CN102313811A - Toxoplasma gondii IgG antibody colloidal gold immunity chromatography detection reagent strip and its preparation method - Google Patents
Toxoplasma gondii IgG antibody colloidal gold immunity chromatography detection reagent strip and its preparation method Download PDFInfo
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Abstract
A Toxoplasma gondii IgG antibody colloidal gold immunity chromatography detection reagent strip and its preparation method relate to a Toxoplasma gondii IgG antibody detection reagent and provide a Toxoplasma gondii IgG antibody colloidal gold immunity chromatography detection reagent strip and its preparation method. The reagent strip provided by the invention is provided with a vector board, a sampling pad, a colloidal gold pad, a nitric acid fiber film, a Toxoplasma gondii IgG antibody detection line, a contrast line and an absorption pad. The preparation method provided by the invention comprises the following steps of: preparation of Toxoplasma gondii recombinant antigen SAG1(P30), SAG2(P22), ROP2 and GRA7; spotting of the cellulose nitrate membrane; preparation of colloidal gold; marking of colloidal gold with SAG1(P30), SAG2(P22), ROP2 and GRA7; and preparation of the immunity chromatography detection reagent strip. The detection method provided by the invention requires little amount of specimen and no special apparatus; a 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 IgG antibody detectable, especially relate to Toxoplasma Gondi IgG antibody quick detection reagent bar that a kind of employing colloidal gold immunochromatographimethod technology (immunochromatography) carries out 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.
Existing 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.At present, detect IgG antibody 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 IgG antibody colloidal gold immunochromatographiassay assay reagent bar and preparation method thereof.
Said Toxoplasma Gondi IgG antibody colloidal gold immunochromatographiassay assay reagent bar is provided with carrier board, application of sample pad, collaurum pad, nitrocellulose membrane (NC film), Toxoplasma Gondi IgG antibody detection line, control line and absorption pad; Said application of sample pad, collaurum pad, nitrocellulose membrane and absorption pad stick on the carrier board upper surface successively; One end of said application of sample pad is located on the end of collaurum pad; The other end of collaurum pad is located on the end of nitrocellulose membrane; One end of absorption pad is located on the other end of nitrocellulose membrane, and Toxoplasma Gondi IgG antibody detection line and control line are located on the nitrocellulose membrane successively; Encapsulate anti-human IgG specific fragment γ chain monoclonal antibody at Toxoplasma Gondi IgG 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.
Said carrier board can adopt the PVC plate.
The preparation method of said Toxoplasma Gondi IgG antibody colloidal gold immunochromatographiassay assay reagent 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 nitrocellulose filter IgG detection line, encapsulate anti-human IgG specific fragment γ chain monoclonal antibody, the IgG antibody that the control line place on nitrocellulose filter encapsulates goat-anti toxoplasma antigen SAG1 (P30), SAG2 (P22), ROP2 and GRA7 dries;
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 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 10 000r/min; Abandon supernatant, will precipitate with the TBS damping fluid and be dissolved to 10ml, 4 ℃, the centrifugal 1h of 10 000r/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
Application of sample pad, collaurum pad, nitrocellulose membrane and absorption pad are sticked on the carrier board upper surface successively; One end of application of sample pad is located on the end of collaurum pad; The other end of collaurum pad is located on the end of nitrocellulose membrane; One end of absorption pad is located on the other end of nitrocellulose membrane, and Toxoplasma Gondi IgG antibody detection line and control line are located on the nitrocellulose membrane successively; Encapsulate anti-human IgG specific fragment γ chain monoclonal antibody at Toxoplasma Gondi IgG 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; Be cut into strip with cutting cutter, get Toxoplasma Gondi IgG antibody colloidal gold immunochromatographiassay assay reagent bar.
In step 2) in; The concentration of said anti-human IgG specific fragment γ chain monoclonal antibody can be 1～4mg/mL; The IgG antibody of goat-anti toxoplasma antigen SAG1 (P30), SAG2 (P22), ROP2 and GRA7 can be by volume 1: 1: 1 by anti-SAG1 (P30) antibody, anti-SAG2 (P22) antibody, anti-ROP2 antibody and GRA7 antibody: 1 mixes, and its final concentration can be 1～4mg/mL; The two point sample amount can be 1 μ L/cm.
In step 3), the percent concentration of said trisodium citrate can be 2%.
In step 4); SAG2 (P22) antigen 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, the GRA7 mixed antigen volume ratio of the ROP2 antigen of SAG2 (P22) antigen of SAG1 (P30) antigen of the colloid gold label of right colloid gold label, colloid gold label, colloid gold label and colloid gold label can be by 1: (0.2～5): (0.2～5): mix (0.2～5); The temperature of said oven dry can be 37 ℃.
The invention provides a kind of employing colloidal gold immunochromatographimethod technology and set up Toxoplasma Gondi IgG antibody quick detection reagent bar, can be used for the detection of Toxoplasma Gondi IgG antibody in the samples such as whole blood, serum, blood plasma and cerebrospinal fluid.The required specimen amount of this detection method 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 IgG antibody colloidal gold immunochromatographiassay assay reagent bar embodiment according to the invention is formed synoptic diagram.
Fig. 2 is the experimental result pattern diagram.In Fig. 2, A is the synoptic diagram before using, and B is invalid test (product quality problem), the negative result of C, and D is arc worm-IgG positive findings; 6 is the Toxoplasma Gondi IgG antibody detection line, and 7 is control line.
Embodiment
Following examples will combine accompanying drawing that the present invention is further described.
Referring to Fig. 1, Toxoplasma Gondi IgG antibody colloidal gold immunochromatographiassay assay reagent bar embodiment according to the invention is provided with carrier board 1, application of sample pad 2, collaurum pad 3, nitrocellulose membrane (NC film) 4, Toxoplasma Gondi IgG antibody detection line 6, control line 7 and absorption pad 8.
Said application of sample pad 2, collaurum pad 3, nitrocellulose membrane 4 and absorption pad 8 stick on carrier board 1 upper surface successively; One end of application of sample pad 2 is located on the end of collaurum pad 3; The other end of collaurum pad 3 is located on the end of nitrocellulose membrane 4; One end of absorption pad 8 is located on the other end of nitrocellulose membrane 4, and Toxoplasma Gondi IgG antibody detection line 6 is located on the nitrocellulose membrane 4 with control line 7 successively; Encapsulate anti-human IgG specific fragment γ chain monoclonal antibody at Toxoplasma Gondi IgG 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.
Said carrier board adopts the PVC plate.
The preparation method of said Toxoplasma Gondi IgG antibody colloidal gold immunochromatographiassay assay reagent 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 nitrocellulose filter IgG detection line, encapsulate anti-human IgG specific fragment γ chain monoclonal antibody, encapsulate goat-anti toxoplasma antigen SAG1 (P30), SAG2 (P22), ROP2 and GRA7 IgG antibody at the control line place, dry; The concentration of said anti-human IgG specific fragment γ chain monoclonal antibody is 1～4mg/mL; The IgG antibody of goat-anti toxoplasma antigen SAG1 (P30), SAG2 (P22), ROP2 and GRA7 was 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 1～4mg/mL; The two point sample amount is 1 μ L/cm.
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; The concentration of said trisodium citrate is 2%.
4) mark of collaurum and toxoplasma antigen SAG1 (P30), SAG2 (P22), ROP2 and GRA7:
The 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 10 000r/min; Abandon supernatant, will precipitate with the TBS damping fluid and be dissolved to 10ml, 4 ℃, the centrifugal 1h of 10 000r/min; Abandon supernatant, deposition is diluted to 1ml with TBS, gets SAG1 (P30) antigen of colloid gold label;
The same operation of mark of collaurum and toxoplasma antigen SAG2 (P22), ROP2 and GRA7, respectively SAG2 (P22), ROP2 and the GRA7 antigen of colloid gold label;
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;
With the GRA7 antigen of the ROP2 antigen of SAG2 (P22) antigen of SAG1 (P30) antigen of colloid gold label, colloid gold label, colloid gold label and colloid gold label with volume ratio (0～5): (0～5): (0～5): after mix (0～5); Be applied on the glass fibre membrane equably; 37 ℃ of oven dry; Be prepared into the collaurum pad, seal subsequent use.
5) preparation immunochromatography detector bar
Application of sample pad, collaurum pad, nitrocellulose membrane and absorption pad are sticked on the carrier board upper surface successively; One end of application of sample pad is located on the end of collaurum pad; The other end of collaurum pad is located on the end of nitrocellulose membrane; One end of absorption pad is located on the other end of nitrocellulose membrane, and Toxoplasma Gondi IgG antibody detection line and control line are located on the nitrocellulose membrane successively; Encapsulate anti-human IgG specific fragment γ chain monoclonal antibody at Toxoplasma Gondi IgG 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; Be cut into strip with cutting cutter, get Toxoplasma Gondi IgG antibody colloidal gold immunochromatographiassay assay reagent bar.
Below provide the clinical samples that adopts Toxoplasma Gondi IgG antibody colloidal gold immunochromatographiassay assay reagent bar to detect the patient:
Get sample to be checked (whole blood, serum, blood plasma, cerebrospinal fluid) 5～40 μ L, application of sample drips 100 μ L physiological saline in Toxoplasma Gondi IgG antibody colloidal gold immunochromatographiassay assay reagent bar sample place, leaves standstill the 20min observations.Only there is an aubergine band to occur, then is judged to feminine gender in the detector bar check plot; All there is an aubergine band to occur at detection zone (T) and check plot, then is 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 2).
Below provide the performance calibrating of Toxoplasma Gondi IgG antibody colloidal gold immunochromatographiassay assay reagent bar:
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 positive different titers of arc worm-IgG adopts the calibrating of Toxoplasma Gondi IgG antibody colloidal gold immunochromatographiassay assay reagent bar 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 negative match-rate with 50 parts of negative control serum calibratings.The clinical samples that definite employing ELISA (import reagent) method of negative control serum is confirmed.
4) criticize interior difference: same batch of Toxoplasma Gondi IgG antibody colloidal gold immunochromatographiassay assay reagent 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.
5) differences between batches: different batches Toxoplasma Gondi IgG antibody colloidal gold immunochromatographiassay assay reagent 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.
6) 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.
7) cross reaction: adopt this Toxoplasma Gondi IgG antibody colloidal gold immunochromatographiassay assay reagent 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.
8) Detection of Stability: use the Arrhenius rule, Toxoplasma Gondi IgG antibody colloidal gold immunochromatographiassay assay reagent bar is placed 37 ℃ detect 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.
Detection of the present invention is carried out on a Toxoplasma Gondi IgG antibody colloidal gold immunochromatographiassay assay reagent bar, realizes the detection of Toxoplasma Gondi IgG antibody through following dual mode:
Mode one: utilize the colloidal gold immunochromatographimethod technology, IgG detection line and control line place encapsulate anti-human IgG specific fragment γ chain monoclonal antibody and goat-anti toxoplasma antigen SAG1 (P30), SAG2 (P22), ROP2 and GRA7 IgG antibody respectively on nitrocellulose filter.After arc worm recombinant antigen SAG1 (P30), SAG2 (P22), ROP2 and the GRA7 of the golden mark of purifying mix in certain proportion, be coated on the all-glass paper in advance; Dried; Be prepared into the gold colloid pad; Be aided with appropriate application of sample pad again, combination Toxoplasma Gondi IgG antibody colloidal gold immunochromatographiassay assay reagent bar (assembling mode is seen Fig. 1).When detecting positive sample, Toxoplasma Gondi IgG antibody combines to form immune complex in the sample with recombinant antigen SAG1 (P30), SAG2 (P22), ROP2 and the GRA7 of colloid gold label.Because the chromatography effect, compound moves forward along the thieving paper direction of absorption pad.During through detection line, 1. arc worm-IgG para-immunity compound combines with the anti-human IgG monoclonal antibody that encapsulates in advance to form " Au-SAG1 (P30) (and/or SAG2 (P22) and/or ROP2 and/or GRA7)-specific anti Toxoplasma Gondi IgG antibody-anti-human IgG monoclonal antibody-solid phase material " centre-fills and condenses colour developing; 2. then enrichment develops the color free gold mark antigen with goat-anti toxoplasma antigen (SAG1 (P30), SAG2 (P22), ROP2 and GRA7) antibodies at the control line place.Negative sample then only develops the color at the control line place.
Mode two; Envelope antigen, the antibody of mode one are exchanged: IgG detection line and control line place encapsulate arc worm recombinant antigen (SAG1 (P30), SAG2 (P22), ROP2 and GRA7 combination) and sheep anti-mouse igg antibody respectively on nitrocellulose filter, and the anti-human IgG specific fragment γ chain monoclonal antibody of golden mark is coated on the all-glass paper in advance.
Below provide specific embodiment.
Embodiment 1
On nitrocellulose filter (NC film) IgG detection line, encapsulate anti-human IgG specific fragment γ chain monoclonal antibody; Encapsulate goat-anti toxoplasma antigen (SAG1 (P30), SAG2 (P22), ROP2 and GRA7) antibody at the control line place; Room temperature is dried, and the sealing room temperature preservation is subsequent use.Wherein, The concentration of anti-human IgG specific fragment γ chain 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; The two 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.Pack Toxoplasma Gondi IgG antibody colloidal gold immunochromatographiassay assay reagent bar in the aluminium foil bag into drying agent, machine seals, and sealing is preserved.
Get sample serum 10 μ L to be checked, application of sample drips 100 μ L physiological saline simultaneously in Toxoplasma Gondi IgG antibody colloidal gold immunochromatographiassay assay reagent bar sample application zone, leaves standstill the 20min observations.Only there is an aubergine band to occur, then is judged to feminine gender in Toxoplasma Gondi IgG antibody colloidal gold immunochromatographiassay assay reagent 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 and check plot, is null result.(see figure 2)
Similar with embodiment 1, difference is that the gold colloid pad only is made up of SAG1 (P30), ROP2 and GRA7, does not contain SAG2 (P22).The result judges identical with embodiment 1.
Similar with embodiment 1, difference is that the gold colloid pad only is made up of SAG2 (P22), ROP2 and GRA7, does not contain SAG1 (P30).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 IgG antibody colloidal gold immunochromatographiassay assay 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 IgG antibody colloidal gold immunochromatographiassay assay reagent bar width does not have and cuts oblique phenomenon at 3 ± 0.1mm.
2) positive sample coincidence rate: 50 parts of positive control serums of arc worm-IgG of confirming through ELISA (import reagent) detection; Adopt the Toxoplasma Gondi IgG antibody colloidal gold immunochromatographiassay assay reagent bar of invention to detect positive 50 parts of arc worm-IgG, positive sample coincidence rate 100%.
3) negative sample coincidence rate: the negative control serum that 50 parts of ELISA (import reagent) are definite, adopt Toxoplasma Gondi IgG antibody colloidal gold immunochromatographiassay assay reagent bar not detect positive sample, negative sample coincidence rate 100%.
4) criticize interior difference: same batch of Toxoplasma Gondi IgG antibody colloidal gold immunochromatographiassay assay reagent bar; Detecting the positive arc worm of the clinical samples of confirming-high, medium and low serum of IgG reference with characteristic positive serum (ELISA (import reagent)) detects; Identical titre testing result shows that the shade of colour band is consistent, and the result that negative serum detects is negative.
5) differences between batches: different batches Toxoplasma Gondi IgG antibody colloidal gold immunochromatographiassay assay reagent bar; Detecting the positive arc worm of the clinical samples of confirming-high, medium and low serum of IgG reference with characteristic positive serum (ELISA (import reagent)) detects; Identical titre testing result shows that the shade of colour band is consistent, and the result that negative serum detects is negative.
6) interference test: testing result does not receive the interference of sample hemolysis (n=50), piarhemia (n=50) and jaundice (n=50).
7) cross reaction: adopt this Toxoplasma Gondi IgG antibody colloidal gold immunochromatographiassay assay reagent bar, carry out the detection of systemic loupus erythematosus (n=30), rheumatoid disease (n=38), autoallergic autoimmunity systemic diseases such as (n=40), do not find cross reaction.
8) Detection of Stability: Toxoplasma Gondi IgG antibody colloidal gold immunochromatographiassay assay reagent bar is placed 37 ℃ detect after 20 days, above each item index does not have marked change.
Claims (8)
1. Toxoplasma Gondi IgG antibody colloidal gold immunochromatographiassay assay reagent bar is characterized in that being provided with carrier board, application of sample pad, collaurum pad, nitrocellulose membrane, Toxoplasma Gondi IgG antibody detection line, control line and absorption pad; Application of sample pad, collaurum pad, nitrocellulose membrane and absorption pad stick on the carrier board upper surface successively; One end of application of sample pad is located on the end of collaurum pad; The other end of collaurum pad is located on the end of nitrocellulose membrane; One end of absorption pad is located on the other end of nitrocellulose membrane, and Toxoplasma Gondi IgG antibody detection line and control line are located on the nitrocellulose membrane successively; Encapsulate anti-human IgG specific fragment γ chain monoclonal antibody at Toxoplasma Gondi IgG 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.
2. Toxoplasma Gondi IgG antibody colloidal gold immunochromatographiassay assay reagent bar as claimed in claim 1 is characterized in that said carrier board is the PVC plate.
3. the preparation method of Toxoplasma Gondi IgG antibody colloidal gold immunochromatographiassay assay reagent bar as claimed in claim 1 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 nitrocellulose filter IgG detection line, encapsulate anti-human IgG specific fragment γ chain monoclonal antibody, encapsulate the IgG antibody of goat-anti toxoplasma antigen SAG1 (P30), SAG2 (P22), ROP2 and GRA7 at the control line place, 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 1m L mixing, 4 ℃, the centrifugal 1h of 10 000r/min; Abandon supernatant, will precipitate with the TBS damping fluid and be dissolved to 10m L, 4 ℃, the centrifugal 1h of 10 000r/min; Abandon supernatant, deposition is diluted to 1mL with TBS, gets SAG1 (P30) antigen of colloid gold label;
(2) collaurum is identical with step (2) 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 colloidal gold mark 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
Application of sample pad, collaurum pad, nitrocellulose membrane and absorption pad are sticked on the carrier board upper surface successively; One end of application of sample pad is located on the end of collaurum pad; The other end of collaurum pad is located on the end of nitrocellulose membrane; One end of absorption pad is located on the other end of nitrocellulose membrane, and Toxoplasma Gondi IgG antibody detection line and control line are located on the nitrocellulose membrane successively; Encapsulate anti-human IgG specific fragment γ chain monoclonal antibody at Toxoplasma Gondi IgG 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; Be cut into strip with cutting cutter, get Toxoplasma Gondi IgG antibody colloidal gold immunochromatographiassay assay reagent bar.
4. the preparation method of Toxoplasma Gondi IgG antibody colloidal gold immunochromatographiassay assay reagent bar as claimed in claim 3 is characterized in that in step 2) in, the concentration of said anti-human IgG specific fragment γ chain monoclonal antibody is 1～4mg/mL.
5. the preparation method of Toxoplasma Gondi IgG antibody colloidal gold immunochromatographiassay assay reagent bar as claimed in claim 3; It is characterized in that in step 2) in; The IgG antibody of said 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.
6. the preparation method of Toxoplasma Gondi IgG antibody colloidal gold immunochromatographiassay assay reagent bar as claimed in claim 3 is characterized in that in step 3), and the percent concentration of said trisodium citrate is 2%.
7. the preparation method of Toxoplasma Gondi IgG antibody detectable bar as claimed in claim 3; 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).
8. the preparation method of Toxoplasma Gondi IgG antibody colloidal gold immunochromatographiassay assay reagent bar as claimed in claim 3 is characterized in that in step 4), and the temperature of said oven dry is 37 ℃.
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Application publication date: 20120111 |