CN1378084A - Immunological microball method for detecting pyloric helicobacterium in stool sample - Google Patents
Immunological microball method for detecting pyloric helicobacterium in stool sample Download PDFInfo
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Abstract
The immunological microsphere method of detecting pyloric helicobacterium in stool sample includes labeling microsphere to form immunological microsphere; suspending the tested stool sample containing pyloric helicobacterium in buffering liquid; mixing the immunological microsphere and the sample buffering liquid or mixing pyloric helicobacterium resisting antibody with the buffering liquid before adding pyloric helicobacterium resisting antigen labeled microsphere; and detecting pyloric helicobacterium in the stool sample. The method has the advantages of low cost, fast and convenient operation and reliable result.
Description
(1) technical field:
The present invention relates to a kind of immunological microball method of measuring helicobacter pylori in the fecal specimens.
(2) background technology:
Helicobacter pylorus Pseudomonas Helicobacterium.It is a kind of bacterium of finding the human gastrointestinal tract, have been found that it and people's gastroduodenal disease as: gastritis, peptic ulcer, cancer of the stomach and other disease have substantial connection.
There have been many different technology to be used for the mensuration of helicobacter pylori.According to the non-invasive of having drawn materials, the methods for clinical diagnosis of helicobacter pylori infections can be divided into two big classes at present: 1. noninvasive test: comprise the breath test of serological method, isotope labeling urea and gastric juice polymerase chain reaction etc.2. traumatic inspection: be gastroscope dependence method, comprise variform, microbiology and Protocols in Molecular Biology etc.
The equal defectiveness of above-mentioned these methods is difficult to satisfy the clinical diagnosis needs.
The methods such as enzyme linked immunological, immuno-enzymatic test, latex agglutination test, Western blot that adopt serological method detect the peripheral blood quiet and beautiful door full bacterium of helicobacter and component such as cytotoxic antibody etc.Because specific antibody just appears in helicobacter pylori infections in the blood after several weeks, also can there be cross reacting antibody (as Cj infection) in the negative patient blood, and behind the helicobacter pylori eradication in the blood antibody for a long time (＞6 months) maintain positive level, have activity to infect so the serology positive can not be affirmed patient fully; The negative infection that can not get rid of the initial stage.Therefore, the serology detection of antibodies can only be used for the epidemiology examination and can not be used for clinical diagnosis, can not be used for observation of curative effect.
Its principle of the breath test of isotope labeling urea (C-13 and C-14 breath test) is the urease that can produce strong activity according to helicobacter pylori, and it decomposes the urea in the gastric juice, produces carbon dioxide and ammonia.With the plain labeled urea of C-13 or C-14 position, allow the oral back of patient be decomposed by the urease of helicobacter pylori, produce the CO of C-13 or C-14, positive patient can detect C-13 (C-13/C-12 ratio) abundance and C-14 (radioactivity) activity in breath, thereby make a definite diagnosis helicobacter pylori infections, be considered to the diagnosis " goldstandard " except that microbe growth at present.But the former needs the gas mass spectrometer, and the latter has certain radioactivity, also needs expensive instruments such as liquid glimmer instrument, and checks that expenses standard is higher, has greatly limited the application of this method.Other bacterium in the intestines and stomach also can secrete urease, when these bacterial contaminations, can produce false positive results.Behind the eradication therapy of helicobacter pylori, urease is suppressed, and this method easily causes false negative result.
The gene diagnosis method, stomach tube is drawn gastric juice or gastroscope extracts gastric juice or biopsy as inserting, and extracts DNA and does polymerase chain reaction and probe hybridization diagnosing helicobacter pylori infection, has the advantage that accuracy is good, detection sensitivity is high.The also Disease-causing gene of bacterial detection such as activity study of vacuolating cytotoxin A gene etc. specifically, but complicated operation, easily pollution, and increased patient's economy spending greatly,, should not advocate as conventional method to having multiple inspection means today with diagnosing helicobacter pylori infection.
Micro-biological process is mainly bacterium separation and Culture technology, is " goldstandard " of diagnosing helicobacter pylori infection, can obtain such as required bacteriums such as antigen preparation, drug sensitive test, somatotype and Study on Pathogenicity simultaneously.But require to have certain anaerobism condition of culture and technology, be difficult for promoting as the routine diagnosis means.
The most frequently used Hp Infect And Diagnose method when rapid urease test is gastrocopy comprises PH indicator method and analytical chemistry method.The biopsy specimens of using, also available gastric juice sample detects more.Also have work to pick up histopathology dyeing, smear staining etc. in addition and directly see methods such as bacterium, this several method all is traumatic inspections, needs gastroscope to draw materials and the judgement experience.Therefore, limited the application of these methods.
In sum, the Heliobacter pylori antigen in bacterial antigens, the especially fecal specimens preferably can be directly checked in the infection of helicobacter pylori.For this reason, U.S. Meridian Diagnostics, Inc. proposes a kind of " immunoassay of helicobacter pylori in the fecal specimens " (Chinese patent application number 97103112.6).This method adopts Heliobacter pylori antigen in the enzyme linked immunosorbent detection technology for detection fecal specimens, have advantages such as highly sensitive, that specificity is good, but the reagent cost height that this method adopted, during operating cost, need expensive microplate reader etc., limited the use of this method.
(3) summary of the invention:
Problem when the objective of the invention is to solve reagent used in the prior art and instrument cost height, operating cost.
General introduction
The invention provides a kind of immunological microball method of measuring helicobacter pylori in the fecal specimens; It comprises:
(a), form immune microsphere the antibody of anti-helicobacter pylori/or Heliobacter pylori antigen mark microballoon;
(b) ight soil that suspection is contained helicobacter pylori is suspended in the sample buffer;
(c) immune microsphere of mark helicobacter pylori antibody mixes with the sample buffer that contains ight soil; Or certain helicobacter pylori antibody mixed with the sample buffer that contains ight soil, add Heliobacter pylori antigen mark microballoon then;
(d) detect the helicobacter pylori that may exist in the ight soil.
In embodiments of the invention, the particle type of microballoon (or particulate) that can be used to mark is a lot, and commonly used has: the microparticle that animal or human's red blood cell, bacterium, multiple macromolecular material aggregate into (as polystyrene latex etc.), resin, bentonite, gelatin particle, activated charcoal, collodion, glass microsphere etc.To adopt latex and red blood cell in the preferred version.
In embodiments of the invention, being used for the antibody of mark microballoon can be polyclonal antibody, also can be various monoclonal antibodies or its mixing.Preferred version will adopt polyclonal antibody.
The method of antibody or antigenic mark microballoon has several: physisorption, chemical crosslinking and indirect method etc.When implementing, should select according to the concrete characteristics of microballoon and the characteristic of antibody.
Fecal specimens preparation: a certain amount of ight soil is diluted in the sample buffer by proper proportion, abundant stirring and evenly mixing, low-speed centrifugal number minute is got supernatant, and is to be measured.Sample diluting liquid wherein should contain protein and washing agent, can reduce nonspecific reaction.For improving sample buffer that detection sensitivity can contain ight soil at the helicobacter pylori processing of concentrating.
The method that detects the helicobacter pylori that may exist in the ight soil has direct method and indirect method.Direct method is meant, with helicobacter pylori antibody mark microballoon, forms immune microsphere; Directly immune microsphere is mixed with the sample buffer that contains ight soil, detect the helicobacter pylori that combines with immune microsphere, as: the change (as color, turbidity, permeability etc.) of damping fluid physics or chemical characteristic, detection immune microsphere mixed the change (concentration of free microballoon) of front and back microballoon quantity and physical characteristics etc. before and after agglutinating reaction, immune garland, detection immune microsphere mixed with damping fluid with damping fluid.Indirect method has agglutination inhibition usually, its principle is the antibodies of the microballoon of Heliobacter pylori antigen mark and a certain amount of anti-this bacterium and agglutinating reaction occurs, when having this bacterial infection, Heliobacter pylori antigen competition and antibodies in the ight soil, thereby the aggegation of inhibition Heliobacter pylori antigen mark microballoon.
The present invention adopts the immune microsphere technology, detects the helicobacter pylori in the fecal specimens.It has advantages such as with low cost, convenient to operation, reliable results.Describe in detail
Assay method of the present invention has used microballoon, it has numerous species, aggregates into particulate (as polystyrene latex etc.), resin, bentonite, gelatin particle, activated charcoal, collodion, glass microsphere of latex etc. as animal or human's red blood cell, bacterium, multiple macromolecular material.To adopt latex and red blood cell in the preferred version, be optimum with latex.
The kind of latex is a lot, and commonly used is polystyrene latex at present, the commercial latex of existing all size on the existing market.The latex diameter that the present invention adopts is excellent with 0.2～1.5 micron.
The present invention has used the antibody of anti-helicobacter pylori, and two kinds of monoclonal antibody and polyclonal antibodies are arranged, and commonly used is polyclonal antibody.These antibody can obtain from the serum of the animal of sensitization.At present both at home and abroad existing many companies can provide the antibody of commercial anti-helicobacter pylori, and price that can less expensive obtains.Chinese patent (application number 97103112.6) has been described the preparation method of helicobacter pylori antibody in detail.Only be summarized as follows at this: Heliobacter pylori antigen is expelled in mammal such as the bodies such as rabbit, sheep, the antigen of the suitable dosage of multiple injection, the demonstration test animal has produced helicobacter pylori antibody, gets experimental animal serum, purifiedly can obtain required antibody.
The method of antibody or antigenic mark microballoon has physisorption and chemical crosslinking etc.Physisorption is a charging property of utilizing the latex particle surface, and the physisorption method is simple to operation, but the firmness of this combination is often relatively poor.Along with the prolongation of sensitization reagent storage life, it is apparent in view to come off.Chemical crosslinking refers to adopt the latex that contains chemical active radical as carrier, antibody or antigen with the chemical bond covalent cross-linking on the latex surface.The stable performance of this chemical crosslinking, long shelf-life, result of use is significantly improved.The surface active groups of latex can be that carboxyl, amino or some have chemically active group, and it can be finished by condensation agent with combining of proteantigen or antibody.The technology that antibody or antigen are combined on the microballoon is well known to those of ordinary skill in the art.
A, physisorphtion: earlier the latex suspension is diluted to debita spissitudo, after antibody or antigen are dissolved in damping fluid, dropwise adds in the latex suspension of dilution, shake and make it fully mixed with distilled water.Can place room temperature or 37 ℃ after the mixing, the insulation certain hour.After absorption was finished, the last adsorbate that is free in the supernatant was removed in centrifugation, and after the supernatant that inclines, precipitation is still recovered suspended state with damping fluid.Add bovine serum albumin(BSA) or gelatin hydrolysate etc., make the adsorptive power on latex surface fully saturated, reduce nonspecific reaction; And answer strictness to avoid in adsorption process, taking place the latex self-solidifying.
B, chemical crosslink technique: the microballoon of band reactive group can adopt chemical crosslink technique to combine with antibody or antigen.Its cross-linking method of different activities group is different.Adopt chemical crosslink technique to combine as Carboxylated Polystyrene latex with antibody or antigen.In order to improve cross-linking effect, introduce " arm " to latex earlier usually.As antibody is directly crosslinked closely on Carboxylated Polystyrene latex, since sterically hindered, hinder it and corresponding antigen to react.Therefore connect " arm " EACA earlier, increasing the distance of aglucon and carrier surface, immune microsphere susceptibility of making like this and specificity all are higher than physisorption, and the stability of reagent also significantly improves.Amino on the latex and the amino that is crosslinked on the thing are finished by the condensation agent carbodiimides.
Fecal specimens preparation: a certain amount of ight soil is diluted in the sample buffer by proper proportion, abundant stirring and evenly mixing, it is centrifugal to carry out routine, as 200-500 rev/min of employing, 3-5 minute, get supernatant, to be measured.The sample buffer that can contain ight soil for the raising detection sensitivity carries out the processing of concentrating such as centrifugal or filtration at helicobacter pylori.This supernatant with greater than 4000 rev/mins speed, is centrifugally removed supernatant more than 10 minutes, will precipitate again break up, mixing, to be measured.Sample diluting liquid wherein should contain protein and washing agent, can reduce nonspecific reaction.Protein component can be selected from hyclone, sheep blood serum, guinea pig serum, horse serum, casein, albumin, gelatin, bovine serum albumin(BSA), human serum albumins etc.Washing agent can contain Tween-20, TritonX-100 etc.
Detect the helicobacter pylori that may exist in the fecal specimens, its method has direct method and indirect method.Direct method is meant, with helicobacter pylori antibody mark microballoon, forms immune microsphere; Immune microsphere is mixed with the sample buffer that contains ight soil, detect the helicobacter pylori that combines with immune microsphere, as: change (concentration of free microballoon) of microballoon quantity and physical characteristics or the like before and after the change (as color, turbidity, permeability etc.) of damping fluid physics or chemical characteristic, detection immune microsphere mixed with damping fluid before and after agglutinating reaction, immune garland, detection immune microsphere mixed with damping fluid.Indirect method has agglutination inhibition usually, its principle is the antibodies of the microballoon of Heliobacter pylori antigen mark and a certain amount of anti-this bacterium and agglutinating reaction occurs, when having this bacterial infection, Heliobacter pylori antigen competition and antibodies in the ight soil, thereby the aggegation of inhibition Heliobacter pylori antigen mark microballoon.Agglutination inhibition comprises:
(a), form immune microsphere the antigenic mark microballoon of helicobacter pylori;
(b) ight soil that suspection is contained helicobacter pylori is suspended in the sample buffer;
(c) a certain amount of helicobacter pylori antibody is mixed with the sample buffer that contains ight soil, add the microballoon of Heliobacter pylori antigen mark after 2-10 minute;
(d) aforesaid liquid fully mixed, shake continuously and got final product observations in 2-10 minute: the negative reaction of agglutinating particle occurs, no helicobacter pylori or amount are described in the excrement sample seldom; Keep the positive reaction of homogeneous latex emulsion shape, illustrate to have helicobacter pylori in the fecal specimens.
Agglutination test divides test tube method and slide method.Slide method is easy and simple to handle, the emulsion reagent of certain volume method for preparing fecal specimens (for improving sensitivity processings of sample will being concentrated usually) and equal-volume helicobacter pylori antibody sensitization shakes continuously and got final product observations in several minutes behind mixing on the slide (being generally black background).The positive reaction of agglutinating particle occurs, illustrate to have helicobacter pylori in the fecal specimens; Keep the negative reaction of homogeneous latex emulsion shape, no helicobacter pylori be described in the excrement sample or measure seldom.
The immune microsphere garland forms the immune microsphere reagent of helicobacter pylori antibody sensitization that test gets certain volume method for preparing fecal specimens and equivalent in small test tube, mixes rearmounted room temperature or 37 ℃ temperature 10-40 minute.With phosphate buffer centrifuge washing 2 times.Get one of last sedimentation cell and put on the microslide, dyeing detects immune garland at microscopically then.Microscopically, the representative configuration of helicobacter pylori is: s shape, sea-gull sigmoid.If there is immune garland to form, then around bacterium, there is immune microsphere to adhere to, the floweriness ring is the same, so claim the immune microsphere rosette formation test.If find the bacterium that is colored of S shape or sea-gull sigmoid, around it, there is the microballoon more than 2～3 to adhere to simultaneously, then represent the rosette formation test positive, can infer in this fecal specimens in view of the above to have helicobacter pylori; As do not find that immune garland forms, no helicobacter pylori then is described in the excrement sample or measures seldom.
Detect the physics of damping fluid or method that chemical characteristic changes commonly used nephelometry arranged.Nephelometry is to form agglutinating particle according to its corresponding antigen of immune particulate of helicobacter pylori antibody sensitization in conjunction with the back, the turbidity of reaction mixture system is diminished, transmitted light strengthens, the change of turbidity and the concentration of helicobacter pylori are funtcional relationship, can measure the amount of helicobacter pylori in the sample thus.Can utilize laboratory spectrophotometer commonly used to measure with the incident light of 340-500nm wavelength.Also available light scattering method agglutinating particle is in the change of the scattered light intensity of a certain angle, and detection sensitivity is higher than the transmitted light nephelometry.Low approximately 1-2 times of the remolding sensitivity particle counting of nephelometry, but can satisfy the quantitative measurement requirement.
Detect immune microsphere mixes front and back microballoon quantity and physical characteristics with damping fluid change.Method commonly used has particle counting.The particle counting ratio juris be according to bag by the immune microsphere of helicobacter pylori antibody, its corresponding antigen reduces in conjunction with the immune microsphere quantity that agglutinating reaction takes place, make to be in disperse state originally.Measured object concentration is high more, and agglutinating reaction is stronger, and free immune microsphere quantity is fewer, by the variation of free state immune microsphere quantity before and after the quantal response, just can make quantitative test to measured object-helicobacter pylori in the sample.This method is easy and simple to handle, and the detection sensitivity height is generally the 0.1-1ng/ml scope; Used instrument and equipment can be general with blood-counter system and the automatic biochemical analyzer DIAS system that clinical labororatory uses always.
(4) description of drawings:
Table 1 is result's contrast that existing enzyme-linked immune detection method and latex agglutination of the present invention detect.
Table 2 detects the result of 4 parts of fecal specimens simultaneously for enzyme-linked immune detection method and latex agglutination of the present invention.
Below the invention will be further described by some indefiniteness embodiment.
(5) specific embodiments:
Embodiment one: physical method sensitization microballoon
Material is prepared: the polystyrene microsphere that diameter is 0.6～1.2 micron (can buy to U.S. SIGMA company), the polyclonal antibody of goat-anti helicobacter pylori, Heliobacter pylori antigen (antigen standard items or soluble antigen) (can buy to U.S. KPL company, the U.S. bioengineering of China Shenzhen crystalline substance company limited).
The damping fluid preparation:
0.27mol/L glycine buffer: 7.51 gram glycocoll, 9.95 gram sodium chloride are dissolved in distilled water, transfer to pH8.2 with 1mol/L NaOH, and distilled water complements to 1 liter.
0.27mol/L glycine buffer+1% human serum albumins: 1 gram human serum albumins is dissolved in 100 milliliters of 0.27mol/L glycine buffers.
0.054mol/L glycine buffer: with distilled water the 0.27mol/L glycine buffer is done dilution in 1: 5 and get final product.
Earlier 1 milliliter 10% latex suspension is diluted to 1-2%, latex is washed hematocrit 3 times (centrifugal 20 minutes of 10000g) with the 0.054mol/L glycine buffer with distilled water.Again be suspended in 10 milliliters of 0.054mol/L glycine buffers then, gradually 1 milligram of antibody or antigen added in the latex suspension of dilution, shake and make it fully mixed.(1% latex suspension 1ml generally can adsorb 0.1～1mg material).Can place room temperature or 37 ℃ of insulations after the mixing, the time was generally 30～60 minutes.After absorption is finished, 10, the last adsorbate that is free in the supernatant is removed in 000g centrifugation 30 minutes, and after the supernatant that inclines, precipitation is recovered suspended state with 0.27mol/L glycine buffer+1% human serum albumins damping fluid.Add 0.04% Sodium azide, in 4 ℃ of preservations, standby.
Embodiment two: chemical crosslink technique sensitization microballoon
Polyacetals base polystyrene microballoon 1% latex of band aldehyde radical adds 0.1 milligram of antibody or antigen for 1 milliliter, adds 10 milliliters of 0.1mol/LpH5.3 acetate buffers.Under the room temperature, container was stirred 10 hours, 10000g is centrifugal 20 minutes then, removes supernatant.Precipitation adds 10 milliliters of pH9.5 1mol/L monoethanolamine/0.1%Tween-20.Stirred 3 hours, 10000g is centrifugal 20 minutes then, removes supernatant.Precipitation adds 10 milliliters of Tris damping fluids, stirs 24 hours, and centrifugal 10000g 20 minutes removes supernatant.Add 0 milliliter of the phosphate buffer 1 that contains 1% human serum albumins, suspend again; Add 0.04% Sodium azide, in 4 ℃ of preservations, standby.
0.1mol/LpH5.3 acetate buffer: 10 milliliters of 0.1mol/L acetic acid, 40 milliliters of 0.1mol/L sodium acetates.
Tris damping fluid: 0.05mol/LTris 0.1mol/ sodium chloride.
Embodiment three: fecal specimens is prepared
Fecal specimens by 1: 4 or dilution in 1: 6, is added suitable fecal specimens in the sample diluting liquid, fully mix.200 rev/mins, centrifugal 3 minutes.Get supernatant, to be measured.When adopting agglutinating reaction and agglutination inhibition to detect helicobacter pylori, be to improve detection sensitivity, the processing of sample can being concentrated with centrifugal 15 minutes of this supernatant 12000g, is removed supernatant, will precipitate again break up, mixing, to be measured.
Sample diluting liquid: 100 milliliters of 0.27mol/L glycine buffers, add human serum albumins 1 gram, add 3 milliliters of Tween-20.Add 0.04% Sodium azide simultaneously.
Embodiment four: latex agglutination test
Agglutination test (employing slide method) 12cm * 16cm glass agglutinating reaction plate (divide 30 lattices of 1cm * 1.5cm on the plate, be generally black background).The emulsion reagent of (about 30-50 microlitre) method for preparing fecal specimens and (about 30-50 microlitre) helicobacter pylori antibody sensitization shakes several minutes (clockwise or counter-clockwise direction) continuously and gets final product observations behind mixing on the slide.The oarse-grained positive reaction of aggegation occurs, illustrate to have helicobacter pylori in the fecal specimens; Keep the negative reaction of homogeneous latex emulsion shape, no helicobacter pylori be described in the excrement sample or measure seldom.
Contrast is provided with: with physiological saline is blank, with the negative stool sample of enzyme linked immunosorbent detection as negative control.
Judged result
" ++ ++ ": coarse particle appears in the whole aggegations of latex particle, and becomes a white edge all around, occurs agglutinating particle in general 1～2 minute.Liquid is limpid to be the extra-heavy positive " ++ ++ ".
" +++": coarse particle appears in the whole aggegations of latex particle.4 all white edges are not too obvious.Agglutinating particle occurred in general 3-4 minute, liquid is more limpid to be strong positive " +++".
" ++ ": agglutinating particle appears in latex.The little muddiness of liquid.Occurring agglutinating particle in general 5-6 minute is than strong positive " ++ ".
"+": agglutinating particle appears in latex, the liquid muddiness, and general 8-10 minute agglutinating particle occurs is the weak positive "+".
"-": not aggegation, evenly muddy negative "-" is white in color.
Positive criterion: allly aggegation occurs and just be judged to the positive.
Stool sample can be made a series of grades in case of necessity and doubly dilute, carry out semiquantitative determination.
Fecal specimens concentrated handle back (two times centrifugal, 200 rev/mins, centrifugal 3 minutes, get supernatant, with centrifugal 15 minutes of this supernatant 12000g, remove supernatant again, to precipitate again break up, mixing, to be measured), latex agglutination test sensitivity and enzyme-linked immunoassay method are similar.
By the Heliobacter pylori antigen of dose known amounts, be mixed with calibration object, adopt Chinese patent (application number 97103112.6) described enzyme-linked immune detection method and latex agglutination of the present invention to detect simultaneously, the results are shown in Table 1.
Two kinds of methods detect 4 parts of fecal specimens simultaneously, the results are shown in Table 2.
Sensitivity and enzyme-linked immunoassay method with fecal specimens latex agglutination test of doing after the processing of concentrating is suitable as can be seen from table 1,2.
Embodiment five, agglutination inhibition:
The sample buffer that contain ight soil of suitable dilution 50 microlitre helicobacter pylori antibodies with embodiment three preparations fully mixed, add the immune microsphere of equal-volume Heliobacter pylori antigen mark then; Aforesaid liquid is fully mixed, shakes continuously several minutes (clockwise or counter-clockwise direction) get final product observations.
The result judges: the negative reaction of agglutinating particle occurs, no helicobacter pylori be described in the excrement sample or measure seldom; Keep the positive reaction of homogeneous latex emulsion shape, illustrate to have helicobacter pylori in the fecal specimens.
Agglutination inhibition is than latex agglutination test susceptibility height
Embodiment six, immune microsphere garland form test
The immune microsphere reagent of getting (50 a microlitre) embodiment three preparation fecal specimens and (50 a microlitre) helicobacter pylori antibody sensitization mixed rearmounted 37 ℃ of intermittent oscillatings 30～60 minutes in small test tube.Use phosphate buffer with 4000 rev/mins of washings in centrifugal 20 minutes 2 times then.Get one of last sedimentation cell and put on the microslide, add a methylene blue solution simultaneously, dyeing added cover glass in 3～15 minutes; Under high power lens or oily mirror, detect immune garland.Microscopically, helicobacter pylori is dyed blueness by methylene blue, and its typical form is: s shape, sea-gull sigmoid.If there is immune garland to form, then around this bacterium, there is microballoon to adhere to, the floweriness ring is the same, so claim the immune microsphere rosette formation test.At microscopically, dye blue bacterium if find the quilt of S shape or sea-gull sigmoid, around it, there is the microballoon more than 2～3 to adhere to simultaneously, then represent the rosette formation test positive, there is helicobacter pylori in this fecal specimens; As do not find that immune garland forms, no helicobacter pylori then is described in the excrement sample or measures seldom.
This method is all responsive than latex agglutination test and enzyme linked immunological.
The method that is used for helicobacter pylori dyeing has a variety of, and this is that this research field technician is known, as Gram, silver dyeing, methylene blue dyeing.Only select a kind of more convenient colouring method at this.
The methylene blue dye liquor: methylene blue (methylene blue) 1.0g, boric acid 0.5g, adding distil water is to 100ml.
Embodiment seven, turbidimetry
Use and adopt turbidimetry to detect the fecal specimens helicobacter pylori on the RA-50 semi-automatic biochemical analyzer.
Adopt the antibody labeling to thinner microballoon (about diameter 0.2 micron) of aforementioned physisorption method with anti-helicobacter pylori.
Heliobacter pylori antigen standard items (can buy to Shenzhen brilliant U.S. bio-engineering corporation), the typical curve preparation: (pH7.4) is diluted to 1.1 * 10 with the helicobacter pylori standard items with the 0.1mol/L glycine buffer
6, 3 * 10
6, 9 * 10
7, 1.9 * 10
7/ milliliter follows these steps to operation, makes typical curve.
Method of operating: embodiment three prepared fecal specimens (getting supernatant after only once centrifugal) 200 microlitres are added immune microsphere 100 microlitres, abundant mixing, 30 ℃ of water-baths 1 minute, the RA-50 semi-automatic biochemical analyzer, those long end-point methods of 340NM are measured, and instrument calculates the printing measurement result according to the typical curve of internal memory.Add standard items and immune microsphere during standard curve determination, after the same water-bath, reading.For avoiding the turbidity of fecal specimens own to disturb, should establish the sample contrast.
The remolding sensitivity enzyme-linked immunoassay method of nephelometry is low, but can satisfy the quantitative measurement requirement.
Claims (11)
1, a kind of immunological microball method of measuring helicobacter pylori in the fecal specimens, it comprises:
(a), form immune microsphere the antibody of anti-helicobacter pylori/or Heliobacter pylori antigen mark microballoon;
(b) ight soil that suspection is contained helicobacter pylori is suspended in the sample buffer;
(c) immune microsphere of mark helicobacter pylori antibody mixes with the sample buffer that contains ight soil; Or helicobacter pylori antibody mixed with the sample buffer that contains ight soil, add the microballoon of Heliobacter pylori antigen mark then;
(d) detect the helicobacter pylori that may exist in the ight soil
2, according to the method for claim 1, being used for the particle of microballoon of mark has: one or more that animal or human's red blood cell, bacterium, multiple high molecular polymerization particle, resin, bentonite, gelatin particle, activated charcoal, collodion, glass granules are total, will adopt latex or red blood cell in the preferred version.
3, according to the method for claim 1, the sample buffer that will contain ight soil is at the helicobacter pylori processing of concentrating.
4 methods according to claim 3 is characterized in that: this method of handling that concentrates is that the damping fluid that will contain fecal specimens carries out the centrifugal processing of concentrating.
5, should contain protein and washing agent according to the sample diluting liquid that the process of claim 1 wherein.Protein component is selected from one or more of hyclone, sheep blood serum, guinea pig serum, horse serum, casein, albumin, gelatin, bovine serum albumin(BSA), human serum albumins, and washing agent contains Tween-20 or Triton X-100.
6, the immunological microball method of helicobacter pylori in the mensuration fecal specimens according to claim 1; It comprises:
(a), form immune microsphere the antibody labeling microballoon of anti-helicobacter pylori;
(b) ight soil that suspection is contained helicobacter pylori is suspended in the sample buffer;
(c) immune microsphere of mark helicobacter pylori antibody mixes with the sample buffer that contains ight soil;
(d) detect the helicobacter pylori that combines with immune microsphere;
7, according to claim 6 method, it is characterized in that: the method that detects the helicobacter pylori that combines with immune microsphere is: above-mentioned mixing material is shaken continuously get final product observations more than 1 minute: the positive reaction of agglutinating particle occurs, illustrate to have helicobacter pylori in the fecal specimens; Keep the negative reaction of homogeneous latex emulsion shape, no helicobacter pylori be described in the excrement sample or measure seldom.
8, according to claim 6 method, it is characterized in that: the method that detects the helicobacter pylori combine with immune microsphere is: above-mentioned mixing material was put room temperature or 37 ℃ of insulations more than 5 minutes, get a part of liquid and dye; Detect immune garland at microscopically, immune microsphere be adsorbed on helicobacter pylori around form garland, so garland exists and then can infer indirectly and have helicobacter pylori in the sample; Otherwise then there are not helicobacter pylori or amount seldom.
9, according to claim 6 method, it is characterized in that: the method that detects the helicobacter pylori that combines with immune microsphere is: detect immune microsphere mixes front and back damping fluid physics or chemical characteristic with damping fluid change, thereby may have helicobacter pylori in the supposition fecal specimens.
10, according to claim 6 method, it is characterized in that: the method that detects the helicobacter pylori that combines with immune microsphere is: detect immune microsphere mixes front and back microballoon quantity and physical characteristics with damping fluid change, thereby may have helicobacter pylori in the supposition fecal specimens.
11, the immunological microball method of helicobacter pylori in the mensuration fecal specimens according to claim 1, it comprises:
(a), form immune microsphere the antigenic mark microballoon of helicobacter pylori;
(b) ight soil that suspection is contained helicobacter pylori is suspended in the sample buffer;
(c) the liquid 30-60 μ l that will contain helicobacter pylori antibody mixes with isopyknic sample buffer that contains ight soil, adds the microballoon of Heliobacter pylori antigen mark after 2-10 minute;
(d) aforesaid liquid fully mixed, shake continuously and get final product observations more than 1 minute: the negative reaction of agglutinating particle occurs, no helicobacter pylori or amount are described in the excrement sample seldom; Keep the negative reaction of homogeneous latex emulsion shape, illustrate to have helicobacter pylori in the fecal specimens.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021157464A CN1173183C (en) | 2002-04-19 | 2002-04-19 | Immunological microball method for detecting pyloric helicobacterium in stool sample |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
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| CNB021157464A CN1173183C (en) | 2002-04-19 | 2002-04-19 | Immunological microball method for detecting pyloric helicobacterium in stool sample |
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| Publication Number | Publication Date |
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| CN1378084A true CN1378084A (en) | 2002-11-06 |
| CN1173183C CN1173183C (en) | 2004-10-27 |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100425991C (en) * | 2004-02-25 | 2008-10-15 | 中国科学院动物研究所 | Immunization microsphere in use for detecting SARS antibody, preparation method and application |
| CN100504391C (en) * | 2004-02-25 | 2009-06-24 | 中国科学院动物研究所 | Immune microsphere for detecting SARS antigen and preparation method |
| CN102539756A (en) * | 2011-12-23 | 2012-07-04 | 王滔 | Method for testing immune microspheres of pylori helicobacter pylori in gastric mucosa samples |
| CN106053787A (en) * | 2016-06-06 | 2016-10-26 | 上海师范大学 | Fluorescent immunochromatography test strip for detecting furazolidone metabolites as well as preparation and application |
| CN109061138A (en) * | 2018-09-03 | 2018-12-21 | 万绵水 | For detecting the kit of d-dimer in joint fluid |
| CN109342722A (en) * | 2018-09-30 | 2019-02-15 | 深圳市鸿美诊断技术有限公司 | It is a kind of for quantitative determining the preparation method of Heliobacter pylori antigen reagent in excrement |
| CN113671182A (en) * | 2020-05-13 | 2021-11-19 | 洛阳中科生物芯片技术有限公司 | Antibody joint detection kit for gD and gE proteins of porcine pseudorabies virus, and preparation method and application thereof |
-
2002
- 2002-04-19 CN CNB021157464A patent/CN1173183C/en not_active Expired - Fee Related
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100425991C (en) * | 2004-02-25 | 2008-10-15 | 中国科学院动物研究所 | Immunization microsphere in use for detecting SARS antibody, preparation method and application |
| CN100504391C (en) * | 2004-02-25 | 2009-06-24 | 中国科学院动物研究所 | Immune microsphere for detecting SARS antigen and preparation method |
| CN102539756A (en) * | 2011-12-23 | 2012-07-04 | 王滔 | Method for testing immune microspheres of pylori helicobacter pylori in gastric mucosa samples |
| CN106053787A (en) * | 2016-06-06 | 2016-10-26 | 上海师范大学 | Fluorescent immunochromatography test strip for detecting furazolidone metabolites as well as preparation and application |
| CN109061138A (en) * | 2018-09-03 | 2018-12-21 | 万绵水 | For detecting the kit of d-dimer in joint fluid |
| CN109342722A (en) * | 2018-09-30 | 2019-02-15 | 深圳市鸿美诊断技术有限公司 | It is a kind of for quantitative determining the preparation method of Heliobacter pylori antigen reagent in excrement |
| CN113671182A (en) * | 2020-05-13 | 2021-11-19 | 洛阳中科生物芯片技术有限公司 | Antibody joint detection kit for gD and gE proteins of porcine pseudorabies virus, and preparation method and application thereof |
| CN113671182B (en) * | 2020-05-13 | 2023-11-28 | 洛阳中科生物芯片技术有限公司 | Antibody joint inspection kit for porcine pseudorabies virus gD and gE proteins and preparation method and application thereof |
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| Publication number | Publication date |
|---|---|
| CN1173183C (en) | 2004-10-27 |
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Assignee: Fujian Newland Life Technologies (Holdings) Co., Ltd. Assignor: Wang Tao Contract fulfillment period: 2009.11.16 to 2015.11.16 contract change Contract record no.: 2009350000263 Denomination of invention: Immunological microball method for detecting pyloric helicobacterium in stool sample Granted publication date: 20041027 License type: Exclusive license Record date: 20091222 |
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