 400 μ L, in triplicate.

6.3 final constant volumes are to 500 μ L.

Three, Sample dilution preparation

NaH is added in purified water ₂pO ₄, Na ₂hPO ₄, preparation 100ml phosphate buffer, add bovine serum albumin, Tween-20, ProClin 300, orchil, regulate pH value to 7.4-7.6.

Four, the preparation of pepsinogen Cgene standard items

Preparing 3 bottles of PG I standard items, is antiseptic containing 0.1%ProCline 300.The concentration often criticizing PG I titer in kit is 25 μ g/L, 100 μ g/L, 200 μ g/L.

Five, the preparation of enzyme mark liquid

Preparation 15mL HRP marks anti-PG I monoclonal antibody, is kept at 0.02%methylosothiazolone, in 0.02%bromonitrodioxne stabilizing agent and 0.002%active isothiazoloneization antiseptic.

Six, the preparation of cleansing solution

The concentrated phosphoric acid salt buffer of preparation 20ml (using after 20 times of dilutions) is antiseptic containing polysorbas20 and 0.1%ProCline-300.

Seven, the configuration of substrate solution

1. prepare substrate buffer solution

1.1 take 4.542g Tris, take 0.458g sodium borate, and constant volume, to 300ml, regulates pH to 8.7-9.0.

2. prepare substrate A liquid

2.1 take 0.023g carbamide peroxide, take 0.024g 4-Morpholinopyridine (stabilizing agent).

2.2 add sodium borate buffer liquid constant volume to 100ml with Tris-HCl, room temperature preservation.

3. prepare substrate B liquid

3.1 take 0.088g luminol, take 0.051g 3-(10-phenothiazinyl) propane-1-sulfonate (reinforcing agent).

3.2 add sodium borate buffer liquid constant volume to 100ml with Tris-HCl.

The B liquid prepared wraps up with masking foil by 3.3, room temperature preservation.

Eight, kit provided by the invention, comprises following component:

Reagent name	Loading amount	Use-pattern
			Bag is by the immunomagnetic beads of pepsinogen Cgene monoclonal antibody	3ml	Direct use
Cleansing solution	20ml	Use after 20 times of dilutions
			Sample dilution	100ml	Direct use
Pepsinogen Cgene standard items	1.5ml, 3 bottles	Direct use
			Enzyme mark liquid	15ml	Direct use
Chemoluminescent substrate	15ml	Direct use

The present invention has following innovation:

1. the invention provides a kind of pepsinogen Cgene chemiluminescence detection kit, can be used for atrophic gastritis diagnosis and early stage gastric cancer screening.

2. the capture antibody used and enzyme labelled antibody are monoclonal antibody, and be that the affinity of reaction is higher, non-specific adsorption reduces, and the production differences between batches of monoclonal antibody are relatively little, more easily ensure product batch between stable.

Accompanying drawing explanation

The Trendline of the values of chemiluminescence that Fig. 1: 1mg magnetic bead bag is obtained by PG I antigen for variable concentrations after 0.0308mgPG I monoclonal antibody.

Embodiment,

The first step: wrap by the preparation of the immunomagnetic beads of pepsinogen Cgene monoclonal antibody

One, the preparation of magnetic bead activation buffer:

1. the preparation of first wash buffer:

1.1 take the MES hygrate of 0.1952g in the pure water of 100ml, adjustment pH to 5.0.

1.2 add 50 μ l Tween-20s, are made into 0.01mol/L MES damping fluid, adjustment pH to 5.0.

2. the preparation of coupling buffer:

2.1 take 0.38138g Na ₂b ₄o ₇10H ₂o is dissolved in 40ml pure water, obtains the borax soln of 0.05mol/L.

2.2 take 0.1237g H ₃bO ₃be dissolved in 10ml pure water, obtain the BAS of 0.2mol/L.

2.3 Na getting 18ml 0.05mol/L ₂b ₄o ₇the H of solution and 2ml 0.2mol/L ₃bO ₃solution mixes, and is settled to 80ml and obtains 0.05mol/L borate buffer.

3. the preparation of whole washing lotion:

3.1 take 2.78g Na ₂hPO ₄be dissolved in 100ml pure water and obtain 0.2mol/L Na ₂hPO ₄solution.

3.2 take 1.2g NaH ₂pO ₄be dissolved in 50ml pure water and obtain 0.2mol/L NaH ₂pO ₄solution.

3.3 get 81ml 0.2mol/LNa ₂hPO ₄solution and 19ml 0.2mol/L NaH ₂pO ₄solution mixes, and obtains 0.2mol/L PB buffer solution and obtains 0.2mol/LPB buffer solution.

3.3 get 50ml 0.2mol/L PB damping fluid, and 2 times are diluted to 100ml, add 0.9gNaCl, and the BSA of 0.5g0.5%-1%, 0.02-0.03% biological preservative, 0.05% polysorbas20, fully dissolve mixing.

4. the preparation of confining liquid:

4.1 get 100ml Tris damping fluid, during the pH of solution is adjusted to 8.5-9.0.

4.2 add 2%BSA to it.

The preparation of 5.EDC solution and NHS solution.

5.1 1-(3-the dimethylamino-propyl)-3-ethyl carbodiimide taking 25mg/ml, are dissolved in the first wash buffer of 1ml, are mixed with the EDC solution of 25mg/ml.

5.2 N-hydroxy-succinamides taking 25mg/ml are dissolved in first wash buffer, are configured to 25mg/mlNHS solution.

Two, the operation steps of magnetic bead activation buffer:

1. previous cleaning:

1.1 get 100 μ l magnetic beads, are diluted to the magnetic bead concentration of 10mg/ml, therefrom draw 100 μ l.

1.2 Magneto separate framves are separated, remove supernatant.

1.3 wash three times, Magneto separate with first washing lotion 250 μ l, remove supernatant.

2. activate:

2.1 get the magnetic bead that previous cleaning completes adds 150 μ l MES, then adds the NHS solution of 50 μ l EDC and 50 μ l, fully mixes.The lower 25 DEG C of activation 30min of room temperature.

2.2 have activated Magneto separate removes supernatant, with the resuspended washing of 250 μ l coupling buffer three times, divides supernatant of leaving away.

3. dilute anti-PG I monoclonal antibody:

Anti-PG I monoclonal antibody is taken out from-20 DEG C of refrigerators, after it melts, takes out 40 μ l, be diluted to 400 μ l with borate buffer 1:10.

5. coupling:

4.1 add the magnetic bead after the resuspended activation of antibody of the variable concentrations of 400 μ l coupling liquid dilutions, fully mix.

4.2 37 DEG C of coupling 2h, 37 DEG C of shaking tables rock constant temperature blending.

5. close

5.1 magnetic beads coupling terminated are separated on Magneto separate frame, remove supernatant.

5.2 add 400 μ l confining liquids, and 1h closed by 37 DEG C of shaking tables.

Supernatant is removed after 5.3 closed end.

6. wash preservation eventually:

6.1 magnetic beads closed with the whole wash liquid of 400 μ l, in triplicate.

6.3 final constant volumes are to 500 μ l.

Second step: Sample dilution is prepared

NaH is added in purified water ₂pO ₄, Na ₂hPO ₄, preparation 100ml phosphate buffer, add bovine serum albumin, Tween-20, ProClin 300, orchil, regulate pH value to 7.4-7.6.

3rd step: the preparation of pepsinogen Cgene standard items

Preparing 3 bottles of PG I standard items, is antiseptic containing 0.1%ProCline 300.The concentration often criticizing PG I titer in kit is 25 μ g/L, 100 μ g/L, 200 μ g/L.

4th step: the preparation of enzyme mark liquid

Preparation 15mL HRP marks anti-PG I monoclonal antibody, is kept at 0.02%methylosothiazolone, in 0.02%bromonitrodioxne stabilizing agent and 0.002%active isothiazoloneization antiseptic.

5th step: the preparation of cleansing solution

The concentrated phosphoric acid salt buffer of preparation 20ml (using after 20 times of dilutions) is antiseptic containing Tween-20 and 0.1%ProCline-300.

6th step: the configuration of substrate solution

1. prepare substrate buffer solution

1.1 take 4.542g Tris, take 0.458g sodium borate, and constant volume, to 300ml, regulates pH to 8.7-9.0.

2. prepare substrate A liquid

2.1 take 0.023g carbamide peroxide, take 0.024g 4-Morpholinopyridine (stabilizing agent).

2.2 add sodium borate buffer liquid constant volume to 100ml with Tris-HCl, room temperature preservation.

3. prepare substrate B liquid

3.1 take 0.088g luminol, take 0.051g 3-(10-phenothiazinyl) propane-1-sulfonate.

3.2 add sodium borate buffer liquid constant volume to 100ml with Tris-HCl.

The B liquid prepared wraps up with masking foil by 3.3, room temperature preservation.

7th step: the present embodiment Full-automatic chemiluminescence detecting instrument detection mode, step is as follows:

1. instrument washing

10-15 washing is carried out to the measuring chamber of instrument and cleaning station, treat that its background value declines, and the measurement of substrate luminous value reaches steady, measurement can be started.

2. application of sample and immune response

2.1 by the magnetic bead of q.s, and enzyme mark anti-PG I antibody adds in the kit of Full-automatic chemiluminescence detector successively.

2.2 add 50-100 μ l serum or plasma sample standard items in 4ml test tube, put into the sample disk of Full-automatic chemiluminescence apparatus device successively.

2.3 primary first-order equations draw 30 μ l PG I antibody magnetic bead reagent, and enzyme labelled antibody once adds 100 μ l, and antigen titer adds 80 μ l.Mix rear 37 DEG C of incubation 30min.

3. start instrument, detect whole process and comprise: application of sample, hatch, washing, measure.

4. washing reaction cup: mechanical arm by incubation slot reaction cup clip to the cleaning station of automation illumination instrument, be magnetic-adsorption under cleaning station groove, instrument suction nozzle is drawn by supernatant, and a tube head is ejection cleansing solution, and cleaning disc turns around common washing 6 times.

7., after application of sample terminates, after 37 DEG C of incubation grooves hatch 30min, reaction cup is clipped to cleaning station by mechanical arm, washing reaction cup: be magnetic-adsorption under cleaning station groove, instrument suction nozzle is drawn by supernatant, and tube head is ejection cleansing solution, and cleaning disc turns around common washing 6 times.

8. add luminous substrate working fluid: add 100 μ l substrate solutions in two first backward reaction cup of needle tubing, prepare to measure after hatching 100s.

9. read luminous value: transfer to measuring chamber by reaction cup from cleaning station, measure, obtain numerical value.

8th step: clinical sample detects

Fig. 1 is the Trendline of the values of chemiluminescence that 1mg magnetic bead bag is obtained by PG I antigen for variable concentrations after 0.0308mgPG I monoclonal antibody.

1. detection scheme

It is human serum or blood plasma that this reagent detects sample.

Take a blood sample and within first 10 hours, should keep on an empty stomach.Gather venous samples can to non-additive serum plastic test tube or have in EDTA or anticoagulant heparin pipe, the test tube 5 ~ 6 times of turning upside down in time is to mix sample.When test sample book is serum, condensation (at least 30 minutes) need be placed at room temperature (20 ~ 25 DEG C).Serum condenses afterwards and blood plasma uses centrifugal method (as with plastic test tube, accelerating to 2000G, centrifugal 10 ~ 15 minutes) to be separated immediately.Plasma/serum sample can refrigerate in refrigerator (2 ~ 8 DEG C), as needed the storage of longer time should freezing (being suitable for-70 ~-20 DEG C of storages).Sample should mix after thawing.Avoid multigelation.Avoid using containing hemolysin, grease or dirty impure sample.

Sample picks up from the normal health check-up of 1000 example, blood donor.Serum sample physical examination result is all without liver, brain, kidney, disease of digestive tract, and without transfusion and major operation history in half a year, women is not in the gestational period and lactation.Measured value is carried out statistical analysis, draws normal serum term of reference: PG I is in 70-165 μ g/L scope.

2. kit performance index of the present invention

Sensitivity: minimumly detect value 0.1 μ g/L; Accuracy: variation within batch CV%<10.0%, batch variation CV%<15.0%; Linear coefficient: r>0.9900; The range of linearity: 1.00 ~ 200 μ g/L.

Claims (4)

1. a pepsinogen Cgene enzyme-catalyzed chemical luminescence detection kit, it is characterized in that, this kit comprises enzyme mark liquid, pepsinogen Cgene standard items, wraps by the immunomagnetic beads of pepsinogen Cgene monoclonal antibody, Sample dilution, Chemoluminescent substrate, cleansing solution.

2. according to a kind of pepsinogen Cgene enzyme-catalyzed chemical luminescence detection kit according to claim 1, it is characterized in that, pepsinogen Cgene monoclonal antibody is coated on magnetic bead microsphere surface, through catching pepsinogen Cgene in sample and after adding enzyme mark pepsinogen Cgene monoclonal antibody reagent, forming solid phase-antibody-antigene-enzyme labelled antibody sandwich immunoassay compound.

3. according to a kind of pepsinogen Cgene enzyme-catalyzed chemical luminescence detection kit according to claim 1, it is characterized in that, described bag is the magnetic microsphere containing being marked with former I monoclonal antibody of antipepsin by the immunomagnetic beads of pepsinogen Cgene monoclonal antibody;

In described enzyme mark liquid, the enzyme of enzyme labelled antibody is horseradish peroxidase;

Described cleansing solution is the damping fluid containing Tween-20;

Described chemical luminous substrate solution is enzyme-catalyzed chemical luminescence substrate solution.

4. as claim 1-3 lets alone the kit as described in, it is characterized in that, described bag is obtained as follows by the immunomagnetic beads of pepsinogen Cgene monoclonal antibody:

(1) preparation of first wash buffer:

Take the MES hygrate of 0.1952g in the pure water of 100ml, adjustment pH to 5.0;

Add 50 μ l Tween-20s, be made into 0.01mol/L MES damping fluid, adjustment pH to 5.0;

(2) preparation of coupling buffer:

Take 0.38138g Na ₂b ₄o ₇10H ₂o is dissolved in 40ml pure water, obtains the borax soln of 0.05mol/L;

Take 0.1237g H ₃bO ₃be dissolved in 10ml pure water, obtain the BAS of 0.2mol/L;

Get the Na of 18ml 0.05mol/L ₂b ₄o ₇the H of solution and 2ml 0.2mol/L ₃bO ₃solution mixes, and is settled to 80ml and obtains 0.05mol/L borate buffer;

(3) preparation of whole washing lotion:

Take 2.78g Na ₂hPO ₄be dissolved in 100ml pure water and obtain 0.2mol/L Na ₂hPO ₄solution;

Take 1.2g NaH ₂pO ₄be dissolved in 50ml pure water and obtain 0.2mol/L NaH ₂pO ₄solution;

Get 81ml 0.2mol/LNa ₂hPO ₄solution and 19ml 0.2mol/L NaH ₂pO ₄solution mixes, and obtains 0.2mol/L PB buffer solution and obtains 0.2mol/LPB buffer solution;

Get 50ml 0.2mol/L PB damping fluid, 2 times are diluted to 100ml, add 0.9gNaCl, and the BSA of 0.5g0.5%-1%, 0.02-0.03% biological preservative, 0.05% polysorbas20, fully dissolve mixing;

(4) preparation of confining liquid:

Get 100ml Tris damping fluid, the pH of solution is adjusted between 8.5-9.0;

Add 2%BSA wherein;

(5) preparation of EDC solution and NHS solution:

Take the EDC of 25mg/ml, be dissolved in the first wash buffer of 1ml, be mixed with the EDC solution of 25mg/ml;

The N-hydroxy-succinamide taking 25mg/ml is dissolved in first wash buffer, is configured to 25mg/ml NHS solution;

(6) magnetic bead bag is by process:

A, previous cleaning: get 100 μ l magnetic beads, be diluted to the magnetic bead concentration of 10mg/ml, therefrom draw 100 μ l; Magneto separate frame is separated, removes supernatant; Wash three times, Magneto separate with first washing lotion 250 μ l, remove supernatant;

B, activation: get the magnetic bead that previous cleaning completes and add 150 μ lMES, then add the NHS solution of 50 μ l EDC and 50 μ l, fully mix; The lower 25 DEG C of activation 30min of room temperature, have activated Magneto separate and have removed supernatant, with the resuspended washing of 250 μ l coupling buffer three times, divided supernatant of leaving away;

C, dilute anti-G-17 monoclonal antibody:

Anti-G-17 monoclonal antibody taken out from-20 DEG C of refrigerators, after it melts, take out 20 μ l, be diluted to 400 μ l with coupling buffer 1:20, the antibody concentration after dilution is 0.0397mg/ml;

D, coupling:

Add the magnetic bead after the resuspended activation of anti-G-17 monoclonal antibody of 400 μ l coupling buffer dilutions, fully mix, its magnetic bead bag is 1mg magnetic bead by ratio: 0.0159mg antibody;

37 DEG C of coupling 2h, 37 DEG C of shaking tables rock constant temperature blending;

E, close:

Magnetic bead coupling terminated is separated on Magneto separate frame, removes supernatant;

Add 400 μ l confining liquids, 1h closed by 37 DEG C of shaking tables;

Close after terminating and remove supernatant;

F, wash preservation eventually:

With the magnetic bead that the whole wash liquid of 400 μ l has been closed, in triplicate, final constant volume is to 500 μ l.