CN105785016A

CN105785016A - Double-tagging time resolution fluoroimmunoassay reagent kit based on PG magnetic particle

Info

Publication number: CN105785016A
Application number: CN201610308504.9A
Authority: CN
Inventors: 黄飚; 范俊; 周彬; 张艺; 张珏; 郭明明; 邓黎莉; 朱岚
Original assignee: Jiangsu Institute of Nuclear Medicine
Current assignee: Jiangsu Institute of Nuclear Medicine
Priority date: 2016-05-11
Filing date: 2016-05-11
Publication date: 2016-07-20

Abstract

The invention relates to a double-tagging time resolution fluoroimmunoassay reagent kit based on a PG magnetic particle and a preparing method and detecting method thereof and belongs to the technical field of immune detection analyzing techniques and nanometer biological techniques. The reagent kit comprises a reaction buffer solution, a concentrated cleaning solution, an enhancing solution, a magnetic particle solution coated with PGI monoclonal antibody, a magnetic particle solution coated with PGI monoclonal antibody, a PG calibrator solution, an europium-labeled PGII monoclonal antibody solution and a samarium-labeled PGI monoclonal antibody solution which are independently packaged. The reagent kit can provide a double-tagging reaction system close to a homogeneous phase, can guarantee that the content of PGI and the content of PGII can be detected at the same time, enables a detecting result of PGI and PGII in the same sample liquid to be high in accuracy and small in error, greatly shortens reaction time, shortens detection time, improves efficiency, improves detection sensitivity and specificity and brings great convenience to clinical application.

Description

Double labelling time resolved fluoro-immunoassay test kit based on PG magnetic particle

Technical field

The invention belongs to immune detection analytical technology and field of nano biotechnology, be specifically related to a kind of double labelling time resolved fluoro-immunoassay test kit based on PG magnetic particle and preparation method thereof and detection method.

Background technology

Pepsinogen (Pesinogen, PG) it is pepsic inactive precursor in gastric juice, human pepsinogen can be divided into the pepsinogen group that two kinds of biochemistry is different with immunocompetence feature: pepsinogen Cgene (is called for short PG I) and pepsinogen II (being called for short PGII), PG I is main to be secreted by chief cell and the mucous neck cell of fundic gland, major part enters gastral cavity, enter blood circulation on a small quantity, PGII derives from full gastric gland and distal duodenum BrunnerShi gland, and the PGII of gastric mucosa synthesis is about the 25% of total amount.PGII major part enters digestive tract, enters blood circulation on a small quantity, and therefore PG is referred to as " pointer of reflection stomach state and function ", and the change of its level can directly reflect the change of gastric mucosa and duodenum state and cell quantity.Domestic and international clinical research is pointed out, PG is bigger with the dependency of gastric mucosa pathological changes, mensuration PG content helps and detects duodenal ulcer, atrophic gastritis, gastritis, other digestive tract disease such as gastric cancer, need for Clinical detection and health check-up examination, PG detects as noninvasive method, reduce patient suffering, easy, economical, there is generaI investigation and be worth.

The pepsinogen (PG) development course to disease of stomach, generally can be expressed as: superficial gastritis gastric mucosal erosion ulcer atrophic gastritis gastric cancer, and Other diseases has good diagnosis and screening effect.Pepsinogen Cgene/II Test paper box, for detecting the content of pepsinogen Cgene/II in serum or blood plasma, has simplicity, quick advantage, it is to avoid the inconvenience of the infringement to human body of the X-ray and gastroscope.nullBut pepsinogen Test paper box of the prior art is all single detection box，Namely can only individually pepsinogen I or pepsinogen I I be detected single，And in practical operation，Sometimes need the pepsinogen I in detection same sample liquid and II，To obtain ratio or other ratio PGI/PGII of the two of the content of pepsinogen I in same sample liquid and pepsinogen II，If PGI/PGII ratio is lower than 6，Then the risk of atrophic gastritis and gastric cancer increases，According to the detection that pepsinogen I and pepsinogen II are independent，Detection process has error unavoidably，If pepsinogen I produces a positive error，Pepsinogen I I produces a negative error，The situation that error is amplified is there will be when calculating PGI/PGII ratio，Error is there is in the detection gained ratio that will cause pepsinogen I and pepsinogen II with actual ratio，Cause result inaccurate.

For the detection of pepsinogen, clinical conventional method includes at present: Immunoturbidimetry, euzymelinked immunosorbent assay (ELISA) (ELISA), chemoluminescence method (CLIA) and Timed resolved fluoroimmunoassay (TRFIA).Immunoturbidimetry, simple to operate, it is possible to full-automatic, but, its sensitivity is not high, it is impossible to realize accurate quantification.CLIA method is based on import reagent, relatively costly, and technology maturity is relatively weak at home.Although ELISA method and TRFIA method can accurate quantitative analysis, but operating process is complicated, and be not suitable for single part and small batch detection uses, and currently with the Time-resolved fluoroimmunoassay detection for pepsinogen, it is only capable of individually detection pepsinogen I or pepsinogen I I.As the test kit disclosed in a kind of pepsinogen I I time-resolved fluoroimmunoassay detection kit disclosed in Chinese patent literature CN202854147U is only capable of detection pepsinogen I I.

But, also do not propose at present a kind of to utilize Time-resolved fluoroimmunoassay test kit detecting PGI and PGII simultaneously and preparation method thereof and detection method, for this, double labelling Time-resolved fluoroimmunoassay is combined by the present invention with magnetic particle, provide a kind of double labelling time resolved fluoro-immunoassay test kit based on PG magnetic particle and preparation method thereof and detection method, PGI and the PGII level in sample can not only be detected simultaneously, but also ensure that testing result is more accurately and reliably, there is higher detection sensitivity and specificity, and reached preferably performance parameter.

Summary of the invention

Therefore, the technical problem to be solved in the present invention is in that in prior art can not detect PGI and PGII simultaneously, calculated PGI/PGII ratio error is big, testing result accuracy is low, and it is long to detect the time, sensitivity, poor specificity, efficiency is low, should not carry out a large amount of sample examination, and then provide a kind of and can detect PGI and PGII, testing result accurately and reliably and sensitivity, specificity is high and the detection time the is short double labelling time resolved fluoro-immunoassay test kit based on PG magnetic particle and detection method thereof and purposes simultaneously.

For this, the present invention provides a kind of double labelling time resolved fluoro-immunoassay test kit based on PG magnetic particle, including the reaction buffer of respective independent packaging, concentrated cleaning solutions, enhancing liquid, is coated the magnetic particle solution of PGI monoclonal antibody, is coated the PGI monoclonal antibody solution of the magnetic particle solution of PGII monoclonal antibody, the calibration object solution of PG, the PGII monoclonal antibody solution of europium labelling and samarium labelling.

The described double labelling time resolved fluoro-immunoassay test kit based on PG magnetic particle, the PGII monoclonal antibody solution of described europium labelling is with Eu³⁺-N₂-[P-Carbimide .-benzyl]-diethylenetriamine tetraacethyl sodium labelling PGII monoclonal antibody preparation obtains, described PGII monoclonal antibody and described Eu³⁺-N₂The mass ratio of-[P-Carbimide .-benzyl]-diethylenetriamine tetraacethyl sodium is 5:1；

The PGI monoclonal antibody solution of described samarium labelling is with Sm³⁺-N₂-[P-Carbimide .-benzyl]-diethylenetriamine tetraacethyl sodium labelling PGI monoclonal antibody preparation obtains, described PGI monoclonal antibody and described Sm³⁺-N₂The mass ratio of-[P-Carbimide .-benzyl]-diethylenetriamine tetraacethyl sodium is 5:1；

The magnetic particle solution of the described PGI of being coated monoclonal antibody is connected with described PGI monoclonal antibody through the magnetic particle of the activation that diameter is 1-300 nanometer, wash, close, wash and prepare；

The magnetic particle solution of the described PGII of being coated monoclonal antibody is connected with described PGII monoclonal antibody through the magnetic particle of the activation that diameter is 1-300 nanometer, wash, close, wash and prepare.

The described double labelling time resolved fluoro-immunoassay test kit based on PG magnetic particle, described concentrated cleaning solutions is the Tween-20 of NaCl, the 27.74g/L of Tris, the 312.43g/L containing 12.1lg/L, the TritonX-100 of 1g/L, the buffer solution being 7.8 with salt acid for adjusting pH；

The described liquid that strengthens is be the glacial acetic acid of 3.6 ‰ containing volumetric concentration, the sodium acetate of 0.5g/L, the β-naphthoyltrifluoroacetone of 0.05g/L, the mixed solution of the trioctyl phosphine oxide of 0.03g/L and TritonX-100 that volumetric concentration is 1 ‰；

The preparation of described reaction buffer is 6.06g trishydroxymethylaminomethane and 8.8gNaCl is dissolved in deionized water, is 7.8 with salt acid for adjusting pH value, is subsequently adding the NaN of BSA, 1g that mass concentration is >=98% of 60g₃；

The calibration object solution of described PG is with the NaN of BSA and the 1g/L containing 2g/L₃The pH7.8Tris-HCl of 50mmol/L, described PG psma ligand is made containing the mixing calibration object solution that described PGI antigen concentration is 0ng/mL, 10ng/mL, 50ng/mL, 100ng/mL, 200ng/mL, 300ng/mL and concentration series that described PGII antigen concentration is 0ng/mL, 5ng/mL, 10ng/mL, 15ng/mL, 25ng/mL, 40ng/mL by reaction buffer.

The described double labelling time resolved fluoro-immunoassay test kit based on PG magnetic particle, the formula of every described test kit is:

Described concentrated cleaning solutions, 40mL；

Described enhancing liquid, 30mL；

Described reaction buffer, 50mL；

The PGII monoclonal antibody solution of described europium labelling, 0.01-0.1mg/mL, 2mL；

The PGI monoclonal antibody solution of described samarium labelling, 0.01-0.1mg/mL, 2mL；

The calibration object solution of 6 × described PG, every bottle of 1mL；

The described magnetic particle solution being coated PGI monoclonal antibody, 0.05-0.1mg/mL, 2mL；

The described magnetic particle solution being coated PGII monoclonal antibody, 0.05-0.1mg/mL, 2mL.

The invention provides a kind of method preparing the described double labelling time resolved fluoro-immunoassay test kit based on PG magnetic particle, including be made by method any one or a few:

The preparation of the calibration object solution of described PG: take the NaN of PGII antigenic solution BSA and the 1g/L containing 2g/L of PGI antigenic solution that concentration is 1mg/mL and 1mg/mL₃The Tris-Hcl reaction buffer that pH is 7.8 of 50mmol/L be configured to containing the mixing calibration object solution that described PGI antigen concentration is 0ng/mL, 10ng/mL, 50ng/mL, 100ng/mL, 200ng/mL, 300ng/mL and concentration series that described PGII antigen concentration is 0ng/mL, 5ng/mL, 10ng/mL, 15ng/mL, 25ng/mL, 40ng/mL, carry out subpackage, lyophilizing, 4 DEG C of preservations；Or

The preparation of the described magnetic particle solution being coated PGI monoclonal antibody: the ferroso-ferric oxide microsphere mass concentration that cut-off footpath is 1-300nm is the glutaraldehyde activated of 5-10%, after room temperature mixes 4 hours, clean 1-3 time with the PBS that the pH of 0.05mol/L is 7.2, then suspend with above-mentioned buffer solution, make the magnetic particle suspension that concentration is 100mg/mL；Take magnetic particle suspension described in 100 μ L, it is subsequently adding the PBS that pH is 7.2 of the 0.05mol/L of 1mL and the PGI monoclonal antibody of 50 μ g, incubation is mixed 2 hours in 25 DEG C, then Magnetic Isolation is carried out, retain magnetic particle, clean 1-3 time with the PBS that the pH of 0.05mol/L is 7.2, close 30 minutes at 25 DEG C with the PBS that pH is 7.2 containing the 0.0lmol/L that mass concentration is 5%BSA of 1mL, clean 1-3 time with PBS that the pH of 0.05mol/L is 7.2, with being 0.5%BSA containing mass concentration and mass concentration is the NaN of 0.1%₃The Tris-HCl buffer that pH is 7.2 of 0.05mol/L resuspended, subpackage, preserve at being placed in 2-8 DEG C；Or

The preparation of the described magnetic particle solution being coated PGII monoclonal antibody: the ferroso-ferric oxide microsphere concentration that cut-off footpath is 1-300nm is the glutaraldehyde activated of 5-10%, after room temperature mixes 4 hours, clean 1-3 time with the PBS that the pH of 0.05mol/L is 7.2, then suspend with above-mentioned buffer solution, make the magnetic particle suspension that concentration is 100mg/mL；Take magnetic particle suspension described in 100 μ L, it is subsequently adding the PBS that pH is 7.2 of the 0.05mol/L of 1mL and the PGII monoclonal antibody of 50 μ g, incubation is mixed 2 hours in 25 DEG C, then Magnetic Isolation is carried out, retain magnetic particle, clean 1-3 time with the PBS that the pH of 0.05mol/L is 7.2, close 30 minutes at 25 DEG C with the PBS that pH is 7.2 containing the 0.0lmol/L that mass concentration is 5%BSA of 1mL, clean 1-3 time with PBS that the pH of 0.05mol/L is 7.2, with being 0.5%BSA containing mass concentration and mass concentration is 0.1%NaN₃The Tris-HCl buffer that pH is 7.2 of 0.05mol/L resuspended, subpackage, preserve at being placed in 2-8 DEG C, to obtain final product；Or

The preparation of the PGI monoclonal antibody solution of described samarium labelling: take PGI antibody 1mL, through the conversion buffered condition of PD-10 post, eluent is the Na that pH is 8.5 of the 50mmol/L containing 0.155mol/LNaCl₂CO₃-NaHCO₃Buffer, collect protein peak, obtaining the PGI monoclonal antibody solution being concentrated into 2g/L, take in the described PGI monoclonal antibody solution 500 μ L described isothiocycmatobenzyl diethylenetriamines tetraacethyl samarium sodium adding 0.2mg, 25 DEG C of magnetic agitation are reacted 20 hours, then reactant liquor is transferred to and carry out chromatography with on the SepharoseCL-6B post of the Tris buffer balance that the concentration that pH is 7.8 is 80mmol/L in advance, collect protein peak, dilution, carry out subpackage, vacuum lyophilization, is placed in-20 DEG C of preservations；Or

The preparation of the PGII monoclonal antibody solution of described europium labelling: take PGII antibody 1mL, through the conversion buffered condition of PD-10 post, eluent is the Na that pH is 8.5 of the 50mmol/L containing 0.155mol/LNaCl₂CO₃-NaHCO₃Buffer, collect protein peak, obtaining the PGII monoclonal antibody solution being concentrated into 2g/L, take the described PGII monoclonal antibody solution 500 μ L isothiocycmatobenzyl diethylenetriamines tetraacethyl europium sodium adding 0.2mg, 25 DEG C of magnetic agitation are reacted 20 hours, reactant liquor is transferred to and carries out chromatography with on the SepharoseCL-6B post of the Tris buffer balance that the concentration that pH is 7.8 is 80mmol/L in advance, collect protein peak, dilution, carry out subpackage, vacuum lyophilization, is placed in-20 DEG C of preservations；Or

The preparation of described enhancing liquid: take the TritonX-100 of 3.6mL glacial acetic acid, 0.5g sodium acetate, 0.05g β-naphthoyltrifluoroacetone, 0.03g trioctyl phosphine oxide and 1mL, mixing, add deionized water and be settled to 1000mL, subpackage, preserve at being placed in 2-8 DEG C, to obtain final product；Or

The preparation of described reaction buffer: take 6.06g trishydroxymethylaminomethane and 8.8gNaCl is dissolved in deionized water, regulating pH value with hydrochloric acid is 7.8, the mass concentration being subsequently adding 60g is >=NaN of 98%BSA, 1g₃, add deionized water subsequently and be settled to 1L, subpackage, preserve at being placed in 2-8 DEG C, to obtain final product；Or

The preparation of described concentrated cleaning solutions: the TritonX-100 taking Tween-20 and the 1g of NaCl, the 27.74g of Tris, the 312.43g of 12.1lg adds in deionized water, after being completely dissolved, adjust pH to 7.8 with hydrochloric acid, then add deionized water and be settled to 1L, subpackage, preserves at being placed in 2-8 DEG C, to obtain final product.

The invention provides a kind of utilization described to comprise the steps based on the method detecting PGI and PGII while the double labelling time resolved fluoro-immunoassay test kit of PG magnetic particle,

S1, the calibration object solution taking described PG respectively and testing sample, mix with the magnetic particle solution of the described PGI of being coated monoclonal antibody, the magnetic particle solution being coated PGII monoclonal antibody and reaction buffer, then hatch, standby；

S2, the solution after hatching in S1 step is carried out Magnetic Isolation, then inhale and abandon supernatant, be subsequently adding described concentrated cleaning solutions and be carried out, after mixing, described solution is carried out Magnetic Isolation, then inhale and abandon supernatant, repeat above-mentioned cleaning step；

S3, the PGII monoclonal antibody solution of the PGI monoclonal antibody solution of samarium labelling diluted with described reaction buffer and europium labelling is added in the solution after the cleaning in step S2, carry out oscillation incubation, then it is carried out with described concentrated cleaning solutions, after mixing, carry out Magnetic Isolation, then inhale and abandon supernatant, repeat above-mentioned cleaning step, it is subsequently adding described enhancing liquid, oscillating reactions；

S4, employing time-resolved fluorescence immunoassay instrument measure the fluorescent value of europium and samarium in the enhanced solution in S3 step respectively, obtain the calibration object solution of described PG and the fluorescent value of testing sample respectively, then the concentration of PGI antigen and the concentration of described PGII antigen and the self-corresponding fluorescent value drawing standard curve of Qi Ge according to the calibration object solution of described PG respectively, then the corresponding fluorescent value of described testing sample is substituted in corresponding described PGI standard curve or PGII standard curve, calculate and obtain the content of PGI and PGII in described testing sample.

Described method, comprises the steps,

S1, the calibration object solution taking described PG respectively and testing sample 48-52 μ L, mix with the magnetic particle solution 4-6 μ L of the described PGI of being coated monoclonal antibody, the magnetic particle solution 4-6 μ L and reaction buffer 140-160 μ L that are coated PGII monoclonal antibody, it is added in micropore or the Ep pipe of clear microplate, in 23-27 DEG C of oscillation incubation 4-6 minute；

S2, the described clear microplate in S1 step or the solution in Ep pipe are carried out the Magnetic Isolation 25-35 second, then inhale and abandon supernatant, each micropore or Ep pipe add 290-310 μ L be carried out by the described concentrated cleaning solutions of 1:48-52 dilution proportion, after mixing, solution in described clear microplate or Ep pipe is carried out the Magnetic Isolation 18-22 second, then inhale and abandon supernatant, repeat above-mentioned cleaning step 5-6 time；

nullS3、The PGII monoclonal antibody solution 90-110 μ L of the PGI monoclonal anti 90-110 μ L and europium labelling that press the samarium labelling of 1:48-52 dilution proportion with described reaction buffer is added the described clear microplate in step S2 or in Ep pipe，23-27 DEG C of oscillation incubation 4-6 minute，Then in each micropore or Ep pipe, add 290-310 μ L be carried out by the described concentrated cleaning solutions of 1:48-52 dilution proportion，After mixing，Solution in described clear microplate or Ep pipe is carried out the Magnetic Isolation 18-22 second，Then inhale and abandon supernatant，Repeat above-mentioned cleaning step 5-6 time，Then liquid is strengthened to micropore in described clear microplate or in Ep pipe described in addition 190-210 μ L，In 23-27 DEG C of oscillating reactions 2.5-3.5min；

Described method, comprises the steps,

S1, the calibration object solution taking described PG respectively and testing sample 50 μ L, mix with the magnetic particle solution 5 μ L of the described PGI of being coated monoclonal antibody, the magnetic particle solution 5 μ L and reaction buffer 150 μ L that are coated PGII monoclonal antibody, it is added in micropore or the Ep pipe of clear microplate, in 25 DEG C of oscillation incubations 5 minutes；

S2, the described clear microplate in S1 step or the solution in Ep pipe are carried out Magnetic Isolation 30 seconds, then inhale and abandon supernatant, each micropore or Ep pipe add 300 μ L be carried out by the described concentrated cleaning solutions of 1:50 dilution proportion, after mixing, described clear microplate or Ep pipe are carried out Magnetic Isolation 20 seconds, then inhale and abandon supernatant, repeat above-mentioned cleaning step 5-6 time；

S3, the PGII monoclonal antibody solution 100 μ L of the PGI monoclonal anti 100 μ L and europium labelling that press the samarium labelling of 1:50 dilution proportion with reaction buffer is added the described clear microplate in step S2 step or in Ep pipe, 25 DEG C of oscillation incubations 5 minutes, then in each micropore or Ep pipe, add 300 μ L to be carried out by the described concentrated cleaning solutions of 1:50 dilution proportion, after mixing, solution in described clear microplate or Ep pipe is carried out Magnetic Isolation 20 seconds, then inhale and abandon supernatant, repeat above-mentioned cleaning step 5-6 time, then to micropore in described clear microplate or Ep pipe adds described in 200 μ L enhancing liquid, in 25 DEG C of oscillating reactions 3min；

Described method, in described S1 step, described testing sample also includes pre-treatment step, takes testing sample and is centrifuged 5 minutes in 3000rpm, then takes upper liquid and be the described testing sample for detecting.

The invention provides described test kit or the described method purposes in detection PGII and/or PGI.

The technique scheme of the present invention has the advantage that compared to existing technology

null(1) the double labelling time resolved fluoro-immunoassay test kit based on PG magnetic particle of the present invention，Reaction buffer including respective independent packaging、Concentrated cleaning solutions、Strengthen liquid、It is coated the magnetic particle solution of PGI monoclonal antibody、It is coated the magnetic particle solution of PGII monoclonal antibody、The calibration object solution of PG、The PGII monoclonal antibody solution of europium labelling and the PGI monoclonal antibody solution of samarium labelling，Be combined with PGI antigen by the coated PGI monoclonal antibody of the PGI monoclonal antibody of rare earth ion samarium (Sm) labelling and magnetic particle，Form Sm labelling PGI antibody-PGI antigen-magnetic particle and be coated the sandwich immunoassay complex of PGI antibody，Be combined with antigen with the coated PGII monoclonal antibody of the PGII monoclonal antibody of rare earth ion europium (Eu) labelling and magnetic particle，Form Eu labelling PGII antibody-PGII antigen-magnetic particle and be coated the sandwich immunoassay complex of PGII antibody，Two kinds of immune complex Direct precipitations in externally-applied magnetic field，Do not need the centrifugal complex that immunoreation can be formed and other separating substances unconjugated，Can detect in 15min simultaneously and obtain the content of PGI and PGII in serum，Overcome radio immunoassay in prior art、Enzyme-linked immunosorbent assay、Singly mark Timed resolved fluoroimmunoassay、Chemoluminescence method can only measure PGI respectively、The accumulation of error calculating PGII/PGI ratio again after PGII and bring，Testing result is accurately and reliably，Particularly raising PGII/PGI ratio parameter detection accuracy had outstanding advantage，Except having the highly sensitive of TRFIA technology、The storage time is long、"dead" pollution、Outside the plurality of advantages such as standard curve range width，Enrichment and magnetic particle also by immunity magnetic particle fully spread the characteristic that combined surface area is expanded in a liquid，It is greatly shortened the response time，Improve detection sensitivity，Magnetic particle is connected by chemical group orientation with antibody simultaneously，Greatly reduce the consumption of pairing antibody and significantly improve the precision of detection，Realize automatization simultaneously，Overcome the board-like TRFIA technology of conventional microporous to need sample accumulation to arrive some could to detect，Achieve the instant detection of sample，Efficiency is high；

nullThe efficient double labelling time-resolved fluorescence technology that this test kit is is carrier with nano magnetic microgranule: magnetic particle is connected with the free amino group of PG monoclonal antibody，Form the immune magnetic particle being coated with PG monoclonal antibody，This immunity magnetic particle relies on the specific surface area of its super large " can catch " a large amount of PG antigen antibody complex with Eu or Sm labelling at short notice，Form the sandwich immunoassay complex of immunomagnetic beads-PG antigen-Eu or Sm labelling PG antibody，With strengthening liquid by Eu and Sm from after complex disintegrates down，A kind of micell is formed with the chelating chelating strengthened in liquid，Micel can send very strong transmitting wavelength (615nm and 643nm) and life-span (820 μ s and 880 μ s) different fluorescence under the exciting of ultraviolet light，Signal enhances million times and two signals are easily distinguished，The content of PGI and PGII antigen in serum can be detected simultaneously，One-shot measurement obtains PGI simultaneously、Three results of PGII and PGI/PGII，Due to consistent，The ratio of PGI/PGII is more accurate.

(2) the double labelling time resolved fluoro-immunoassay test kit based on PG magnetic particle of the present invention, the PGII monoclonal antibody solution of described europium labelling is with Eu³⁺-N₂-[P-Carbimide .-benzyl]-diethylenetriamine tetraacethyl sodium labelling PGII monoclonal antibody preparation obtains, described PGII monoclonal antibody and described Eu³⁺-N₂The mass ratio of-[P-Carbimide .-benzyl]-diethylenetriamine tetraacethyl sodium is 5:1；The PGI monoclonal antibody solution of described samarium labelling is with Sm³⁺-N₂-[P-Carbimide .-benzyl]-diethylenetriamine tetraacethyl sodium labelling PGI monoclonal antibody preparation obtains, described PGI monoclonal antibody and described Sm³⁺-N₂The mass ratio of-[P-Carbimide .-benzyl]-diethylenetriamine tetraacethyl sodium is 5:1；The magnetic particle of the described PGI/PGII of being coated monoclonal antibody is connected with described PG monoclonal antibody through the magnetic particle of the activation that diameter is 1-300 nanometer, wash, close, wash and prepare；By adopting lanthanide series samarium (Sm³⁺) and europium (Eu³⁺) distinguish labelling PGI and PGII antibody, fluorescence intensity is high, and the emission spectrum peak narrow range of fluorescence, is class line spectrum, and detection specificity is good；The stokes displacement of exciting light and transmitting light is big, and exciting light can not directly arrive optoelectronic receiver, solves the stray light of exciting light, and sensitivity and the specificity of detection are greatly improved；Fluorescence lifetime is long, and by delaying the measurement time, the decay of short-life background fluorescence re-records specific fluorescence, avoids the interference of background completely after disappearing.

(3) method of the double labelling time resolved fluoro-immunoassay test kit based on PG magnetic particle described in preparation of the present invention, in the preparation of the calibration object solution of described PG, by reasonably adjusting the calibration object series concentration of described PG so that the standard curve of drafting more can measure the content of PGI and/or PGII accurately；In the described preparation of magnetic particle solution being coated PGI or PGII monoclonal antibody, by the glutaraldehyde activated ferroso-ferric oxide microsphere using mass concentration to be 5-10%, substantially increase the connection of magnetic particle and PGI or PGII monoclonal antibody, improve efficiency；In the preparation of PGI or the PGII monoclonal antibody of described samarium labelling, by using Na₂CO₃-NaHCO₃Buffer solution elution, collects the monoclonal antibody solution obtained purer, is conducive to the labelling of PGI or PGII monoclonal antibody.

Accompanying drawing explanation

In order to be illustrated more clearly that the specific embodiment of the invention or technical scheme of the prior art, the accompanying drawing used required in detailed description of the invention or description of the prior art will be briefly described below, apparently, accompanying drawing in the following describes is some embodiments of the present invention, for those of ordinary skill in the art, under the premise not paying creative work, it is also possible to obtain other accompanying drawing according to these accompanying drawings.

Fig. 1 is the PGI canonical plotting described in the embodiment of the present invention 2；

Fig. 2 is the PGII canonical plotting described in the embodiment of the present invention 2.

Detailed description of the invention

What relate to reagent or instrument in following embodiment is commercially available prod, and the product of different manufacturers can't bring significant difference, wherein Eu on technique effect³⁺-N₂-[P-Carbimide .-benzyl]-diethylenetriamine tetraacethyl sodium, Sm³⁺-N₂-[P-Carbimide .-benzyl]-diethylenetriamine tetraacethyl sodium, purchased from PerkinElmerLifeandAnalyticalSciences (Waltham, MA, USA)；

Test serum, takes from Jiang Yuan hospital of Jiangsu Province；

Magnetic particle (ferroso-ferric oxide microsphere), purchased from JSRLifeSciences (Tokyo, Japan)；

PGI antigen, purchased from Wuxi Jiangyuan Industry Company Jiangsu Institute of Nuclear Medicine；

PGII antigen, purchased from Wuxi Jiangyuan Industry Company Jiangsu Institute of Nuclear Medicine；

PGI monoclonal antibody, purchased from Wuxi Jiangyuan Industry Company Jiangsu Institute of Nuclear Medicine；

PGII monoclonal antibody, purchased from Wuxi Jiangyuan Industry Company Jiangsu Institute of Nuclear Medicine；

Tween-20, purchased from Sigma (St.Louis, MO, USA)；

TritonX-100, purchased from Sigma (St.Louis, MO, USA)；

BSA, purchased from Sigma (St.Louis, MO, USA)；

SepharoseCL-6B, purchased from GE company；

Ultraviolet spectrophotometer, purchased from American MD company, model is M5；

Magnetic separator, purchased from Jiaxing Quest Life Science Co., Ltd., model is CFL-1；

PD-10 post, purchased from American GE company；

Time-resolved fluorescence immunoassay instrument, PE company of the U.S., model AutoDELFIA1235.

Embodiment 1

Present embodiments provide a kind of double labelling time resolved fluoro-immunoassay test kit based on PG magnetic particle, including the reaction buffer of respective independent packaging, concentrated cleaning solutions, enhancing liquid, it is coated the magnetic particle solution of PGI monoclonal antibody, is coated the PGI monoclonal antibody solution of the magnetic particle solution of PGII monoclonal antibody, the calibration object solution of PG, the PGII monoclonal antibody solution of europium labelling and samarium labelling, wherein:

The PGII monoclonal antibody solution of described europium labelling is with Eu³⁺-N₂-[P-Carbimide .-benzyl]-diethylenetriamine tetraacethyl sodium labelling PGII monoclonal antibody preparation obtains, described PGII monoclonal antibody and described Eu³⁺-N₂The mass ratio of-[P-Carbimide .-benzyl]-diethylenetriamine tetraacethyl sodium is 5:1；

The magnetic particle solution of the described PGI of being coated monoclonal antibody is connected with described PGI monoclonal antibody through the magnetic particle of the activation that diameter is 1 nanometer, wash, close, wash and prepare；

The magnetic particle solution of the described PGII of being coated monoclonal antibody is connected with described PGII monoclonal antibody through the magnetic particle of the activation that diameter is 1 nanometer, wash, close, wash and prepare；

Described concentrated cleaning solutions is the TritonX-100 of Tween-20, the 1g/L of NaCl, the 27.74g/L of Tris, the 312.43g/L containing 12.1lg/L, the buffer solution being 7.8 with salt acid for adjusting pH；

The described liquid that strengthens is be the glacial acetic acid of 3.6 ‰ containing volumetric concentration, the sodium acetate of 0.5g/L, the β-naphthoyltrifluoroacetone of 0.05g/L, the trioctyl phosphine oxide of 0.03g/L and and the mixed solution of TritonX-100 that volumetric concentration is 1 ‰；

Described reaction buffer is the trishydroxymethylaminomethane containing 6.06g/L, the BSA of NaCl, the 60g/L of 8.8g/L, and volumetric concentration is the NaN of 1g/L₃, the buffer solution being 7.8 with salt acid for adjusting pH；

The calibration object solution of described PG is with the NaN of BSA and the 1g/L containing 2g/L₃The pH7.8Tris-Hcl reaction buffer of 50mmol/L described PG psma ligand is made containing the mixing calibration object solution that described PGI antigen concentration is 0ng/mL, 10ng/mL, 50ng/mL, 100ng/mL, 200ng/mL, 300ng/mL and concentration series that described PGII antigen concentration is 0ng/mL, 5ng/mL, 10ng/mL, 15ng/mL, 25ng/mL, 40ng/mL.

The formula of every described test kit is:

Described concentrated cleaning solutions, 40mL；

Described enhancing liquid, 30mL；

Described reaction buffer, 50mL；

The PGII monoclonal antibody solution of described europium labelling, 0.05mg, 2mL；

The PGI monoclonal antibody solution of described samarium labelling, 0.05mg, 2mL；

The calibration object solution of 6 × described PG, every bottle of 1mL；

The described magnetic particle solution being coated PGI monoclonal antibody, 0.08mg/mL, 2mL；

The described magnetic particle solution being coated PGII monoclonal antibody, 0.08mg/mL, 2mL.

The present embodiment additionally provides a kind of method preparing mentioned reagent box, specifically includes following steps:

The preparation of the calibration object solution of described PG: take the NaN of PGII antigenic solution BSA and the 1g/L containing 2g/L of PGI antigenic solution that concentration is 1mg/mL and 1mg/mL₃The Tris-Hcl reaction buffer that pH is 7.8 of 50mmol/L be configured to containing the mixing calibration object solution that described PGI antigen concentration is 0ng/mL, 10ng/mL, 50ng/mL, 100ng/mL, 200ng/mL, 300ng/mL and concentration series that described PGII antigen concentration is 0ng/mL, 5ng/mL, 10ng/mL, 15ng/mL, 25ng/mL, 40ng/mL, carry out subpackage, every bottle of 1mL, lyophilizing, 4 DEG C of preservations, to obtain final product；

The preparation of the described magnetic particle solution being coated PGI monoclonal antibody: cut-off footpath is the ferroso-ferric oxide microsphere concentration of 1nm be 5% glutaraldehyde activated, after room temperature mixes 4 hours, clean 1-3 time with the PBS that the pH of 0.05mol/L is 7.2, then suspend with above-mentioned buffer solution, make the magnetic particle suspension that concentration is 100mg/mL；Take magnetic particle suspension described in 100 μ L, it is subsequently adding the PBS that pH is 7.2 of the 0.05mol/L of 1mL and the PGI monoclonal antibody of 50 μ g, incubation is mixed 2 hours in 25 DEG C, then Magnetic Isolation is carried out, retain magnetic particle, clean 1-3 time with the PBS that the pH of 0.05mol/L is 7.2, close 30 minutes at 25 DEG C with the PBS that pH is 7.2 containing the 0.0lmol/L that mass concentration is 5%BSA of 1mL, clean 1-3 time with the PBS that the pH of 0.05mol/L is 7.2, with 1mL be 0.5%BSA containing mass concentration and mass concentration is 0.1%NaN₃The Tris-HCl buffer that pH is 7.2 of 0.05mol/L resuspended, make the described magnetic particle solution being coated PGI monoclonal antibody that concentration is 0.08mg/mL, preserve at being placed in 2-8 DEG C, to obtain final product；

The preparation of the described magnetic particle solution being coated PGII monoclonal antibody: cut-off footpath is the ferroso-ferric oxide microsphere concentration of 1-300nm be 5% glutaraldehyde activated, after room temperature mixes 4 hours, clean 1-3 time with the PBS that the pH of 0.05mol/L is 7.2, then suspend with above-mentioned buffer solution, make the magnetic particle suspension that concentration is 100mg/mL；Take magnetic particle suspension described in 100 μ L, it is subsequently adding the PBS that pH is 7.2 of the 0.05mol/L of 1mL and the PGII monoclonal antibody of 50 μ g, incubation is mixed 2 hours in 25 DEG C, then Magnetic Isolation is carried out, retain magnetic particle, clean 1-3 time with the PBS that the pH of 0.05mol/L is 7.2, close 30 minutes at 25 DEG C with the PBS that pH is 7.2 containing the 0.0lmol/L that mass concentration is 5%BSA of 1mL, clean 1-3 time with the PBS that the pH of 0.05mol/L is 7.2, with 1mL be 0.5%BSA containing mass concentration and mass concentration is the NaN of 0.1%₃The Tris-HCl buffer that pH is 7.2 of 0.05mol/L resuspended, make the described magnetic particle solution being coated PGII monoclonal antibody that concentration is 0.08mg/mL, preserve at being placed in 2-8 DEG C, to obtain final product；Or

The preparation of the PGI monoclonal antibody solution of described samarium labelling: take described PGI monoclonal antibody antibody 1mL, through the conversion buffered condition of PD-10 post, eluent is the Na that pH is 8.5 of the 50mmol/L containing 0.155mol/LNaCl₂CO₃-NaHCO₃Buffer, collect protein peak, obtain the PGI monoclonal antibody solution being concentrated into 2g/L, take in the described PGI monoclonal antibody solution 500 μ L described isothiocycmatobenzyl diethylenetriamines tetraacethyl samarium sodium adding 0.2mg, 25 DEG C of magnetic agitation are reacted 20 hours, then reactant liquor is transferred to and carry out chromatography with on the SepharoseCL-6B post of the Tris buffer balance that the concentration that pH is 7.8 is 80mmol/L in advance, collect protein peak, being diluted to concentration is 0.08mg/mL, carry out subpackage, every bottle of 2mL, vacuum lyophilization, it is placed in-20 DEG C of preservations, to obtain final product；

The preparation of the PGII monoclonal antibody solution of described europium labelling takes PGII antibody 1mL, and through the conversion buffered condition of PD-10 post, eluent is the Na that pH is 8.5 of the 50mmol/L containing 0.155mol/LNaCl₂CO₃-NaHCO₃Buffer, collect protein peak, obtain the PGI monoclonal antibody solution being concentrated into 2g/L, take the described PGII monoclonal antibody solution 500 μ L isothiocycmatobenzyl diethylenetriamines tetraacethyl europium sodium adding 0.2mg, 25 DEG C of magnetic agitation are reacted 20 hours, reactant liquor is transferred to and carries out chromatography with on the SepharoseCL-6B post of the Tris buffer balance that the concentration that pH is 7.8 is 80mmol/L in advance, collect protein peak, being diluted to concentration is 0.08mg/mL, carry out subpackage, every bottle of 2mL, vacuum lyophilization, it is placed in-20 DEG C of preservations, to obtain final product；

The preparation of described enhancing liquid: take the TritonX-100 of 3.6mL glacial acetic acid, 0.5g sodium acetate, 0.05g β-naphthoyltrifluoroacetone, 0.03g trioctyl phosphine oxide and 1mL, mixing, add deionized water and be settled to 1000mL, carry out subpackage, every bottle of 30mL, preserves at being placed in 2-8 DEG C, to obtain final product；

The preparation of described reaction buffer: the NaCl taking 6.06g trishydroxymethylaminomethane and 8.8g is dissolved in deionized water, regulating pH value with hydrochloric acid is 7.8, the mass concentration being subsequently adding 60g is >=NaN of 98%BSA and 1g₃, add deionized water subsequently and be settled to 1L, carry out subpackage, every bottle of 50mL, preserve at being placed in 2-8 DEG C, to obtain final product；

The preparation of described concentrated cleaning solutions: take the Tris of 12.1lg, 312.43g NaCl, 27.74g the TritonX-100 of Tween-20 and 1g add in deionized water, after being completely dissolved, adjust pH to 7.8 with hydrochloric acid, then add deionized water and be settled to 1L, carry out subpackage, every bottle of 40mL, preserves at being placed in 2-8 DEG C, to obtain final product.

Embodiment 2

The magnetic particle solution of the described PGI of being coated monoclonal antibody is connected with described PGI monoclonal antibody through the magnetic particle of the activation that diameter is 300 nanometers, wash, close, wash and prepare；

The magnetic particle solution of the described PGII of being coated monoclonal antibody is connected with described PGII monoclonal antibody through the magnetic particle of the activation that diameter is 300 nanometers, wash, close, wash and prepare；

The formula of every described test kit is:

Described concentrated cleaning solutions, 40mL；

Described enhancing liquid, 30mL；

Described reaction buffer, 50mL；

The PGII monoclonal antibody solution of described europium labelling, 0.1mg；2mL

The PGI monoclonal antibody solution of described samarium labelling, 0.1mg；2mL

The calibration object solution of 6 × described PG, every bottle of 1mL；

The described magnetic particle solution being coated PGI monoclonal antibody, 0.05mg/mL, 2mL；

The described magnetic particle solution being coated PGII monoclonal antibody, 0.05mg/mL, 2mL.

The preparation of the described magnetic particle solution being coated PGI monoclonal antibody: cut-off footpath is the ferroso-ferric oxide microsphere mass concentration of 300nm be 10% glutaraldehyde activated, after room temperature mixes 4 hours, clean 1-3 time with the PBS that the pH of 0.05mol/L is 7.2, then suspend with above-mentioned buffer solution, make the magnetic particle suspension that concentration is 100mg/mL；Take magnetic particle suspension described in 100 μ L, it is subsequently adding the PBS that pH is 7.2 of the 0.05mol/L of 1mL and the PGI monoclonal antibody of 50 μ g, incubation is mixed 2 hours in 25 DEG C, then Magnetic Isolation is carried out, retain magnetic particle, clean 1-3 time with the PBS that the pH of 0.05mol/L is 7.2, close 30 minutes at 25 DEG C with the PBS that pH is 7.2 containing the 0.0lmol/L that mass concentration is 5%BSA of 1mL, clean 1-3 time with the PBS that the pH of 0.05mol/L is 7.2, with 1mL be 0.5%BSA containing mass concentration and mass concentration is 0.1%NaN₃The Tris-HCl buffer that pH is 7.2 of 0.05mol/L resuspended, make the described magnetic particle solution being coated PGI monoclonal antibody that concentration is 0.1mg/mL, preserve at being placed in 2-8 DEG C, to obtain final product；

The preparation of the described magnetic particle solution being coated PGII monoclonal antibody: cut-off footpath is the ferroso-ferric oxide microsphere mass concentration of 300nm be 10% glutaraldehyde activated, after room temperature mixes 4 hours, clean 1-3 time with the PBS that the pH of 0.05mol/L is 7.2, then suspend with above-mentioned buffer solution, make the magnetic particle suspension that concentration is 100mg/mL；Take magnetic particle suspension described in 100 μ L, it is subsequently adding the PBS that pH is 7.2 of the 0.05mol/L of 1mL and the PGII monoclonal antibody of 50 μ g, incubation is mixed 2 hours in 25 DEG C, then Magnetic Isolation is carried out, retain magnetic particle, clean 1-3 time with the PBS that the pH of 0.05mol/L is 7.2, close 30 minutes at 25 DEG C with the PBS that pH is 7.2 containing the 0.0lmol/L that mass concentration is 5%BSA of 1mL, clean 1-3 time with the PBS that the pH of 0.05mol/L is 7.2, with 1mL be 0.5%BSA containing mass concentration and mass concentration is 0.1%NaN₃The Tris-HCl buffer that pH is 7.2 of 0.05mol/L resuspended, make the described magnetic particle solution being coated PGII monoclonal antibody that concentration is 0.1mg/mL, preserve at being placed in 2-8 DEG C, to obtain final product；

The preparation of the PGI monoclonal antibody solution of described samarium labelling: take described PGI monoclonal antibody 1mL, through the conversion buffered condition of PD-10 post, eluent is the Na that pH is 8.5 of the 50mmol/L containing 0.155mol/LNaCl₂CO₃-NaHCO₃Buffer, collect protein peak, obtain the PGI monoclonal antibody solution being concentrated into 2g/L, take in the described PGI monoclonal antibody solution 500 μ L described isothiocycmatobenzyl diethylenetriamines tetraacethyl samarium sodium adding 0.2mg, 25 DEG C of magnetic agitation are reacted 20 hours, then reactant liquor is transferred to and carry out chromatography with on the SepharoseCL-6B post of the Tris buffer balance that the concentration that pH is 7.8 is 80mmol/L in advance, collect protein peak, being diluted to concentration is 0.05mg/mL, carry out subpackage, every bottle of 2mL, vacuum lyophilization, it is placed in-20 DEG C of preservations, to obtain final product；

The preparation of the PGII monoclonal antibody solution of described europium labelling takes PGII antibody 1mL, and through the conversion buffered condition of PD-10 post, eluent is the Na that pH is 8.5 of the 50mmol/L containing 0.155mol/LNaCl₂CO₃-NaHCO₃Buffer, collect protein peak, obtain the PGI monoclonal antibody solution being concentrated into 2g/L, take the described PGII monoclonal antibody solution 500 μ L isothiocycmatobenzyl diethylenetriamines tetraacethyl europium sodium adding 0.2mg, 25 DEG C of magnetic agitation are reacted 20 hours, reactant liquor is transferred to and carries out chromatography with on the SepharoseCL-6B post of the Tris buffer balance that the concentration that pH is 7.8 is 80mmol/L in advance, collect protein peak, being diluted to concentration is 0.05mg/mL, carry out subpackage, every bottle of 2mL, vacuum lyophilization, it is placed in-20 DEG C of preservations, to obtain final product；

The preparation of described reaction buffer: take 6.06g trishydroxymethylaminomethane and 8.8gNaCl is dissolved in deionized water, regulating pH value with hydrochloric acid is 7.8, the mass concentration being subsequently adding 60g is >=NaN of 98%BSA and 1g₃, add deionized water subsequently and be settled to 1L, carry out subpackage, every bottle of 50mL, preserve at being placed in 2-8 DEG C, to obtain final product；

Embodiment 3

The magnetic particle solution of the described PGI of being coated monoclonal antibody is connected with described PGI monoclonal antibody through the magnetic particle of the activation that diameter is 200 nanometers, wash, close, wash and prepare；

The magnetic particle solution of the described PGII of being coated monoclonal antibody is connected with described PGII monoclonal antibody through the magnetic particle of the activation that diameter is 200 nanometers, wash, close, wash and prepare；

The formula of every described test kit is:

Described concentrated cleaning solutions, 40mL；

Described enhancing liquid, 30mL；

Described reaction buffer, 50mL；

The PGII monoclonal antibody solution of described europium labelling, 0.01mg；2mL

The PGI monoclonal antibody solution of described samarium labelling, 0.01mg；2mL

The calibration object solution of 6 × described PG, every bottle of 1mL；

The described magnetic particle solution being coated PGI monoclonal antibody, 0.1mg/mL, 2mL；

The described magnetic particle solution being coated PGII monoclonal antibody, 0.1mg/mL, 2mL.

The preparation of the calibration object solution of described PG: take the NaN of PGII antigenic solution BSA and the 1g/L containing 2g/L of PGI antigenic solution that concentration is 1mg/mL and 1mg/mL₃The Tris-Hcl reaction buffer that pH is 7.8 of 50mmol/L be configured to containing the mixing calibration object solution that described PGI antigen concentration is 0ng/mL, 10ng/mL, 50ng/mL, 100ng/mL, 200ng/mL, 300ng/mL and concentration series that described PGII antigen concentration is 0ng/mL, 5ng/mL, 10ng/mL, 15ng/mL, 25ng/mL, 40ng/mL, carry out subpackage, every bottle of 1mL, lyophilizing, 4 DEG C of preservations, to obtain final product；Or

The preparation of the described magnetic particle solution being coated PGI monoclonal antibody: cut-off footpath is the ferroso-ferric oxide microsphere mass concentration of 200nm be 8% glutaraldehyde activated, after room temperature mixes 4 hours, clean 1-3 time with the PBS that the pH of 0.05mol/L is 7.2, then suspend with above-mentioned buffer solution, make the magnetic particle suspension that concentration is 100mg/mL；Take magnetic particle suspension described in 100 μ L, it is subsequently adding the PBS that pH is 7.2 of the 0.05mol/L of 1mL and the PGI monoclonal antibody of 50 μ g, incubation is mixed 2 hours in 25 DEG C, then Magnetic Isolation is carried out, retain magnetic particle, clean 1-3 time with the PBS that the pH of 0.05mol/L is 7.2, close 30 minutes at 25 DEG C with the PBS that pH is 7.2 containing the 0.0lmol/L that mass concentration is 5%BSA of 1mL, clean 1-3 time with the PBS that the pH of 0.05mol/L is 7.2, with 1mL be 0.5%BSA containing mass concentration and mass concentration is 0.1%NaN₃The Tris-HCl buffer that pH is 7.2 of 0.05mol/L resuspended, make the described magnetic particle solution being coated PGI monoclonal antibody that concentration is 0.01mg/mL, preserve at being placed in 2-8 DEG C, to obtain final product；

The preparation of the described magnetic particle solution being coated PGII monoclonal antibody: cut-off footpath is the ferroso-ferric oxide microsphere mass concentration of 200nm be 8% glutaraldehyde activated, after room temperature mixes 4 hours, clean 1-3 time with the PBS that the pH of 0.05mol/L is 7.2, then suspend with above-mentioned buffer solution, make the magnetic particle suspension that concentration is 100mg/mL；Take magnetic particle suspension described in 100 μ L, it is subsequently adding the PBS that pH is 7.2 of the 0.05mol/L of 1mL and the PGII monoclonal antibody of 50 μ g, incubation is mixed 2 hours in 25 DEG C, then Magnetic Isolation is carried out, retain magnetic particle, clean 1-3 time with the PBS that the pH of 0.05mol/L is 7.2, close 30 minutes at 25 DEG C with the PBS that pH is 7.2 containing the 0.0lmol/L that mass concentration is 5%BSA of 1mL, clean 1-3 time with the PBS that the pH of 0.05mol/L is 7.2, with 1mL be 0.5%BSA containing mass concentration and mass concentration is 0.1%NaN₃The Tris-HCl buffer that pH is 7.2 of 0.05mol/L resuspended, make the described magnetic particle solution being coated PGII monoclonal antibody that concentration is 0.01mg/mL, preserve at being placed in 2-8 DEG C, to obtain final product；

The preparation of the PGI monoclonal antibody solution of described samarium labelling: take PGI monoclonal antibody solution 1mL, through the conversion buffered condition of PD-10 post, eluent is the Na that pH is 8.5 of the 50mmol/L containing 0.155mol/LNaCl₂CO₃-NaHCO₃Buffer, collect protein peak, obtain the PGI monoclonal antibody solution being concentrated into 2g/L, take in the described PGI monoclonal antibody solution 500 μ L described isothiocycmatobenzyl diethylenetriamines tetraacethyl samarium sodium adding 0.2mg, 25 DEG C of magnetic agitation are reacted 20 hours, then reactant liquor is transferred to and carry out chromatography with on the SepharoseCL-6B post of the Tris buffer balance that the concentration that pH is 7.8 is 80mmol/L in advance, collect protein peak, being diluted to concentration is 0.1mg/mL, carry out subpackage, every bottle of 2mL, vacuum lyophilization, it is placed in-20 DEG C of preservations, to obtain final product；

The preparation of the PGII monoclonal antibody solution of described europium labelling: take PGII monoclonal antibody solution 1mL, through the conversion buffered condition of PD-10 post, eluent is the Na that pH is 8.5 of the 50mmol/L containing 0.155mol/LNaCl₂CO₃-NaHCO₃Buffer, collect protein peak, obtain the PGI monoclonal antibody solution being concentrated into 2g/L, take the described PGII monoclonal antibody solution 500 μ L isothiocycmatobenzyl diethylenetriamines tetraacethyl europium sodium adding 0.2mg, 25 DEG C of magnetic agitation are reacted 20 hours, reactant liquor is transferred to and carries out chromatography with on the SepharoseCL-6B post of the Tris buffer balance that the concentration that pH is 7.8 is 80mmol/L in advance, collect protein peak, being diluted to concentration is 0.1mg/mL, carry out subpackage, every bottle of 2mL, vacuum lyophilization, it is placed in-20 DEG C of preservations, to obtain final product；

The preparation of described reaction buffer: take 6.06g trishydroxymethylaminomethane and 8.8gNaCl is dissolved in deionized water, regulating pH value with hydrochloric acid is 7.8, the mass concentration being subsequently adding 60g is >=NaN of 98%BSA and 1g₃, add deionized water subsequently and be settled to 1L, carry out subpackage, every bottle of 50mL, preserve at being placed in 2-8 DEG C, to obtain final product；Or

Embodiment 4

Utilize the test kit described in embodiment 1 to detect pepsinogen I (PGI) and the method for pepsinogen II (PGII) content simultaneously, comprise the steps:

S1, the calibration object solution taking described PG respectively and testing sample 48 μ L, mix with the magnetic particle solution 6 μ L of the described PGI of being coated monoclonal antibody, the magnetic particle solution 6 μ L and reaction buffer 140 μ L that are coated PGII monoclonal antibody, it is added in micropore or the Ep pipe of clear microplate, in 27 DEG C of oscillation incubations 4 minutes；Wherein, take testing sample and be centrifuged 5 minutes in 3000rpm, then take upper liquid and be the described testing sample for detecting；

S2, the described clear microplate in S1 step or the solution in Ep pipe are carried out Magnetic Isolation 35 seconds, then inhale and abandon supernatant, each micropore or Ep pipe add 290 μ L be carried out by the described concentrated cleaning solutions of 1:52 dilution proportion, after mixing, solution in described clear microplate or Ep pipe is carried out Magnetic Isolation 18 seconds, then inhale and abandon supernatant, repeat above-mentioned cleaning step 5-6 time；

S3, the PGII monoclonal antibody solution 90 μ L of the PGI monoclonal antibody solution 90 μ L and europium labelling that press the samarium labelling of 1:52 dilution proportion with described reaction buffer is added the described clear microplate in step S2 or in Ep pipe, 27 DEG C of oscillation incubations 4 minutes, then in each micropore or Ep pipe, add 310 μ L to be carried out by the described concentrated cleaning solutions of 1:52 dilution proportion, after mixing, solution in described clear microplate or Ep pipe is carried out Magnetic Isolation 18 seconds, then inhale and abandon supernatant, repeat above-mentioned cleaning step 5-6 time, then to micropore in described clear microplate or Ep pipe adds described in 210 μ L enhancing liquid, in 23 DEG C of oscillating reactions 3.5min；

S4, employing time-resolved fluorescence immunoassay instrument measure the fluorescent value of europium and samarium in the enhanced solution in S3 step respectively, obtain respectively described PG calibration object solution and the fluorescent value of testing sample, then the concentration of PGI antigen and the concentration of described PGII antigen and the self-corresponding fluorescent value drawing standard curve of Qi Ge according to the calibration object solution of described PG respectively, then the corresponding fluorescent value of described testing sample is substituted in corresponding described PGI standard curve or PGII standard curve, calculate and obtain the content of PGI and PGII in described testing sample.

Embodiment 5

Utilize the test kit described in embodiment 2 to detect pepsinogen I (PGI) and the method for pepsinogen II (PGII) content simultaneously, comprise the steps:

S1, the calibration object solution taking described PG respectively and testing sample 52 μ L, mix with the magnetic particle solution 4 μ L of the described PGI of being coated monoclonal antibody, the magnetic particle solution 4 μ L and reaction buffer 160 μ L that are coated PGII monoclonal antibody, it is added in micropore or the Ep pipe of clear microplate, in 23 DEG C of oscillation incubations 6 minutes；Wherein, take testing sample and be centrifuged 5 minutes in 3000rpm, then take upper liquid and be the described testing sample for detecting；

S2, the described clear microplate in S1 step or the solution in Ep pipe are carried out Magnetic Isolation 25 seconds, then inhale and abandon supernatant, each micropore or Ep pipe add 310 μ L be carried out by the described concentrated cleaning solutions of 1:48 dilution proportion, after mixing, solution in described clear microplate or Ep pipe is carried out Magnetic Isolation 22 seconds, then inhale and abandon supernatant, repeat above-mentioned cleaning step 5-6 time；

S3, the PGII monoclonal antibody solution 110 μ L of the PGI monoclonal antibody solution 110 μ L and europium labelling that press the samarium labelling of 1:48 dilution proportion with described reaction buffer is added the described clear microplate in step S2 or in Ep pipe, 23 DEG C of oscillation incubations 6 minutes, then in each micropore or Ep pipe, add 290 μ L to be carried out by the described concentrated cleaning solutions of 1:48 dilution proportion, after mixing, solution in described clear microplate or Ep pipe is carried out Magnetic Isolation 22 seconds, then inhale and abandon supernatant, repeat above-mentioned cleaning step 5-6 time, then to micropore in described clear microplate or Ep pipe adds described in 190 μ L enhancing liquid, in 27 DEG C of oscillating reactions 2.5min；

Embodiment 6

Utilize the test kit described in embodiment 3 to detect pepsinogen I (PGI) and the method for pepsinogen II (PGII) content simultaneously, comprise the steps:

S1, the calibration object solution taking described PG respectively and testing sample 50 μ L, mix with the magnetic particle solution 5 μ L of the described PGI of being coated monoclonal antibody, the magnetic particle solution 5 μ L and reaction buffer 150 μ L that are coated PGII monoclonal antibody, it is added in micropore or the Ep pipe of clear microplate, in 25 DEG C of oscillation incubations 5 minutes；Wherein, take testing sample and be centrifuged 5 minutes in 3000rpm, then take upper liquid and be the described testing sample for detecting；

S3, the PGII monoclonal antibody solution 100 μ L of the PGI monoclonal antibody solution 100 μ L and europium labelling that press the samarium labelling of 1:50 dilution proportion with reaction buffer is added the described clear microplate in step S2 step or in Ep pipe, 25 DEG C of oscillation incubations 5 minutes, then in each micropore or Ep pipe, 300 μ L are added by the described concentrated cleaning solutions of 1:50 dilution proportion, after mixing, described clear microplate or Ep pipe are carried out Magnetic Isolation 20 seconds, then inhale and abandon supernatant, repeat above-mentioned cleaning step 5-6 time, then to micropore in described clear microplate or Ep pipe adds described in 200 μ L enhancing liquid, in 25 DEG C of oscillating reactions 3min；

nullS4、Time-resolved fluorescence immunoassay instrument is adopted to measure the fluorescent value of europium and samarium in the enhanced solution in S3 step respectively，Obtain the calibration object solution of described PG and the fluorescent value of testing sample respectively，Then according to the calibration object solution concentration of described PG and corresponding fluorescent value drawing standard curve，The concentration respectively 0ng/mL of calibration object solution PGI、10ng/mL、50ng/mL、100ng/mL、200ng/mL、300ng/mL，Each concentration does 5 Duplicate Samples，Testing result is in Table 1，Testing result is with fluorescence signal value for vertical coordinate，The calibration object solution concentration of PGI is abscissa，Set up equation fit standard curve，PGI standard curve is shown in Fig. 1，The concentration respectively 0ng/mL of calibration object solution PGII、5ng/mL、10ng/mL、15ng/mL、25ng/mL、40ng/mL，Each concentration does 5 Duplicate Samples，Testing result is in Table 2，Testing result is with fluorescence signal value for vertical coordinate，The calibration object solution concentration of PGII is abscissa，Set up equation fit standard curve，PGII standard curve is shown in Fig. 2，Then the corresponding fluorescent value that the detection of described testing sample obtains is substituted in corresponding described PGI standard curve or PGII standard curve，Calculate and obtain in described testing sample the content of PGI and PGII in Table 3.

The testing result of table 1PGI calibration object

The testing result of table 2PGII calibration object

Table 3 pattern detection result

Comparative example

Comparison with commercially available pepsinogen test kit

(in described sample, PGI concentration range is 40-300ng/mL to be respectively adopted the method detection sample in commercially available pepsinogen time resolution immunofluorescent reagent box (TRFIA) (Wuxi Jiangyuan Industry Company Jiangsu Institute of Nuclear Medicine) and embodiment 6, PGII concentration range is 7-60ng/mL) in triplicate, the testing result correctness of the test kit of the checking present invention, result is shown in table 4 below.

Table 4 pattern detection result

The error of comparing embodiment 6 and the detection of commercially available pepsinogen time resolution immunofluorescent reagent box is respectively less than 10%, and the testing result of this reagent is accurately and reliably.Compared to comparative example, double labelling time resolved fluoro-immunoassay test kit based on PG magnetic particle of the present invention, has the advantage that 1) highly sensitive, the sensitivity of PGI is 0.9ng/mL, the sensitivity of PGII is 0.08ng/mL, and serum (slurry) sample need not dilute；2) broad quantum, PGI sample concentration value can accurately detect between 0.9-300ng/mL；3) the detection time is short, and from sample incubation to detection, about 25min completes；4) sample requirements is few, and one time loading only needs 50 μ L.

Obviously, above-described embodiment is only for clearly demonstrating example, and is not the restriction to embodiment.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here without also cannot all of embodiment be given exhaustive.And the apparent change thus extended out or variation are still among the protection domain of the invention.

Claims

1. based on the double labelling time resolved fluoro-immunoassay test kit of PG magnetic particle, it is characterized in that, including the reaction buffer of respective independent packaging, concentrated cleaning solutions, enhancing liquid, be coated the magnetic particle solution of PGI monoclonal antibody, be coated the PGI monoclonal antibody solution of the magnetic particle solution of PGII monoclonal antibody, the calibration object solution of PG, the PGII monoclonal antibody solution of europium labelling and samarium labelling.

2. the double labelling time resolved fluoro-immunoassay test kit based on PG magnetic particle according to claim 1, it is characterised in that

3. the double labelling time resolved fluoro-immunoassay test kit based on PG magnetic particle according to claim 2, it is characterised in that

4. the double labelling time resolved fluoro-immunoassay test kit based on PG magnetic particle according to any one of claim 1-3, it is characterised in that the formula of every described test kit is:

Described concentrated cleaning solutions, 40mL；

Described enhancing liquid, 30mL；

Described reaction buffer, 50mL；

The calibration object solution of 6 × described PG, every bottle of 1mL；

5. the method for the double labelling time resolved fluoro-immunoassay test kit based on PG magnetic particle prepared described in any one of claim 1-4, it is characterised in that include being made by method any one or a few:

6. one kind utilize described in any one of claim 1-4 based on while the double labelling time resolved fluoro-immunoassay test kit of PG magnetic particle detect PGI and PGII method, it is characterised in that comprise the steps,

7. method according to claim 6, it is characterised in that comprise the steps,

8. the method according to claim 6 or 7, it is characterised in that comprise the steps,

9. the method according to any one of claim 6-8, it is characterised in that in described S1 step, described testing sample also includes pre-treatment step, takes testing sample and is centrifuged 5 minutes in 3000rpm, then take upper liquid and be the described testing sample for detecting.

10. the test kit described in any one of claim 1-4 or the purposes in detection PGI and/or PGII of the method described in any one of claim 6-9.