Immunochromatographic assay test paper for diagnosing psittacosis chlamydia infection and preparation method thereof

Immunochromatographic assay test paper for diagnosing psittacosis chlamydia infection and preparation method thereof

  • CN 1,866,015 B
  • Filed: 06/06/2006
  • Issued: 05/09/2012
  • Est. Priority Date: 06/06/2006
  • Status: Active Grant
First Claim
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1. an immune chromatography test paper that detects chlamydia psitacci infection comprises the sample pad of closely connecting successively, gold mark pad, tunica fibrosa and the adsorptive pads that contains staphylococcus aureus protein A mark colloidal gold probe or anti-chicken IgG mark colloidal gold probe;

  • Said tunica fibrosa is coated with detection line and the nature controlling line that is separated from each other, and said detection line is a Cps MOMP, and said nature controlling line is antibody that can combine with staphylococcus aureus protein A or the antibody that can combine with anti-chicken IgG;

    When said gold mark pad contained staphylococcus aureus protein A mark colloidal gold probe, said nature controlling line was the antibody that can combine with staphylococcus aureus protein A;

    When said gold mark pad contained anti-chicken IgG mark colloidal gold probe, said nature controlling line was the antibody that can combine with anti-chicken IgG;

    The preparation method of the immune chromatography test paper of said detection chlamydia psitacci infection may further comprise the steps;

    1) Cps MOMP is sprayed onto on the tunica fibrosa, encapsulates a zone of tunica fibrosa, obtain detection line;

    The antibody-solutions that can combine with staphylococcus aureus protein A or can be sprayed onto on the tunica fibrosa with the antibody-solutions that anti-chicken IgG combines encapsulates another zone of tunica fibrosa, obtains nature controlling line;

    37 ℃

    of dryings 0.5~

    1.0 hour stick on it on adsorptive pads apart from the nearer end of nature controlling line then;

    2) preparation staphylococcus aureus protein A or anti-chicken IgG mark colloidal gold probe;

    Staphylococcus aureus protein A or anti-chicken IgG mark colloidal gold probe are dissolved in the sodium tetraborate solution that mass percent concentration is 0.25-0.4%;

    Making its whole mass percent concentration is 5-10%;

    Then glass fibre membrane, resin are immersed respectively in the above-mentioned probe solution, obtain gold mark pad, at-20 ℃



    -50 ℃

    after freezing 5~

    8 hours;

    After freezing the draining, respectively with its stick on tunica fibrosa that step

         1) obtains apart from a nature controlling line end far away;

    3) in step

         2) in the other end of gold mark pad paste sample pad again, obtain detecting the immune chromatography test paper of chlamydia psitacci infection;

    Said staphylococcus aureus protein A or anti-chicken igg antibody mark colloidal gold probe;

    Preparation as follows;

    it is 0.01% that water dissolved chlorine auric acid makes its final concentration;

    Boil the every 100ml chlorauric acid solution in back and carry out following operation;

    the trisodium citrate aqueous solution 1.6-1.8ml of adding 1%;

    Continued to boil 10-15 minute, and returned to 100ml, use K after being cooled to room temperature with pure water 2CO 3Transfer pH to 6-7;

    Stir and add 0.7mg staphylococcus aureus protein A or 0.8mg mouse-anti chicken IgG;

    The polyglycol (20000) that adds 2ml 10% behind the 20min-30min continues to stir 20min-30min, the centrifugal 25min-35min of 9000rpm-12000rpm;

    Abandon supernatant, deposition is staphylococcus aureus protein A mark colloidal gold probe or mouse-anti chicken IgG mark colloidal gold probe;

    Said percent concentration is a mass percent concentration;

    The preparation method of said Cps MOMP is following;

    Design primer, amplification have the nucleotide sequence fragment of sequence 1 in the sequence table that the patent No. is 02146790.0 patent of invention, wherein have Nco I and Hind III enzyme recognition site on the primer;

    Then;

    With amplified fragments with Nco I and Hind III double digestion;

    Be inserted into the recombinant vector of the encoding gene that obtains containing Cps MOMP between Nco I and the Hind III enzyme recognition site of carrier pET32a (+);

    Change this recombinant vector over to recipient bacterium BL21 (DE3), screening obtains expressing the engineering bacteria with protein of the amino acid residue sequence of sequence 2 in the sequence table that the patent No. is 02146790.0 patent of invention;

    The above-mentioned engineering bacteria that obtains is seeded in the LB flat board, cultivated 20~

    24 hours for 37 ℃

    , the single colonies typical inoculation of picking 10ml LB meat soup, 37 ℃

    of shaking table concussions were cultivated 10~

    12 hours, taked 1;

    10 enlarged culture method until getting into fermentation reactor;

    Fermentation medium is 2 * YT nutrient culture media, and the volume ratio with 1;

    50 during fermentation is inoculated;

    The control temperature is 37 ℃

    in the fermentation, stirs revolution 350~

    500rpm, throughput 1L nutrient culture media 1L/min air, and fermentation reactor is with 1mol/L NaOH and 30%NaH 2PO 4Regulate fermentation liquor pH7.0;

    Record fermentation liquor absorbance, when fermentation liquor A value reaches 0.8, adding the derivant isopropyl-, to make its final concentration be 0.02mmol/L, and downward modulation temperature to 26 ℃

    , stirring revolution and throughput are constant;

    After inducing 3-4 hour, whole fermentation ends;

    The extraction of Cps MOMP and purifying;

    zymocyte liquid was collected bacterial sediment with the centrifugal 10-15 of 8000g-9000g minute, added the 1ml pure water by every g weight in wet base thalline, put into refiner, made thalline homogenate under hypotonic environment broken;

    Collect homogenate and be rough MOMP antigen;

    Get rough MOMP antigen 1 00 μ

    l and carry out the SDS-PAGE analysis by conventional method, the result has a tangible protein band at the 51Kd place, and protein content accounts for total protein concentration more than 50%;

    Get rough Cps MOMP, 4 ℃

    the centrifugal 10-15 of 8000g-9000g minute, abandon deposition;

    Collect supernatant, supernatant was abandoned supernatant in 4 ℃

    of centrifugal 10-15 of 10000g-12000g minutes;

    Collecting precipitation occlusion body, occlusion body are washed 3 times with PBS again, the 4 ℃

    of centrifugal 10-15 of 10000g-12000g minute collecting precipitation occlusion bodies;

    Contain 8mol/L urea PBS 30ml by every g weight in wet base occlusion body adding, blow and beat to occlusion body dissolving and albuminous degeneration for 37 ℃

    , collect sex change MOMP in bag filter;

    With the PBS dislysate 1000ml dialysis of the urea content that successively decreases, different dislysates are respectively dialysed and were carried out protein renaturation in 4 hours, collect refolded protein liquid respectively;

    4 ℃

    the centrifugal 10-15 of 10000g-12000g minute, abandon deposition, collect the MOMP antigen after supernatant is purifying;

    The method for coating of said tunica fibrosa is following;

    Dilute said Cps MOMP to 2~

    3mg/ml with 0.01M pH 7.2 phosphate buffers respectively and be used to encapsulate detection line;

    Diluting goat anti-human igg 2.5mg/ml or sheep anti-mouse igg 2-3mg/ml respectively with the PBS of 0.01M pH 7.2 is respectively applied for and encapsulates nature controlling line;

    Be sprayed on respectively on the thick nitrocellulose filter of 2.5mm with the XYZ3000 of BIODOT company Membrane jetter, form the detection line and the nature controlling line that are separated from each other, 37 ℃

    of dry 2.5h process two kinds of NC films that nature controlling line is respectively goat anti-human igg or sheep anti-mouse igg.

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