CN102313812A - Toxoplasma gondii IgM antibody and total immunoglobulin combined detection reagent strip and its preparation method - Google Patents
Toxoplasma gondii IgM antibody and total immunoglobulin combined detection reagent strip and its preparation method Download PDFInfo
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Abstract
A Toxoplasma gondii IgM antibody and total immunoglobulin combined detection reagent strip and its preparation method relate to a Toxoplasma gondii IgM antibody and total immunoglobulin combined detection reagent. A preparation method provided by the invention comprises the following steps of: arranging two reagent strips which are both equipped with a vector board, a sampling pad, a colloidal gold pad, a nitric acid fiber film, a contrast line and an absorption pad; arranging a Toxoplasma gondii IgM antibody detection line and a total immunoglobulin detection line on the two reagent strips; preparing Toxoplasma gondii recombinant antigen SAG1(P30), SAG2(P22), ROP2 and GRA7; carrying out spotting of the cellulose nitrate membrane; preparing colloidal gold; carrying out marking of colloidal gold with SAG1(P30), SAG2(P22), ROP2 and GRA7; and preparing the immunity chromatography detector strip. The detection reagent strip provided by the invention is used for the detection of Toxoplasma gondii IgM antibody and total immunoglobulin in whole blood, serum, blood plasma and cerebrospinal fluid specimen. The detection provided by the invention requires little amount of specimen and no special apparatus; the detection result is directly read by naked eyes; in addition, the detection is simple, rapid, accurate and reliable with strong specificity and high sensitivity, requires low cost, and is widely applied.
Description
Technical field
The present invention relates to a kind of Toxoplasma Gondi IgM antibody and total antibody combined detectable, especially relate to Toxoplasma Gondi IgM antibody that a kind of employing colloidal gold immunochromatographimethod technology (immunochromatography) carries out and total antibody combined detectable bar and preparation method thereof.
Background technology
Toxoplasmosis (Toxoplasmosis) is that the people beast of a kind of serious harm human health of causing of toxoplasma gondii (Toxoplasmagondii) suffers from parasitic disease altogether; The arc worm of its pathogen can colonize in the karyocyte of people and multiple animal, the general susceptible of crowd and animal.This disease is distributed widely in all over the world, according to statistics the whole world have approximately 1,000,000,000 people by arch insect infection ([1] Xi Linlin, Li Wei. arc worm pathogenesis, virulence and genotype progress [J]. Chinese Pathogen Biology magazine, 2009,4 (11): 859-861.).Toxoplasmosis distributes very extensive in China; All there are the report of arch insect infection in each province, the whole nation, city, autonomous region; The average infection rate of China's normal population about 4%～9% ([2] Liu Min, Chen Xiaoguang. the analysis on epidemic of Chinese population toxoplasmosis [J]. parasite and medical insect journal, 2010; 17 (3): 131-134), special population such as tumor patient, mental patient, birth defect infant, immunosupress, immunodeficiency patient infection lead higher.Normal adult of thumping majority immunologic function or children are normal asymptomatic or light symptoms is only arranged after by arch insect infection fortunately, and multipotency self-healing and obtain permanent immunity.But after the fetus of congenital infection, children and immunodeficiency person were infected, then prognosis was very serious.It is an important intercurrent disease that causes the AIDS patient dead.Human immunodeficiency virus (HIV) the infected has 20%～80% to merge arch insect infection approximately, the more important thing is that it also is one of important pathogen that causes human congenital malformation, defective, feeblemindedness, stillborn foetus, premature labor.
Toxoplasmosis diagnostic method comprise directly and carry out etiological diagnosis from arc worms of separate tissue such as internal organs, blood, cerebrospinal fluid, need several days even a few time-of-week, waste time and energy, in practical application, be worth little.Conventional sense mainly is to use various serological methods clinically; Detect its special IgG and IgM ([3] Zhou Bin, Zhang Jue, Wang Ke; Deng. the foundation and the Preliminary Clinical [J] thereof of Toxoplasma Gondi IgG and IgM antibody double-tagging time resolved fluoro-immunoassay. Chinese laboratory medicine magazine; 2010,33 (010): 957-959.), serological method can Diagnosis of Congenital, acute and chronic toxoplasmosis; But because the IgG titre of its resisting toxoplasmosis of human or animal of most of immunodeficiencies can not rise or high IgM titre can not occur, so can not diagnose toxoplasmosis with serological method to the human or animal who suffers from immunodeficiency effectively.In addition, serum antibody titer just can progressively descend after antigen disappears the long duration, so can not be applied to treatment effectiveness evaluation.Detect IgG antibody at present and have higher false positive, and problems such as the general poor sensitivity of IgM antibody test ([4] Han Jingyun, Liu Qian, Guo Jian. the technical progress of arch insect infection laboratory diagnosis [J]. laboratory medicine, 2009,24 (5): 393-395.).Therefore, the early diagnosis of disease is helped little, is badly in need of setting up a kind of high susceptibility that has, and inexpensive, quick, simple to operate, do not need specific installation, be more suitable in clinical method of early diagnosis.
The diagnosis of toxoplasmosis and epidemiology survey mainly depend on serological test; Mostly normal human's toxoplasma gondii infection is subclinical infection; When immunity of organisms descended, polypide bred in a large number, invaded and removed exo-erythrocytic each histocyte endoparasitism; Cause the extensive inflammation of various tissues, thereby clinical symptoms occurs.Can inducing producing specificity antibody behind the arc worm of human infection.Infecting early stage IgM antibody increases; IgM fades away after infecting 4 months; High concentration IgG antibody ([5] KASPER D C appears after infecting 1 month; PRUSA A R, HAYDE M, et al.Evaluation of the vitros eciq immunodiagnostic system for detection of anti-toxoplasma immunoglobulin g and immunoglobulin m antibodies for confirmatory testing for acute toxoplasma gondii infection in pregnant women [J] .Journal of clinical microbiology; 2009,47 (1): 164-168.).Therefore, Toxoplasma Gondi IgG antibody is an important indicator of toxoplasmosis diagnosis and epidemiology survey.
Early stage serological method uses arch insect circulating antigen.The arch insect circulating antigen of research and diagnosis usefulness is to obtain with the arch insect infection mouse peritoneal, antigen amount few and impure (often being mixed with host protein) big, that obtain that this method spends, so false positive also happens occasionally.Along with the clone in succession who reaches toxoplasma antigen that popularizes of Protocols in Molecular Biology, it is more and more that recombinant antigen is applied to arc worm experiment.Surface antigen (SAG1 (P30), SAG2 (P22), SAG3 (P43), SAG4 (P18)), polypide clava antigen (ROP1, ROP2), dense granule albumen ([6] Xiong Meihua such as (GRA1, GRA7) of the arc worm that research at present is many; Wang Xiuzhen; Liu Luxia; Deng. the extraction of toxoplasma tachyzoite antigen and analysis of protein [J]. Chinese preventing and treating verminosis magazine, 2001,14 (3): 237-238.).The recombinant antigen that adopts recombinant DNA technology to prepare can overcome the shortcoming of complete toxoplasma antigen, can prepare endless special recombinant toxoplasma antigen fast, economically.
The toxoplasma antibody detection method comprises ELISA, Western-blot etc., and its specificity is all higher.Yet, in the face of severe anti-system form, not only need special detection means accurately, also need a kind of more simple and efficient reagent to come examination, so that with the disease prevention and control countermeasure is provided for clinical.
Summary of the invention
The purpose of this invention is to provide a kind of Toxoplasma Gondi IgM antibody and total antibody combined detectable bar and preparation method thereof.
Toxoplasma Gondi IgM antibody according to the invention and total antibody combined detectable bar are provided with the 1st reagent strip and the 2nd reagent strip;
Said the 1st reagent strip is provided with the 1st carrier board, the 1st application of sample pad, the 1st collaurum pad, the 1st nitrocellulose membrane (NC film), Toxoplasma Gondi IgM antibody detection line, the 1st control line and the 1st absorption pad; The 1st application of sample pad, the 1st collaurum pad, the 1st nitrocellulose membrane and the 1st absorption pad stick on the 1st carrier board upper surface successively; One end of the 1st application of sample pad is located on the end of the 1st collaurum pad; The other end of the 1st collaurum pad is located on the end of the 1st nitrocellulose membrane; One end of the 1st absorption pad is located on the other end of the 1st nitrocellulose membrane, and Toxoplasma Gondi IgM antibody detection line and the 1st control line are located on the 1st nitrocellulose membrane successively; Encapsulate anti-people IgM specific fragment μ chain monoclonal antibody at Toxoplasma Gondi IgM antibody detection line place, encapsulate the IgG antibody of goat-anti toxoplasma antigen SAG1 (P30), SAG2 (P22), ROP2 and GRA7 at the 1st control line place;
Said the 2nd reagent strip is provided with the 2nd carrier board, the 2nd application of sample pad, the 2nd collaurum pad, the 2nd nitrocellulose membrane (NC film), total antibody detection line, the 2nd control line and the 2nd absorption pad; The 2nd application of sample pad, the 2nd collaurum pad, the 2nd nitrocellulose membrane and the 2nd absorption pad stick on the 2nd carrier board upper surface successively; One end of the 2nd application of sample pad is located on the end of the 2nd collaurum pad; The other end of the 2nd collaurum pad is located on the end of the 2nd nitrocellulose membrane; One end of the 2nd absorption pad is located on the other end of the 2nd nitrocellulose membrane, and total antibody detection line and the 2nd control line are located on the 2nd nitrocellulose membrane successively; Encapsulate anti-people Ig monoclonal antibody at total antibody detection line place, encapsulate the IgG antibody of goat-anti toxoplasma antigen SAG1 (P30), SAG2 (P22), ROP2 and GRA7 at the 2nd control line place.
The useable glass tunica fibrosa is connected between the 1st application of sample pad of said the 1st reagent strip and the 2nd application of sample pad of the 2nd reagent strip, puts into kit (claiming the single hole application of sample) again, also can connect or puts up a bridge (claiming the diplopore application of sample) without glass fibre membrane.
Said the 1st carrier board and the 2nd carrier board can adopt the PVC plate.
The preparation method of said Toxoplasma Gondi IgM antibody and total antibody combined detectable bar may further comprise the steps:
1) preparation arc worm recombinant antigen SAG1 (P30), SAG2 (P22), ROP2 and GRA7
Adopt gene clone technology, the DNA of pcr amplification coding toxoplasma antigen, and make its expression in the insertion Escherichia coli, get arc worm recombinant antigen SAG1 (P30), SAG2 (P22), ROP2 and GRA7;
2) point sample of nitrocellulose filter
On the Toxoplasma Gondi IgM antibody detection line, encapsulate anti-people IgM specific fragment μ chain monoclonal antibody; Encapsulate anti-people Ig monoclonal antibody at total antibody detection line place; Encapsulate the IgG antibody of goat-anti toxoplasma antigen SAG1 (P30), SAG2 (P22), ROP2 and GRA7 at the control line place of the 1st reagent strip and the 2nd reagent strip respectively, dry;
3) preparation collaurum
Adopt the trisodium citrate method of reducing to prepare the 25nm collaurum; Getting 1% gold chloride 1mL joins in the 100mL deionization distilled water; The gold chloride concentration that obtains is 0.01%, places the flask of band condensing unit to be heated to boiling, adds 1% trisodium citrate aqueous solution 2.0mL under the magnetic force heated and stirred; Continue heating till solution is vinicolor, cooling is placed in the brown bottle 4 ℃ of refrigerators and preserves subsequent use;
4) mark of collaurum and SAG1 (P30), SAG2 (P22), ROP2 and GRA7
(1) mark of collaurum and toxoplasma antigen SAG1 (P30): get collaurum 10ml, transfer to pH5.4, add 100 μ g SAG1 (P30), mixing with 0.1mol/L NaOH; Place 5min, add 5%BSA 1ml mixing, 4 ℃, the centrifugal 1h of 10000r/min; Abandon supernatant, will precipitate with the TBS damping fluid and be dissolved to 10ml, 4 ℃, the centrifugal 1h of 10000r/min; Abandon supernatant, deposition is diluted to 1ml with TBS, gets SAG1 (P30) antigen of colloid gold label;
(2) collaurum is identical with step (1) with the labeling method of toxoplasma antigen SAG2 (P22), ROP2 and GRA7, gets SAG2 (P22), ROP2 and the GRA7 antigen of colloid gold label respectively;
(3) after the GRA7 mixed antigen with the ROP2 antigen of the SAG2 (P22) of SAG1 (P30) antigen of colloid gold label, colloid gold label, colloid gold label and colloid gold label, be applied to equably on the glass fibre membrane, oven dry is prepared into the collaurum pad;
5) preparation immunochromatography detector bar
The 1st reagent strip is provided with the 1st carrier board, the 1st application of sample pad, the 1st collaurum pad, the 1st nitrocellulose membrane (NC film), Toxoplasma Gondi IgM antibody detection line, the 1st control line and the 1st absorption pad; The 1st application of sample pad, the 1st collaurum pad, the 1st nitrocellulose membrane and the 1st absorption pad stick on the 1st carrier board upper surface successively; One end of the 1st application of sample pad is located on the end of the 1st collaurum pad; The other end of the 1st collaurum pad is located on the end of the 1st nitrocellulose membrane; One end of the 1st absorption pad is located on the other end of the 1st nitrocellulose membrane, and Toxoplasma Gondi IgM antibody detection line and the 1st control line are located on the 1st nitrocellulose membrane successively; Encapsulate anti-people IgM specific fragment μ chain monoclonal antibody at Toxoplasma Gondi IgM antibody detection line place; Encapsulate the IgG antibody of goat-anti toxoplasma antigen SAG1 (P30), SAG2 (P22), ROP2 and GRA7 at the 1st control line place; Be cut into strip with cutting cutter, get the 1st reagent strip of Toxoplasma Gondi IgM antibody and total antibody combined detectable bar;
The 2nd reagent strip is provided with the 2nd carrier board, the 2nd application of sample pad, the 2nd collaurum pad, the 2nd nitrocellulose membrane (NC film), total antibody detection line, the 2nd control line and the 2nd absorption pad; The 2nd application of sample pad, the 2nd collaurum pad, the 2nd nitrocellulose membrane and the 2nd absorption pad stick on the 2nd carrier board upper surface successively; One end of the 2nd application of sample pad is located on the end of the 2nd collaurum pad; The other end of the 2nd collaurum pad is located on the end of the 2nd nitrocellulose membrane; One end of the 2nd absorption pad is located on the other end of the 2nd nitrocellulose membrane, and total antibody detection line and the 2nd control line are located on the 2nd nitrocellulose membrane successively; Encapsulate anti-people Ig monoclonal antibody at total antibody detection line place; Encapsulate the IgG antibody of goat-anti toxoplasma antigen SAG1 (P30), SAG2 (P22), ROP2 and GRA7 at the 2nd control line place; Be cut into strip with cutting cutter, get the 2nd reagent strip of Toxoplasma Gondi IgM antibody and total antibody combined detectable bar.
In step 2) in; The concentration of said anti-people Ig monoclonal antibody can be 1～4mg/mL; The concentration of anti-people IgM specific fragment μ chain monoclonal antibody can be 1～4mg/mL; The IgG antibody of goat-anti toxoplasma antigen SAG1 (P30), SAG2 (P22), ROP2 and GRA7 was by anti-SAG1 (P30) antibody, anti-SAG2 (P22) antibody, anti-ROP2 antibody and GRA7 antibody 1: 1: 1 by volume: 1 mixes, and its final concentration is 1～4mg/mL; Three's point sample amount is 1 μ L/cm.
In step 3), the percent concentration of said trisodium citrate can be 2%.
In step 4); The SAG2 (P22) of said SAG1 (P30) antigen, colloid gold label, the ROP2 antigen of colloid gold label and the GRA7 mixed antigen of colloid gold label with colloid gold label, SAG1 (P30) antigen, the SAG2 (P22) of colloid gold label, the ROP2 antigen of colloid gold label and the GRA7 mixed antigen volume ratio of colloid gold label of the colloid gold label of right colloid gold label are 1: (0.2～5): (0.2～5): mix (0.2～5); The temperature of said oven dry can be 37 ℃.
Available junctional membrane (can adopt glass fibre membrane) is connected the 1st reagent strip (claiming the single hole application of sample) with the 2nd reagent strip on the 1st application of sample pad and the 2nd application of sample pad; Also can connect or put up a bridge (title diplopore application of sample) without junctional membrane; Can Toxoplasma Gondi IgM antibody and total antibody combined detectable bar be put into box, make test card, pack in the aluminium foil bag with drying agent; Machine seals, and sealing is preserved.
The invention provides a kind of employing colloidal gold immunochromatographimethod technology and set up Toxoplasma Gondi IgM antibody and total antibody combined quick detection reagent, can be used for Toxoplasma Gondi IgM antibody and total detection of antibodies in the samples such as whole blood, serum, blood plasma and cerebrospinal fluid.Specimen amount required during detection is minimum, does not need specific apparatus, the direct sentence read result of naked eyes, and detect easy fast, high specificity, highly sensitive, accurately and reliably, cost is low, is widely used.
Description of drawings
Fig. 1 is that the structure of Toxoplasma Gondi IgM antibody according to the invention and total antibody combined detectable bar embodiment is formed synoptic diagram.In Fig. 1, A is the 1st reagent strip, and B is the 2nd reagent strip, and 1 is junctional membrane (can adopt glass fibre membrane).
Fig. 2 is the single hole application of sample assembling synoptic diagram of Toxoplasma Gondi IgM antibody according to the invention and total antibody combined detectable bar embodiment.In Fig. 2, A is the 1st reagent strip, and B is the 2nd reagent strip.
Fig. 3 is the diplopore application of sample assembling synoptic diagram of Toxoplasma Gondi IgM antibody according to the invention and total antibody combined detectable bar embodiment.In Fig. 3, A is the 1st reagent strip, and B is the 2nd reagent strip.
Fig. 4 is the experimental result pattern diagram.In Fig. 3, (1) is the synoptic diagram before using, and (2)～(4) are invalid test (product quality problem), and (5) are the total antibody of arc worm, the IgM antibody positive, and (6) are the total antibody positive of arc worm, the IgM negative antibody; A is the 1st reagent strip, and B is the 2nd reagent strip; T is a detection line, and C is a control line.
Embodiment
Following examples will combine accompanying drawing that the present invention is further described.
Referring to Fig. 1～3, Toxoplasma Gondi IgM antibody according to the invention and total antibody combined detectable bar embodiment are provided with the 1st reagent strip A and the 2nd reagent strip B;
The 1st reagent strip A is provided with the 1st carrier board A1, the 1st application of sample pad A2, the 1st collaurum pad A3, the 1st nitrocellulose membrane (NC film) A4, Toxoplasma Gondi IgM antibody detection line A6, the 1st control line A7 and the 1st absorption pad A8; The 1st application of sample pad A2, the 1st collaurum pad A3, the 1st nitrocellulose membrane A4 and the 1st absorption pad A8 stick on the 1st carrier board A1 upper surface successively; The end of the 1st application of sample pad A2 is located on the end of the 1st collaurum pad A3; The other end of the 1st collaurum pad A3 is located on the end of the 1st nitrocellulose membrane A4; The end of the 1st absorption pad A8 is located on the other end of the 1st nitrocellulose membrane A4, and Toxoplasma Gondi IgM antibody detection line A6 and the 1st control line A7 are located on the 1st nitrocellulose membrane A4 successively; Encapsulate anti-people IgM specific fragment μ chain monoclonal antibody at Toxoplasma Gondi IgM antibody detection line A6 place, encapsulate the IgG antibody of goat-anti toxoplasma antigen SAG1 (P30), SAG2 (P22), ROP2 and GRA7 at the 1st control line A7 place.
The 2nd reagent strip B is provided with the 2nd carrier board B1, the 2nd application of sample pad B2, the 2nd collaurum pad B3, the 2nd nitrocellulose membrane (NC film) B4, total antibody detection line B6, the 2nd control line B7 and the 2nd absorption pad B8; The 2nd application of sample pad B2, the 2nd collaurum pad B3, the 2nd nitrocellulose membrane B4 and the 2nd absorption pad B8 stick on the 2nd carrier board B1 upper surface successively; The end of the 2nd application of sample pad B2 is located on the end of the 2nd collaurum pad B3; The other end of the 2nd collaurum pad B3 is located on the end of the 2nd nitrocellulose membrane B4; The end of the 2nd absorption pad B8 is located on the other end of the 2nd nitrocellulose membrane B4, and total antibody detection line B6 and the 2nd control line B7 are located on the 2nd nitrocellulose membrane B4 successively; Encapsulate anti-people Ig monoclonal antibody at total antibody detection line B6 place, encapsulate the IgG antibody of goat-anti toxoplasma antigen SAG1 (P30), SAG2 (P22), ROP2 and GRA7 at the 2nd control line B7 place.
Available junctional membrane (can adopt glass fibre membrane) is connected between the 1st application of sample pad A2 of the 1st reagent strip A and the 2nd application of sample pad B2 of the 2nd reagent strip B, puts into kit (claiming the single hole application of sample) again, also can connect or puts up a bridge (claiming the diplopore application of sample) without junctional membrane.
Said the 1st carrier board A1 and the 2nd carrier board B1 can adopt the PVC plate.
The preparation method of said Toxoplasma Gondi IgM antibody and total antibody combined detectable bar may further comprise the steps:
1) preparation arc worm recombinant antigen SAG1 (P30), SAG2 (P22), ROP2 and GRA7
Adopt gene clone technology, the DNA of pcr amplification coding toxoplasma antigen, and make its expression in the insertion Escherichia coli, get arc worm recombinant antigen SAG1 (P30), SAG2 (P22), ROP2 and GRA7.
2) point sample of nitrocellulose filter
On the Toxoplasma Gondi IgM antibody detection line, encapsulate anti-people IgM specific fragment μ chain monoclonal antibody; Encapsulate anti-people Ig monoclonal antibody at total antibody detection line place; Dry; The concentration of said anti-people Ig monoclonal antibody can be 1～4mg/mL; The concentration of anti-people IgM specific fragment μ chain monoclonal antibody can be 1～4mg/mL, and the IgG antibody of goat-anti toxoplasma antigen SAG1 (P30), SAG2 (P22), ROP2 and GRA7 was by anti-SAG1 (P30) antibody, anti-SAG2 (P22) antibody, anti-ROP2 antibody and GRA7 antibody 1: 1: 1 by volume: 1 mixes, and its final concentration is 1～4mg/mL; Three's point sample amount is 1 μ L/cm.
3) preparation collaurum
Adopt the trisodium citrate method of reducing to prepare the 25nm collaurum; Get 1% gold chloride 1mL and join in the 100mL deionization distilled water, the gold chloride concentration that obtains is 0.01%, places the flask of band condensing unit to be heated to boiling; Add 1% trisodium citrate aqueous solution 2.0mL under the magnetic force heated and stirred; Continue heating till solution is vinicolor, cooling is placed in the brown bottle 4 ℃ of refrigerators and preserves subsequent usely, and the concentration of said trisodium citrate can be 2%.
4) mark of collaurum and toxoplasma antigen SAG1 (P30), SAG2 (P22), ROP2 and GRA7
(1) mark of collaurum and toxoplasma antigen SAG1 (P30): get collaurum 10ml, transfer to pH5.4, add 100 μ g SAG1 (P30), mixing with 0.1mol/L NaOH; Place 5min, add 5%BSA 1ml mixing, 4 ℃, the centrifugal 1h of 10000r/min; Abandon supernatant, will precipitate with the TBS damping fluid and be dissolved to 10ml, 4 ℃, the centrifugal 1h of 10000r/min; Abandon supernatant, deposition is diluted to 1ml with TBS, gets SAG1 (P30) antigen of colloid gold label;
(2) collaurum is identical with step (1) with the labeling method of toxoplasma antigen SAG2 (P22), ROP2 and GRA7, gets SAG2 (P22), ROP2 and the GRA7 antigen of colloid gold label respectively;
(3) after the GRA7 mixed antigen with the ROP2 antigen of the SAG2 (P22) of SAG1 (P30) antigen of colloid gold label, colloid gold label, colloid gold label and colloid gold label, be applied to equably on the glass fibre membrane, oven dry is prepared into the collaurum pad; The SAG2 (P22) of said SAG1 (P30) antigen, colloid gold label, the ROP2 antigen of colloid gold label and the GRA7 mixed antigen of colloid gold label with colloid gold label, SAG1 (P30) antigen, the SAG2 (P22) of colloid gold label, the ROP2 antigen of colloid gold label and the GRA7 mixed antigen volume ratio of colloid gold label of the colloid gold label of right colloid gold label are 1: (0.2～5): (0.2～5): mix (0.2～5); The temperature of said oven dry can be 37 ℃.
5) preparation immunochromatography detector bar
The 1st reagent strip is provided with the 1st carrier board, the 1st application of sample pad, the 1st collaurum pad, the 1st nitrocellulose membrane (NC film), Toxoplasma Gondi IgM antibody detection line, the 1st control line and the 1st absorption pad; The 1st application of sample pad, the 1st collaurum pad, the 1st nitrocellulose membrane and the 1st absorption pad stick on the 1st carrier board upper surface successively; One end of the 1st application of sample pad is located on the end of the 1st collaurum pad; The other end of the 1st collaurum pad is located on the end of the 1st nitrocellulose membrane; One end of the 1st absorption pad is located on the other end of the 1st nitrocellulose membrane, and Toxoplasma Gondi IgM antibody detection line and the 1st control line are located on the 1st nitrocellulose membrane successively; Encapsulate anti-people IgM specific fragment μ chain monoclonal antibody at Toxoplasma Gondi IgM antibody detection line place; Encapsulate the IgG antibody of goat-anti toxoplasma antigen SAG1 (P30), SAG2 (P22), ROP2 and GRA7 at the 1st control line place; Be cut into strip with cutting cutter, get the 1st reagent strip of Toxoplasma Gondi IgM antibody and total antibody combined detectable bar;
The 2nd reagent strip is provided with the 2nd carrier board, the 2nd application of sample pad, the 2nd collaurum pad, the 2nd nitrocellulose membrane (NC film), total antibody detection line, the 2nd control line and the 2nd absorption pad; The 2nd application of sample pad, the 2nd collaurum pad, the 2nd nitrocellulose membrane and the 2nd absorption pad stick on the 2nd carrier board upper surface successively; One end of the 2nd application of sample pad is located on the end of the 2nd collaurum pad; The other end of the 2nd collaurum pad is located on the end of the 2nd nitrocellulose membrane; One end of the 2nd absorption pad is located on the other end of the 2nd nitrocellulose membrane, and total antibody detection line and the 2nd control line are located on the 2nd nitrocellulose membrane successively; Encapsulate anti-people Ig monoclonal antibody at total antibody detection line place; Encapsulate the IgG antibody of goat-anti toxoplasma antigen SAG1 (P30), SAG2 (P22), ROP2 and GRA7 at the 2nd control line place; Be cut into strip with cutting cutter, get the 2nd reagent strip of Toxoplasma Gondi IgM antibody and total antibody combined detectable bar.
Use junctional membrane (can adopt glass fibre membrane) with the 2nd application of sample pad place the 1st reagent strip to be connected (claiming the single hole application of sample) with the 2nd reagent strip at the 1st application of sample pad again; Also can connect or put up a bridge (title diplopore application of sample) without junctional membrane; Can Toxoplasma Gondi IgM antibody and total antibody combined detectable bar be put into box, make test card, pack in the aluminium foil bag with drying agent; Machine seals, and sealing is preserved.
Below provide immunochromatographyassay assay patient's clinical samples:
Get dilution sample to be checked (whole blood, serum, blood plasma, cerebrospinal fluid) 120 μ L, application of sample leaves standstill the 20min observations in immunochromatography detector bar sample place (single hole application of sample); Or respectively get dilution sample to be checked (whole blood, serum, blood plasma, cerebrospinal fluid) 60 μ L, application of sample leaves standstill the 20min observations in Toxoplasma Gondi IgM antibody and total antibody combined detectable bar sample place.Only respectively there is an aubergine band to occur, then is judged to feminine gender at two Toxoplasma Gondi IgM antibodies and total antibody combined detectable bar check plot; Detection zone T and check plot C at two Toxoplasma Gondi IgM antibodies and total antibody combined detectable bar all have an aubergine band to occur, and then are judged to the positive; After application of sample detected, the aubergine band did not all appear in detection zone and check plot, is the null result (see figure 4).Wherein, sample to be checked (whole blood, serum, blood plasma, cerebrospinal fluid) dilution adopts physiological saline, 0～50 times of extension rate.
Below provide Toxoplasma Gondi IgM antibody and total antibody combined detectable bar performance calibrating:
1) visual examination: white encapsulates smooth, the clean pollution-free spot of reaction film, free from flaw, and adhesive tape does not have and comes unglued, and does not have and cuts oblique phenomenon.
2) positive sample coincidence rate: the positive control serum with the different titers of Toxoplasma Gondi IgM and the total antibody positive of arc worm adopts Toxoplasma Gondi IgM antibodies and total antibody combined detectable bar to examine and determine for each 50 parts, calculates positive coincidence rate.The clinical samples of positive control serum for adopting ELISA (import reagent) method to confirm.
3) negative sample coincidence rate:, calculate negative match-rate with 50 parts of negative control serum calibratings.The clinical samples that definite employing ELISA (import reagent) method of the negative control serum of Toxoplasma Gondi IgM and the total antibody of arc worm is confirmed.
4) sensitivity detects: detect with the indoor quality controlled serum of the Ministry of Public Health, the minimum detectability degree should be less than or equal to 4NCU/mL.
5) criticize interior difference: same batch of Toxoplasma Gondi IgM antibody and total antibody combined detectable bar, detect with characteristic serum, require the positive serum testing result to show that the shade of colour band is consistent, result's feminine gender that negative serum detects.
6) differences between batches: different batches Toxoplasma Gondi IgM antibody and total antibody combined detectable bar, detect with characteristic serum, require the positive serum testing result to show that the shade of colour band is consistent, the result that negative serum detects is negative.
7) interference test: testing result does not receive the interference of sample hemolysis (n=50), piarhemia (n=50) and jaundice (n=50).Serum (or blood plasma) is from the applicant's clinical samples.
8) cross reaction: adopt Toxoplasma Gondi IgM antibody and total antibody combined detectable bar, carry out the detection of systemic loupus erythematosus (n=30), rheumatoid disease (n=30), autoallergic autoimmunity systemic diseases such as (n=30), do not find cross reaction.The serum of autoimmunity systemic disease is from the applicant's clinical definite patient.
9) Detection of Stability: use the Arrhenius rule, Toxoplasma Gondi IgM antibody and total antibody combined detectable bar are placed 37 ℃ of detections after 20 days, above each item index does not have marked change, guarantees that finished product preserves under the drying at room temperature condition, and the term of validity is 18 months.
Below provide specific embodiment.
The 1st reagent strip A encapsulates anti-people IgM specific fragment μ chain monoclonal antibody on nitrocellulose filter (NC film) IgM detection line; Encapsulate anti-people Ig monoclonal antibody on total antibody detection line of the 2nd reagent strip B; C encapsulates the antibody of goat-anti toxoplasma antigen SAG1 (P30), SAG2 (P22), ROP2 and GRA7 at the control line place; Room temperature is dried, and the sealing room temperature preservation is subsequent use.Wherein, The concentration of anti-people IgM specific fragment μ chain monoclonal antibody, anti-people Ig monoclonal antibody is 1mg/mL; Goat-anti toxoplasma antigen (SAG1 (P30), SAG2 (P22), ROP2 and GRA7) IgG antibody by goat-anti toxoplasma antigen (SAG1 (P30), SAG2 (P22), ROP2 and GRA7) IgG antibody by anti-SAG1 (P30)-IgG antibody, anti-SAG2 (P22)-IgG antibody, anti-ROP2-IgG antibody and anti-GRA7-IgG antibody 1: 1: 1 by volume: 1 mixes, and its final concentration is 1mg/mL; Three's point sample amount is 1 μ L/cm.
With arc worm recombinant antigen SAG1 (P30), SAG2 (P22), ROP2 and the GRA7 of the golden mark of purifying with volume ratio 1: 1: 1: 1 mix after, be applied to equably on the all-glass paper, 37 ℃ of oven dry, be prepared into the gold colloid pad, seal subsequent use.The spun glass that immobilised tunica fibrosa is combined with collaurum, thieving paper etc. are combined through PVC adhesive sticker base plate in certain sequence, are cut into the certain width detector bar with cutting cutter.The 1st reagent strip is connected with the 2nd reagent strip at application of sample pad place with all-glass paper again.Or the 1st reagent strip and the 2nd reagent strip put into the plastic casing of corresponding specification, make test card.Pack Toxoplasma Gondi IgM antibody in the aluminium foil bag into total antibody combined detectable bar or detection kit and drying agent, machine seals, and sealing is preserved.
Get the sample serum to be checked 120 μ L of dilution in 1: 10, application of sample leaves standstill the 20min observations in Toxoplasma Gondi IgM antibody and total antibody combined detectable bar sample application zone.Only there is an aubergine band to occur, then is judged to feminine gender at Toxoplasma Gondi IgM antibody and total antibody combined detectable bar check plot; All there is an aubergine band to occur at detection zone and check plot, then is judged to the positive; After application of sample detected, the aubergine band did not all appear in detection zone T and check plot C, is null result.(referring to Fig. 4)
Similar with embodiment 1, difference is that gold colloid pad, the total antibody detection line of arc worm only be made up of SAG1 (P30), SAG2 (P22), ROP2 and GRA7, does not contain SAG1 (P30), SAG2 (P22), ROP2 and GRA7.The result judges identical with embodiment 1.
Similar with embodiment 1, difference is that gold colloid pad, the total antibody detection line of arc worm only be made up of SAG1 (P30), SAG2 (P22), ROP2 and GRA7, does not contain SAG1 (P30), SAG2 (P22), ROP2 and GRA7.The result judges identical with embodiment 1.
Similar with embodiment 1, difference is that sample to be checked is a samples of CSF, and the result judges identical with embodiment 1.
The performance verification test: the scheme by embodiment 1 prepares Toxoplasma Gondi IgM antibody and total antibody combined quick detection reagent bar, carries out performance verification then.
1) visual examination: white encapsulates smooth, the clean pollution-free spot of reaction film, free from flaw, and adhesive tape does not have and comes unglued, and Toxoplasma Gondi IgM antibody and total antibody combined quick detection reagent bar width do not have and cut oblique phenomenon at 3 ± 0.1mm.
2) positive sample coincidence rate: the positive control serum with the different titers of Toxoplasma Gondi IgM and the total antibody positive of arc worm adopts Toxoplasma Gondi IgM antibodies and total antibody combined detectable bar to examine and determine for each 50 parts, calculates positive coincidence rate.The clinical samples that definite employing ELISA (import reagent) method of positive control serum is confirmed.
3) negative sample coincidence rate:, calculate positive coincidence rate with 50 parts of negative control serum calibratings.The clinical samples that definite employing ELISA (import reagent) method of the negative control serum of Toxoplasma Gondi IgM and the total antibody of arc worm is confirmed.
4) sensitivity detects: detect with the indoor quality controlled serum of the Ministry of Public Health, the minimum detectability degree should be less than or equal to 4NCU/mL.
5) criticize interior difference: same batch of Toxoplasma Gondi IgM antibody and total antibody combined detectable bar, detect with characteristic serum, require the positive serum testing result to show that the shade of colour band is consistent, result's feminine gender that negative serum detects.
6) differences between batches: different batches Toxoplasma Gondi IgM antibody and total antibody combined detectable bar, detect with characteristic serum, require the positive serum testing result to show that the shade of colour band is consistent, the result that negative serum detects is negative.
7) interference test: testing result does not receive the interference of sample hemolysis (n=50), piarhemia (n=50) and jaundice (n=50).Serum (or blood plasma) is from the applicant's clinical samples.
8) cross reaction: adopt Toxoplasma Gondi IgM antibody and total antibody combined detectable bar, carry out the detection of systemic loupus erythematosus (n=30), rheumatoid disease (n=30), autoallergic autoimmunity systemic diseases such as (n=30), do not find cross reaction.The serum of autoimmunity systemic disease is from the applicant's clinical definite patient.
9) Detection of Stability: use the Arrhenius rule, Toxoplasma Gondi IgM antibody and total antibody combined detectable bar are placed 37 ℃ of detections after 20 days, above each item index does not have marked change, guarantees that finished product preserves under the drying at room temperature condition, and the term of validity is 18 months.
Claims (10)
1. Toxoplasma Gondi IgM antibody and total antibody combined detectable bar is characterized in that being provided with the 1st reagent strip and the 2nd reagent strip;
The 1st reagent strip is provided with the 1st carrier board, the 1st application of sample pad, the 1st collaurum pad, the 1st nitrocellulose membrane, Toxoplasma Gondi IgM antibody detection line, the 1st control line and the 1st absorption pad; The 1st application of sample pad, the 1st collaurum pad, the 1st nitrocellulose membrane and the 1st absorption pad stick on the 1st carrier board upper surface successively; One end of the 1st application of sample pad is located on the end of the 1st collaurum pad; The other end of the 1st collaurum pad is located on the end of the 1st nitrocellulose membrane; One end of the 1st absorption pad is located on the other end of the 1st nitrocellulose membrane, and Toxoplasma Gondi IgM antibody detection line and the 1st control line are located on the 1st nitrocellulose membrane successively; Encapsulate anti-people IgM specific fragment μ chain monoclonal antibody at Toxoplasma Gondi IgM antibody detection line place, encapsulate the IgG antibody of goat-anti toxoplasma antigen SAG1 (P30), SAG2 (P22), ROP2 and GRA7 at the 1st control line place;
The 2nd reagent strip is provided with the 2nd carrier board, the 2nd application of sample pad, the 2nd collaurum pad, the 2nd nitrocellulose membrane, total antibody detection line, the 2nd control line and the 2nd absorption pad; The 2nd application of sample pad, the 2nd collaurum pad, the 2nd nitrocellulose membrane and the 2nd absorption pad stick on the 2nd carrier board upper surface successively; One end of the 2nd application of sample pad is located on the end of the 2nd collaurum pad; The other end of the 2nd collaurum pad is located on the end of the 2nd nitrocellulose membrane; One end of the 2nd absorption pad is located on the other end of the 2nd nitrocellulose membrane, and total antibody detection line and the 2nd control line are located on the 2nd nitrocellulose membrane successively; Encapsulate anti-people Ig monoclonal antibody at total antibody detection line place, encapsulate the IgG antibody of goat-anti toxoplasma antigen SAG1 (P30), SAG2 (P22), ROP2 and GRA7 at the 2nd control line place.
2. be connected with glass fibre membrane between Toxoplasma Gondi IgM antibody as claimed in claim 1 and the total antibody combined detectable bar, the 1st application of sample pad that it is characterized in that the 1st reagent strip and the 2nd application of sample pad of the 2nd reagent strip.
3. Toxoplasma Gondi IgM antibody as claimed in claim 1 and total antibody combined detectable bar is characterized in that said the 1st carrier board and the 2nd carrier board are the PVC plate.
4. the preparation method of Toxoplasma Gondi IgM antibody as claimed in claim 1 and total antibody combined detectable bar is characterized in that may further comprise the steps:
1) preparation arc worm recombinant antigen SAG1 (P30), SAG2 (P22), ROP2 and GRA7
Adopt gene clone technology, the DNA of pcr amplification coding toxoplasma antigen, and make its expression in the insertion Escherichia coli, get arc worm recombinant antigen SAG1 (P30), SAG2 (P22), ROP2 and GRA7;
2) point sample of nitrocellulose filter
On the Toxoplasma Gondi IgM antibody detection line, encapsulate anti-people IgM specific fragment μ chain monoclonal antibody; Encapsulate anti-people Ig monoclonal antibody at total antibody detection line place; Encapsulate the IgG antibody of goat-anti toxoplasma antigen SAG1 (P30), SAG2 (P22), ROP2 and GRA7 at the control line place of the 1st reagent strip and the 2nd reagent strip respectively, dry;
3) preparation collaurum
Adopt the trisodium citrate method of reducing to prepare the 25nm collaurum; Getting 1% gold chloride 1mL joins in the 100mL deionization distilled water; The gold chloride concentration that obtains is 0.01%, places the flask of band condensing unit to be heated to boiling, adds 1% trisodium citrate aqueous solution 2.0mL under the magnetic force heated and stirred; Continue heating till solution is vinicolor, cooling is placed in the brown bottle 4 ℃ of refrigerators and preserves subsequent use;
4) mark of collaurum and SAG1 (P30), SAG2 (P22), ROP2 and GRA7
(1) mark of collaurum and toxoplasma antigen SAG1 (P30): get collaurum 10m L, transfer to pH5.4, add 100 μ g SAG1 (P30), mixing with 0.1mol/L NaOH; Place 5min, add 5%BSA 1m L mixing, 4 ℃, the centrifugal 1h of 10000r/min; Abandon supernatant, will precipitate with the TBS damping fluid and be dissolved to 10m L, 4 ℃, the centrifugal 1h of 10000r/min; Abandon supernatant, deposition is diluted to 1mL with TBS, gets SAG1 (P30) antigen of colloid gold label;
(2) collaurum is identical with step (1) with the labeling method of toxoplasma antigen SAG2 (P22), ROP2 and GRA7, gets SAG2 (P22), ROP2 and the GRA7 antigen of colloid gold label;
(3) after the GRA7 mixed antigen with SAG1 (P30) antigen, colloid gold label SAG2 (P22), colloid gold label ROP2 and the colloid gold label of colloid gold label, be applied to equably on the glass fibre membrane, oven dry is prepared into the collaurum pad;
5) preparation immunochromatography detector bar
The 1st reagent strip is provided with the 1st carrier board, the 1st application of sample pad, the 1st collaurum pad, the 1st nitrocellulose membrane, Toxoplasma Gondi IgM antibody detection line, the 1st control line and the 1st absorption pad; The 1st application of sample pad, the 1st collaurum pad, the 1st nitrocellulose membrane and the 1st absorption pad stick on the 1st carrier board upper surface successively; One end of the 1st application of sample pad is located on the end of the 1st collaurum pad; The other end of the 1st collaurum pad is located on the end of the 1st nitrocellulose membrane; One end of the 1st absorption pad is located on the other end of the 1st nitrocellulose membrane, and Toxoplasma Gondi IgM antibody detection line and the 1st control line are located on the 1st nitrocellulose membrane successively; Encapsulate anti-people IgM specific fragment μ chain monoclonal antibody at Toxoplasma Gondi IgM antibody detection line place; Encapsulate the IgG antibody of goat-anti toxoplasma antigen SAG1 (P30), SAG2 (P22), ROP2 and GRA7 at the 1st control line place; Be cut into strip with cutting cutter, get the 1st reagent strip of Toxoplasma Gondi IgM antibody and total antibody combined detectable bar;
The 2nd reagent strip is provided with the 2nd carrier board, the 2nd application of sample pad, the 2nd collaurum pad, the 2nd nitrocellulose membrane, total antibody detection line, the 2nd control line and the 2nd absorption pad; The 2nd application of sample pad, the 2nd collaurum pad, the 2nd nitrocellulose membrane and the 2nd absorption pad stick on the 2nd carrier board upper surface successively; One end of the 2nd application of sample pad is located on the end of the 2nd collaurum pad; The other end of the 2nd collaurum pad is located on the end of the 2nd nitrocellulose membrane; One end of the 2nd absorption pad is located on the other end of the 2nd nitrocellulose membrane, and total antibody detection line and the 2nd control line are located on the 2nd nitrocellulose membrane successively; Encapsulate anti-people Ig monoclonal antibody at total antibody detection line place; Encapsulate the IgG antibody of goat-anti toxoplasma antigen SAG1 (P30), SAG2 (P22), ROP2 and GRA7 at the 2nd control line place; Be cut into strip with cutting cutter, get the 2nd reagent strip of Toxoplasma Gondi IgM antibody and total antibody combined detectable bar.
5. the preparation method of Toxoplasma Gondi IgM antibody as claimed in claim 4 and total antibody combined detectable bar is characterized in that in step 2) in, the concentration of said anti-people Ig monoclonal antibody is 1～4mg/mL.
6. the preparation method of Toxoplasma Gondi IgM antibody as claimed in claim 4 and total antibody combined detectable bar is characterized in that in step 2) in, the concentration of anti-people IgM specific fragment μ chain monoclonal antibody is 1～4mg/mL.
7. the preparation method of Toxoplasma Gondi IgM antibody as claimed in claim 4 and total antibody combined detectable bar; It is characterized in that in step 2) in; The IgG antibody of goat-anti toxoplasma antigen SAG1 (P30), SAG2 (P22), ROP2 and GRA7 was by anti-SAG1 (P30) antibody, anti-SAG2 (P22), anti-ROP2 and anti-GRA7 antibody 1: 1: 1 by volume: 1 mixes, and its final concentration is 1～4mg/mL; The point sample amount is 1 μ L/cm.
8. the preparation method of Toxoplasma Gondi IgM antibody as claimed in claim 4 and total antibody combined detectable bar is characterized in that in step 3), and the percent concentration of said trisodium citrate is 2%.
9. the preparation method of Toxoplasma Gondi IgM antibody as claimed in claim 4 and total antibody combined detectable bar; It is characterized in that in step 4); SAG2 (P22) antigen of said SAG1 with colloid gold label (P30) antigen, colloid gold label, the ROP2 antigen of colloid gold label and the GRA7 mixed antigen of colloid gold label, the GRA7 antigen of SAG2 (P22) antigen of the SAG1 of colloid gold label (P30) antigen, colloid gold label, the ROP2 antigen of colloid gold label and colloid gold label are with volume ratio 1: (0.2～5): (0.2～5): mix (0.2～5).
10. the preparation method of Toxoplasma Gondi IgM antibody as claimed in claim 4 and total antibody combined detectable bar is characterized in that in step 4), and the temperature of said oven dry is 37 ℃.
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Application publication date: 20120111 |