CN105652004B - A kind of TMP haptens and its collaurum detection means and preparation method thereof - Google Patents

A kind of TMP haptens and its collaurum detection means and preparation method thereof Download PDF

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CN105652004B
CN105652004B CN201511020191.9A CN201511020191A CN105652004B CN 105652004 B CN105652004 B CN 105652004B CN 201511020191 A CN201511020191 A CN 201511020191A CN 105652004 B CN105652004 B CN 105652004B
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tmp
haptens
monoclonal antibody
collaurum
immunizing antigen
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严义勇
朱海
付辉
李君秀
李细清
毕思远
汪凤林
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Shenzhen Rui Rui Biotechnology Ltd By Share Ltd
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • G01—MEASURING; TESTING
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody

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Abstract

The invention provides a kind of TMP haptens and its collaurum detection means and preparation method thereof, the TMP haptens, for formula（1）Shown chemical structural formula.High using technical scheme, high specificity, detection sensitivity, accuracy is high, reproducible, can more quick, easily detect TMP residual；Any instrument and equipment is not needed, is easy to carry, testing cost is low；Test strips operate using simply without professional person；It is easy that test paper makes, and cost is cheap, meets in food safety detection to TMP residues detection demand.

Description

A kind of TMP haptens and its collaurum detection means and preparation method thereof
Technical field
The invention belongs to technical field of biological, more particularly to a kind of TMP haptens and its collaurum detection dress Put and preparation method thereof.
Background technology
TMP is a kind of lipophilic alkalescent pyrimethamine class bacteriostatic agent.Its antimicrobial spectrum is similar with sulfa drugs, but Antibacterial action is strong compared with sulfa drugs, blue to Escherichia coli, proteus mirabilis, pneumobacillus, staphylococcus saprophyticus, a variety of leather Positive and negative bacteria is effective but invalid to charrin's disease, and minimum inhibitory concentration is often less than 10mg/L, alone easily to cause carefully Bacterium drug resistance, therefore be not used alone typically, mainly form compound preparation with sulfa drug.Therefore it is otherwise known as trimethoprim (TMP), For expanding antimicrobial spectrum, strengthen antibacterial activity.Clinically be used for treat urinary tract infections, enteric infection, respiratory tract infection, bacillary dysentery, Enteritis, typhoid fever, meningitis, tympanitis, epidemic meningitis, septicemia and soft tissue infection etc..It can share with long-acting sulfonamide and be disliked in resistance The preventing and treating of property malaria.Irrational use on veterinary clinic with sulfa drugs, or even abuse, so that Trimethoprim class Residual of the medicine in animal derived food can inevitably occur.Animal of mankind's long-term consumption containing TMP residual Property food and its product, it would be possible to harm is produced to human health.Therefore in order to strengthen to this kind of medicine in animal food Monitoring, it is necessary to establish a kind of accurately, fast and easily TMP residues detection method.
At present, the method for detection TMP mainly has high performance liquid chromatography (HPLC), liquid phase-mass spectrometry both at home and abroad (LC-MASS) method and Enzyme-linked Immunosorbent Assay (ELISA) method.In food safety detection, often first use ELISA primary dcreening operations after again to sun Property sample is confirmed with HPLC or LC-MASS.Instrument equipment safety is complicated in the above method, cost is high, to operator Member's technical requirements are high and can not show result immediately, therefore are not suitable for commodity inspection, epidemic prevention, husbandry sector person to object of suspicion progress Quick on-line checking and monitoring.
The content of the invention
For above technical problem, the invention discloses a kind of TMP haptens and its collaurum detection means and its Preparation method, for the deficiency of existing detection TMP technology, a kind of fast method for detecting TMP residual is established, is made It can more quick, sensitively, and conveniently detect TMP residual, meet the needs of quick detection, provided to food security Important means.
On the other hand, the technical solution adopted by the present invention is：
A kind of TMP haptens, it has the chemical structural formula shown in formula (1)：
The chemical entitled 3- (4- ((2,4- di-amino-pyrimidines -5-) methyl -2,6- Dimethoxyphenyls) third of above-mentioned molecular formula Acid, C16H20N4O5, MASS [M+H] is 349.2, fusing point：214 degree.
As a further improvement on the present invention, described TMP haptens is prepared using following steps：
Step S1：Hydrobromic acid is added in TMP, 2~4h is reacted at 90~110 DEG C, then adds sodium hydroxide Solution is quenched, after cooling separates out crystal, by dissolution of crystals in boiling water, and regulation pH value to neutrality, recrystallization purification；
Step S2：Take the crystal purified in step S1 to be dissolved in DMF (dimethylformamide), add potassium carbonate and bromine third Acid, 40 DEG C are reacted 4~8 hours, are adopted and post was extracted with ethyl acetate is purified to obtain the TMP haptens.
As a further improvement on the present invention, the mol ratio of the TMP and hydrobromic acid is 1：3~12；Preferably, The mass percent concentration of the hydrobromic acid is 48%；It is further preferred that the mol ratio of the TMP and hydrobromic acid is 1：10.
The invention also discloses a kind of TMP immunizing antigen, is prepared into using TMP haptens as described above Arrive, by the TMP haptens and DCC (Dicyclohexylcarbodiimide, dicyclohexylcarbodiimide), NHS (n-hydroxysuccinimide) mixes, and is stirred at 4 DEG C, supernatant is taken after centrifugation；It is 8.0 that human albumin is dissolved in into pH value In PBS solution, stirred after adding DMF, then the supernatant is gradually added into wherein, 12h is reacted at 4 DEG C, after centrifugation, is taken Clear liquid, dialysis purification, obtain the TMP immunizing antigen.
As a further improvement on the present invention, during the dialysis purification, normal saline dialysis are used at 4 DEG C 3 days, daily more Change 3 dialyzates.
The invention also discloses a kind of TMP monoclonal antibody, using TMP immunizing antigen as described above with Immune Balb/c mouse are through cell fusion, and screening obtains secreting the hybridoma of TMP monoclonal antibody, with acquisition Hybridoma is using inducing ascites method is prepared TMP monoclonal antibody in vivo.
Preferably, the TMP monoclonal antibody is prepared using following methods：Exempted from using the TMP Epidemic disease antigen and identify after be immunized 46 week old kunming mices, booster immunization three times after, blood sampling survey potency, treat serum titer no longer on Rise, adjuvant immunity mouse is not added with the antigen of two multiple doses, the lethal mouse of neck is taken off after three days, aseptically takes spleen to prepare Splenocyte, 8 are pressed with eugonic murine myeloma cell:1 ratio is mixed in 50mL centrifuge tubes, adds 30mL serum-frees IPMI1640 culture mediums, 1100r/min are centrifuged and are abandoned supernatant in 5 minutes, and cell mass is gently shaken pine, is placed in 37 C water baths. 1mL50% (percent by volume) PEG-4000 is slowly added into cell, dripped off in 1 minute, while be gently agitated for bottom and sink Form sediment, after standing 1 minute, serum free medium 1mL is slowly at the uniform velocity added along tube wall within first 30 seconds, add 2mL within latter 30 seconds, then quickly Add 27mL and terminate fusion process, 1100r/min is centrifuged 5 minutes, abandons supernatant, is added to after being resuspended with HAT selective mediums It is covered with 96 porocyte culture plates of feeder cells, 37 degrees Celsius, the CO of percent by volume 5%2Under the conditions of cultivate.Changed after 7 days Into HT nutrient solutions, when the hybrid cell quantity in hole reaches more than 300, screened with indirect elisa method, selection strong positive, The hole that inhibition is good, cell growth is vigorous carries out limited dilution cloning, cultivates and detects through the clone of more than 3 times, is in Positive hole inner cell is the hybridoma of secrete monoclonal antibody, and hybridoma is expanded into culture in case monoclonal resists The preparation of body；Then, ascites method production TMP monoclonal antibody is induced using internal.
As a further improvement on the present invention, described the step of inducing ascites method in vivo, is：To mouse peritoneal injection liquid Only, pneumoretroperitoneum injects the hybridoma 3~5 × 10 to paraffin oil 0.5mL/ within 7 days6/ only, after 10 days, treat that mouse web portion is obvious Ascites is collected when expanding, the TMP monoclonal antibody is obtained to purify ascites with caprylic acid-ammonium sulfate precipitation method.
The invention also discloses a kind of TMP collaurum detection means, it includes reaction cup and Test paper, described Test paper includes the sample pad laid successively on bottom plate and bottom plate, nitrocellulose filter and blotting paper；In the reaction cup TMP monoclonal antibody as described above containing colloid gold label, the nitrocellulose filter are provided with detection line and control Line, the detection line are to carry out spraying on nitrocellulose filter using TMP immunizing antigen as described above to be made.
As a further improvement on the present invention, the control line is to carry out spraying using rabbit anti-mouse igg to be made, the detection Line and control line are parallel to each other.Preferably, the detection line and control line distance at least 0.5cm.
Preferably, the rabbit anti-mouse igg is to be prepared by the following：
With carrier protein combination antigen immune NZw, immunizing dose is 50 μ g~100 μ g/ times, and dorsal sc divides Multi-point injection, wherein the carrier protein is conventional carrier protein, preferably human serum albumins, ovalbumin or ox blood is pure Albumen；Head exempts from, and is emulsified with the artificial antigen and equivalent Freund's complete adjuvant of synthesis；Booster immunization, with the artificial antigen of synthesis with Equivalent incomplete Freund's adjuvant emulsifies, continuous immunity 4~5 times, every minor tick 4~8 weeks, 10~15 days after last time is immune, It is surveyed with ELISA method determine potency and reach 105During the above, take a blood sample and separate and collect hyper-immune serum, carried with saturated ammonium sulfate salting out method Take rabbit anti-mouse igg antibody, -20 DEG C freeze it is standby.
The invention also discloses the preparation method of TMP collaurum detection means as described above, and it includes following step Suddenly：
Step A：Hydrobromic acid is added in TMP, 2~4h is reacted at 90~110 DEG C, then adds sodium hydroxide Solution is quenched, and after cooling separates out crystal, by dissolution of crystals in boiling water, it is 7 then to add concentrated ammonia liquor and adjust to pH, and recrystallization carries It is pure；The crystal of purification is dissolved in DMF, adds potassium carbonate and bromo-propionic acid, 40 DEG C are reacted 6 hours, are adopted and are extracted with ethyl acetate Post purifies to obtain the TMP haptens；It is using carbodlimide method that the TMP haptens and carrier proteins Bovine is clear Coupling, is prepared TMP immunizing antigen；
Preferably, the mol ratio of the TMP and hydrobromic acid is 1：3~12；Preferably, the quality of the hydrobromic acid Percent concentration is 48%.It is further preferred that the mol ratio of the TMP and hydrobromic acid is 1：10.
Preferably, the preparation process of the TMP immunizing antigen is by the TMP haptens and DCC, NHS Mixing, stirs at 4 DEG C, supernatant is taken after centrifugation；Human albumin is dissolved in the PBS solution that pH value is 8.0, adds DMF After stir, then the supernatant is gradually added into wherein, 12h is reacted at 4 DEG C, after centrifugation, supernatant is taken, dialysis purification, obtains To the TMP immunizing antigen.
Step B：By the TMP immunizing antigen and immune Balb/c mouse through cell fusion, screening obtains secreting first The hybridoma of oxygen benzyl pyridine monoclonal antibody, ascites is produced with the cell induction mouse of acquisition, obtains TMP after purification Monoclonal antibody；
Step C：Collaurum is prepared with trisodium citrate and gold chloride reaction；By collaurum and the TMP monoclonal Antibody is mixed to form gold labeling antibody, and centrifugation obtains the TMP monoclonal antibody of colloid gold label after redissolving；
Step D：The TMP monoclonal antibody of the obtained colloid gold labels of step C is dispensed into reaction cup, low temperature Dry；The TMP immunizing antigen is carried out on nitrocellulose filter to spray obtained detection line, by rabbit anti-mouse igg in nitre Carry out spraying obtained control line on acid cellulose film；
Step E：Sample pad, nitrocellulose filter and the blotting paper laid successively in the same direction are simultaneously adhered on bottom plate, Assembling obtains the Test paper.
Preferably, the preparation method of the TMP immunizing antigen is：TMP 0.1mmol is taken to be dissolved in 2mLDMF In (dimethylformamide), stirring adds 27.5mg DCC, and (Dicyclohexylcarbodiimide, dicyclohexyl carbon two are sub- Amine) and 14.4mgNHS (n-hydroxysuccinimide).Magnetic agitation reaction overnight, centrifuged supernatant A liquid, weighs people at 4 DEG C The pH that haemocyanin (HSA) 140mg is dissolved in 10mL concentration 0.1mol/L is in 8.0 PBS (phosphate buffer).Add DMF 1mL, stirring and dissolving prepares B liquid, and under magnetic agitation, A liquid is gradually dripped in B liquid, reacts 12h at 4 DEG C.After centrifugation, supernatant is taken, 4 Normal saline dialysis 3d is used at DEG C, changes 3 dialyzates daily.Obtained holoantigen is sub-packed in 0.5mL with 1mg/mL concentration In centrifuge tube, freeze standby in -20 DEG C of refrigerators.
Wherein, it is preferred that the preparation method of the collaurum is preferably：Take 1% (mass percent) chlorauric acid solution 1ml, add 99ml ultra-pure waters into the chlorauric acid solution of final concentration 0.01% (mass percent), after ebuillition of heated, take 1% (quality Percentage) trisodium citrate 1.6ml is disposably rapidly added in the chlorauric acid solution boiled, continue to be heated to solution by faint yellow Switch to it is black-and-blue eventually become shiny red, continue to heat 5min after colour stable, room temperature cooling, supplement dehydration is to original volume.
Preferably, the TMP monoclonal antibody preparation process of the colloid gold label is preferably：It is molten to adjust collaurum Liquid pH value is to 8.0, with constant speed stirrer uniform stirring, while the monoclonal antibody of TMP is added dropwise, is added after 1 hour The suitable PEG of amount of antibody, fully reaction add the suitable BSA of amount of antibody after 30 minutes, after adding, continue stirring 30 minutes. Centrifuged under 9000rpm and obtain within 30 minutes homogeneity gold labeling antibody precipitation, then add PNPB to be resuspended to obtain the first of the colloid gold label Oxygen benzyl pyridine monoclonal antibody.
As a further improvement on the present invention, in addition to by the Test paper glued the test strips of equal in width are cut into, The reaction cup and test strips hermetically drying are preserved.
Cleaning Principle of the present invention be using sample pad formed capillary siphoning effect, make tested substance first with colloid The combination of competition model occurs for the TMP monoclonal antibody of gold mark, as a result, when the TMP monoclonal of colloid gold label During antibody excess, unnecessary TMP monoclonal antibody swimming to detection line, combined and developed the color with TMP immunizing antigen； And the TMP of the colloid gold label combined with detectable substance is more anti-, its V areas binding site tested substance occupies, and can only cross over Detection line swimming, with C positions point and rabbit anti-mouse igg antibody non-specific binding, carries out colorimetric with detection line and obtained to nature controlling line Testing result.
Beneficial effects of the present invention：
First, high using technical scheme, high specificity, detection sensitivity, sensitivity is small up to 2ppb, CV values In 15%, accuracy is high, reproducible, can more quick, sensitively, and conveniently detect TMP residual.
Second, using the TMP collaurum verifying attachment of technical solution of the present invention, it is not necessary to any instrument and equipment, just It is low in carrying, testing cost；Test strips operate using simply without professional person；It is easy that test paper makes, and cost is cheap, storage Convenient, stability is good, can at least preserve six months at room temperature.
3rd, the collaurum detection means of technical scheme, using chromatography type immune colloid gold principle, pass through inspection Detection line and nature controlling line line colorimetric in test paper, the TMP residual quantity come in half-quantitative detection sample, in a short time Rapidly and accurately detect whether sample contains TMP, to determine whether TMP is exceeded, disclosure satisfy that food security To TMP residues detection demand, suitable for meat producing plant and testing agency of government.
Brief description of the drawings
Fig. 1 is the MASS spectrograms of an embodiment of the present invention TMP haptens.
Embodiment
The preferably embodiment of the present invention is described in further detail below.：
Embodiment 1
The preparation of TMP haptens, is prepared using following steps：
Sequentially add 2.5g TMPs in 100mL three-necked bottle, the hydrobromic acid 30mL of 48% mass percent, 100 DEG C reaction 3 hours, the sodium hydroxide solution 6mL for then adding 50% mass percent is quenched.Cooling separate out crystal after, crystal with After be dissolved in 20mL boiling water, then add concentrated ammonia liquor adjust to pH be 7, recrystallization purification.
Take previous step crystal 0.5g to be dissolved in 10 milliliters of DMF, add potassium carbonate 0.5g, and 0.43mL bromo-propionic acids, 40 DEG C of reactions 6 Hour.Ethyl acetate extracted post and purifies to obtain TMP haptens.
Obtained TMP haptens is detected, MASS [M+H] is 349.2, as shown in Figure 1；Its fusing point is 214 Degree.
Embodiment 2
The synthesis of TMP immunizing antigen is prepared using TMP haptens, is prepared using following steps：
TMP haptens 0.1mmol is taken to be dissolved in 2mLDMF, stirring adds 27.5mg DCC and 14.4mgNHS.4℃ Overnight, centrifuged supernatant is A liquid to the reaction of lower magnetic agitation, weighs human albumin (KLH) 140mg and is dissolved in 10mL concentration and is 0.1mol/L pH is in 8.0 PBS.DMF 1mL are added, stirring and dissolving prepares B liquid, and under magnetic agitation, A liquid is gradually dropped B In liquid, 12h is reacted at 4 DEG C.After centrifugation, supernatant is taken, normal saline dialysis 3d is used at 4 DEG C, change 3 dialyzates daily. To holoantigen be sub-packed in 1mg/mL concentration in 0.5mL centrifuge tubes, freeze in -20 DEG C of refrigerators.
Embodiment 3
TMP monoclonal antibody is prepared using TMP immunizing antigen, is prepared using following steps：
Using TMP immunizing antigen and identify after be immunized 46 week old kunming mices, booster immunization three times after, blood sampling Potency is surveyed, treats that serum titer no longer rises, adjuvant immunity mouse is not added with the antigen of two multiple doses, it is lethal small that neck is taken off after three days Mouse, aseptically take spleen to prepare splenocyte, 8 are pressed with eugonic murine myeloma cell:1 ratio is mixed in In 50mL centrifuge tube, 30mL serum-free IPMI1640 culture mediums are added, 1100r/min is centrifuged 5 minutes and abandoned supernatant, by cell The pine that gently shakes is rolled into a ball, is placed in 37 C water baths.The PEG-4000 of the percents by volume of 1mL 50% is slowly added into cell, Dripped off in 1 minute, while be gently agitated for bottom precipitation, after standing 1 minute, slowly at the uniform velocity add serum-free training along tube wall within first 30 seconds Base 1mL is supported, adds 2mL within latter 30 seconds, 27mL is then quickly added into and terminates fusion process, 1100r/min is centrifuged 5 minutes, abandons supernatant, It is added to after being resuspended with HAT selective mediums in 96 porocyte culture plates for being covered with feeder cells, 37 degrees Celsius, volume basis CO than 5%2Under the conditions of cultivate.Change HT nutrient solutions after 7 days into, when the hybrid cell quantity in hole reaches more than 300, use Indirect elisa method screens, and strong positive is selected, the hole progress limited dilution cloning that inhibition is good, cell growth is vigorous, through 3 Clone's culture and detection more than secondary, the hole inner cell being positive is the hybridoma of secrete monoclonal antibody, will be miscellaneous Oncocyte is handed over to expand culture in case the preparation of monoclonal antibody.
TMP monoclonal antibody is produced using ascites method is induced in vivo.Concretely comprise the following steps：Select 4 small through producing Kunming Mouse, only, pneumoretroperitoneum injects hybridoma 3~5 × 10 to intraperitoneal injection saxol 0.5mL/ within 7 days6/ only, after 10 days, treat small Mouse belly collects ascites when substantially expanding.Ascites is purified with caprylic acid-ammonium sulfate precipitation method, through ultraviolet determination TMP list The content of clonal antibody.
Embodiment 4
The preparation of TMP collaurum detection means, comprises the following steps：
(1) preparation of collaurum
1% (mass percent) chlorauric acid solution 1ml is taken, adds 99ml ultra-pure waters into final concentration 0.01% (mass percent) Chlorauric acid solution, after ebuillition of heated, take 1% (mass percent) trisodium citrate 1.6ml to be disposably rapidly added what is boiled In chlorauric acid solution, continue to be heated to solution by it is faint yellow switch to it is black-and-blue eventually become shiny red, continue after colour stable plus Hot 5min, room temperature cooling, supplement dehydration to original volume.
(2) preparation of colloid gold label TMP monoclonal antibody
Colloidal gold solution pH value is adjusted to 8.0, with constant speed stirrer uniform stirring, while TMP Dan Ke is added dropwise Grand antibody, the suitable PEG of amount of antibody being added after 1 hour, fully reaction adds the suitable BSA of amount of antibody after 30 minutes, after adding, Continue stirring 30 minutes.Centrifuged 30 minutes under 9000rpm, obtain the gold labeling antibody precipitation of homogeneity, then add PNPB resuspensions standby With obtaining colloid gold label TMP monoclonal antibody.
(3) preparation of collaurum detection means
TMP immunizing antigen solution is sprayed on nitrocellulose filter and forms detection line T lines, using spraying rabbit-anti mouse IgG forms control line T lines.The detection line and control line are parallel to each other, both distance 0.5cm.
Then, on bottom plate, in the same direction successively by sample pad, it is coated with the detection line T of TMP immunizing antigen Line and be coated with rabbit anti-mouse igg control line T lines nitrocellulose filter and blotting paper overlap adhesion successively, wherein, detection line Close to sample pad, the Test paper glued is cut into the test strips of equal in width close to blotting paper by the control line；Reaction cup Interior addition colloid gold label TMP monoclonal antibody, is freezed；The reaction cup and test strips hermetically drying are preserved.
Embodiment 5
The detection of TMP, method are in sample：
Fresh chicken tissues sample is taken to rub, TMP residual is put through acetonitrile and acetone extraction, n-hexane except fat, sampling In reaction cup, it is incubated at room temperature 10 minutes, is subsequently inserted into test strips, is incubated at room temperature 3 minutes.Test strips are taken out, gently strike off examination Foam-rubber cushion, carry out result interpretation.If T lines show aubergine band with C lines simultaneously, result is feminine gender；If T line colors are than C line Shallow or C lines develop the color and T lines do not develop the color, then result is the positive；If C lines, T lines do not develop the color, detection means has failed.
Embodiment 6
The sensitivity technique of TMP collaurum detection means.
Tested by mark-on, find the sensitivity of TMP collaurum detection means in the present invention up to 2ppb, CV values Less than 15%.
Embodiment 7
The specificity experiments of TMP collaurum detection means.
In the structure of fish muscle of feminine gender, TMP 2ppb, sulfamethyldiazine, sulphadiazine, sulfalene are separately added into Thiadiazoles, madribon, fanasil, sulfapryidine, sulphathiazole, each 20ppb of Huang An Jia Evil Zo. By the operating procedure of embodiment 5, test finds that the present apparatus has excellent selectivity to TMP, with 8 kinds of common sulfonamides Thing no cross reaction.
Embodiment 8
The shelf-life experiment of TMP collaurum detection means.
Shelf-life experiment is done respectively with three batches of products routinely produced, is positioned over indoor room temperature environment and is kept, every 1 month 12 devices are removed, with Quality Control pattern detection, the sample of feminine gender, respectively 1ppb, 2ppb and 4ppb concentration is made respectively, repeats three It is secondary, observed data change, investigate shelf-life durations.Feminine gender colour developing was begun to decline from 14 months, within 1 year product quality without Significant change, it is thus determined that the shelf-life is 1 year.
Above content is to combine specific preferred embodiment further description made for the present invention, it is impossible to is assert The specific implementation of the present invention is confined to these explanations.For general technical staff of the technical field of the invention, On the premise of not departing from present inventive concept, some simple deduction or replace can also be made, should all be considered as belonging to the present invention's Protection domain.

Claims (8)

  1. A kind of 1. TMP haptens, it is characterised in that：It has the chemical structural formula shown in formula (1)：
  2. 2. TMP haptens according to claim 1, it is characterised in that be prepared using following steps：
    Step S1：Hydrobromic acid is added in TMP, 2~4h is reacted at 90~110 DEG C, then adds sodium hydroxide solution It is quenched, after cooling separates out crystal, by dissolution of crystals in boiling water, regulation pH value to neutrality, recrystallization purification；
    Step S2：Take the crystal purified in step S1 to be dissolved in DMF, add potassium carbonate and bromo-propionic acid, reaction 4 at 35~45 DEG C~ 8 hours, adopt and post was extracted with ethyl acetate purifies to obtain the TMP haptens.
  3. 3. TMP haptens according to claim 2, it is characterised in that：Mole of the TMP and hydrobromic acid Than for 1：3~12.
  4. A kind of 4. TMP immunizing antigen, it is characterised in that：Using the TMP described in claims 1 to 3 any one Haptens is prepared, and the TMP haptens is mixed with DCC, NHS, is stirred at 4 DEG C, supernatant is taken after centrifugation； Human albumin is dissolved in the PBS solution that pH value is 8.0, is stirred after adding DMF, the supernatant is then gradually added into it In, 12h is reacted at 4 DEG C, after centrifugation, supernatant is taken, dialysis purification, obtains the TMP immunizing antigen.
  5. A kind of 5. TMP collaurum detection means, it is characterised in that：Including reaction cup and Test paper, the Test paper Including sample pad, nitrocellulose filter and the blotting paper laid successively on bottom plate, and bottom plate；Contain colloid in the reaction cup The TMP monoclonal antibody of gold mark, the nitrocellulose filter are provided with detection line and control line, and the detection line is Spraying is carried out on nitrocellulose filter using the TMP immunizing antigen described in claim 4 to be made；Wherein, the methoxy Benzyl pyridine monoclonal antibody is to be melted using the TMP immunizing antigen described in claim 4 and immune Balb/c mouse through cell Close, screening obtains secreting the hybridoma of TMP monoclonal antibody, is used with the hybridoma of acquisition and induced in vivo Ascites method is prepared.
  6. 6. TMP collaurum detection means according to claim 5, it is characterised in that：The control line is using rabbit Anti- mouse IgG carries out spraying and is made, and the detection line and control line are parallel to each other.
  7. 7. the preparation method of the TMP collaurum detection means described in claim 5 or 6, it is characterised in that it include with Lower step：
    Step A：Hydrobromic acid is added in TMP, 2~4h is reacted at 90~110 DEG C, then adds sodium hydroxide solution It is quenched, after cooling separates out crystal, by dissolution of crystals in boiling water, regulation pH value to neutrality, recrystallization purification；By the crystal of purification It is dissolved in DMF, adds potassium carbonate and bromo-propionic acid, reacted 4~8 hours at 35~45 DEG C, adopts and post purification was extracted with ethyl acetate Obtain the TMP haptens；The TMP haptens and carrier human albumin are coupled using carbodlimide method, TMP immunizing antigen is prepared；
    Step B：By the TMP immunizing antigen and immune Balb/c mouse through cell fusion, screening obtains secreting methoxy benzyl The hybridoma of pyridine monoclonal antibody, ascites is produced with the cell induction mouse of acquisition, obtains TMP Dan Ke after purification Grand antibody；
    Step C：Collaurum is prepared with trisodium citrate and gold chloride reaction；By collaurum and the TMP monoclonal antibody Gold labeling antibody is mixed to form, centrifugation obtains the TMP monoclonal antibody of colloid gold label after redissolving；
    Step D：The TMP monoclonal antibody of the obtained colloid gold labels of step C is dispensed into reaction cup, low temperature drying； The TMP immunizing antigen is carried out on nitrocellulose filter to spray obtained detection line, rabbit anti-mouse igg is fine in nitric acid Tie up and carry out spraying obtained control line on plain film；
    Step E：Sample pad, nitrocellulose filter and the blotting paper laid successively in the same direction are simultaneously adhered on bottom plate, are assembled Obtain the Test paper.
  8. 8. the preparation method of the TMP collaurum detection means described in claim 7, it is characterised in that：Also include gluing The Test paper be cut into the test strips of equal in width, the reaction cup and test strips hermetically drying are preserved.
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