CN109116031A - A kind of detection kit can detect a variety of prenatal and postnatal care pathogen IgG antibodies - Google Patents
A kind of detection kit can detect a variety of prenatal and postnatal care pathogen IgG antibodies Download PDFInfo
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- CN109116031A CN109116031A CN201810823175.0A CN201810823175A CN109116031A CN 109116031 A CN109116031 A CN 109116031A CN 201810823175 A CN201810823175 A CN 201810823175A CN 109116031 A CN109116031 A CN 109116031A
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- microballon
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- G—PHYSICS
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
Abstract
The invention discloses a kind of detection kits that can detect a variety of prenatal and postnatal care pathogen IgG antibodies, the kit includes: multiple sealed reagent bottles that (1) is respectively provided with compounded microbeads suspension, fluorescent marker, Sample dilution, cleaning solution, and (2) separate and concentrate the kit for packing these reagent bottles；Microballon is the polystyrene microsphere through different fluorescent dyeings and coding；Comprising a variety of detection microbeads, standard microballon, with reference to microballon and blank microballon in compounded microbeads suspension, detection microbeads are to be coated with different prenatal and postnatal care pathogen antigens respectively in the bead surface of different coding；Standard microballon includes at least three kinds microballons for combining various concentration gradient human IgG；The microballon of anti-human igg is combined with reference to microballon；Blank microballon is the microballon that any antigen is not added.The present invention can do the detection of multiple prenatal and postnatal care pathogen IgG antibodies to a human serum sample simultaneously, substantially increase the detection efficiency of related pathogen antigen, be of great significance to diagnosis and further treatment.
Description
Technical field
The present invention relates to the detection fields of pathogen IgG antibody, and in particular to can disposably detect a variety of prenatal and postnatal care diseases
The kit of substance IgG antibody.
Background technique
Cytomegalovirus (CMV) infection is extensive, usual non-evident sympton；However, cytomegalovirus can be for a long time
Latent or chronic infection.Since the newborn of relatively frequent disease incidence, infection has serious disease and individual to inhibit to exempt from
Epidemic disease, therefore can determine it for an important human pathogen.
There is no apparent clinical disease when the ewborn infant birth of 95% congenital infection cytomegalovirus.Remaining 5%
Ewborn infant clinical manifestation go out serious disease, as jaundice, hepatosplenomegaly, thrombocytopenic purpura, skull calcification, growth are slow
Slow, pneumonia, brain edema, microcephaly and eye defect.There is the baby of serious symptoms due to secondary in congenital infection cytomegalovirus
Complication after birth will be dead；However, that largely survives then will appear neurotrosis.
Cytomegalovirus can be separated to from saliva, urine, lotion, cervical discharge and sperm.Therefore, virus can be with
It relays through a variety of ways, the virus that spreads through sex intercourse seems to facilitate the virus infection of young man.
Immunosuppressive patient cytomegalovirus infection frequent occurrence, the usually reactivation of latent infection, and may
Life-threatening.These patients include homograft recipient, cancer patient and Immune Deficiency Syndrome (AIDS).It is big and small
Cellular virus infection immunosupress patient's clinical manifestation be pneumonia, cytomegalovirus monocytosis,mononucleosis, hepatitis, pericarditis and
Encephalitis.
Herpes simplex infections are as caused by 2 kinds of visibly different antigenic type HSV-1 and HSV-2.Two kinds of HSV classes
Type is all the pathogen of the mankind.HSV-1 usually causes the infection of oropharynx and eye.HSV-2 causes most reproduction and first
Raw infection.However, tissue signature be not it is absolute, HSV-2 can be separated to occasionally from oropharynx, and the genital infection of 5-10% is
Caused by HSV-1.
Herpes simplex infections are divided into primary infection and superinfection.After primary infection, Sensory nerve's cell is dived
Volt infection, the activation of latent infection lead to superinfection.Superinfection does not have that primary infection is serious, and the infection phase is also shorter.It is single
Pure herpesvirus infection usually occurs at the position initially infected.However, serious local infection or dispersion infection are likely to occur
On the immune person weakened.These people include ewborn infant, exempt to inhibit epidemic disease power treatment patient, such as transplant recipient and cancer
People.
Rubeola is a kind of mild, communicable, viral infection, mainly appears on child and the youth person
On.Rubeola has the feature for the erythema maculopapule fash for occurring two, three days.However, being more than the clinic of 50% rubella-infection person
Symptom is unobvious.The symptom of other rubeolas includes low-heat, slight upper respiratory symptom and pillow inferior gluteal lymph node disease.It is short in young man
Temporary arthralgia and arthritis is common symptom, but complication very serious such as encephalitis or thrombocytopenic purpura are very
It is rare.
Although rubeola is usually benign in the infection of child or adult and is restricted by itself, in most starting for fetus
It may cause within three months spontaneous abortion, dead baby or the inborn defect of baby.Baby is infected in uterus and may have at birth
Obvious shortcoming, it is also possible to normal, it is also possible to occur complication afterwards.
Inborn rubeola symptom have passed through just to be realized for a long time, and classical symptom is congenital heart disease, cataract, sense
Sound merve deafness, the delay of retarded and intrauterine growth.From after rubeolas prevalence in 1964, other clinical manifestation quilts of congenital rubeola
Understanding, including newborn thrombocytopenic purpura, hepatitis, bone disorders and meningoencephalitis.Meanwhile diabetes and progressive
The symptom of the later period performance of these congenital rubella-infections of rubella virus panencephalitis is also found recently.
Rubeola exists all over the world.In the country of not vaccine inoculation, the women of the childbearing age of 10-25% is serum
Reaction negative, it is easy infected.
Toxoplasma is a kind of anti-protozoal helminth widely distributed in the world.Although cat is conclusive host, it
Almost all of mammal and birds can be infected.It is serological statistics indicate that the population quilt of most industrial country 30%
It slowly infects, although the popularity degree of different regions population is different.
There is no symptom after most of case (80-90%) toxoplasma gondii infection.The clinic of general adult acute toxoplasmosis
Show as asymptomatic single lymph node or more lymphadenopathies.Lymphadenopathy may be with fever, uncomfortable and similar infectiousness
Atypical lymphocytosis symptom of monocytosis,mononucleosis.It seldom will appear serious complication in common host,
Such as encephalitis, myocarditis or pneumonia.
Although common host will not usually generate symptom because of toxoplasma gondii infection, to having immune deficiency or immune function
The low host of energy is often fatal.The patient of immune deficiency or immunologic hypofunction can develop as serious dissemination bow
Shape parasitosis or toxoplasmic encephalitis or both all occur.
Symptom can occur in the newborn of about 75% congenital infection.However, nearly all subclinical new
Raw baby will appear vision disorder or nerve sequelae afterwards.The appearance chorioretinitis of about 80-85%, has
Also it will appear the obstacle of vision disorder or spirit, movement.
There is presently no a kind of methods can detect cytomegalovirus in a sample, herpes simplex virus (1 type, 2 simultaneously
Type), the IgG antibody of rubella virus, toxoplasma.
Summary of the invention
The object of the present invention is to provide a kind of detection kit that can detect a variety of prenatal and postnatal care pathogen IgG antibodies, with
Solve drawbacks described above existing for background technique.
The present invention realizes by the following technical solutions:
A kind of detection kit can detect a variety of prenatal and postnatal care pathogen IgG antibodies, the kit include:
(1) multiple sealed reagents of compounded microbeads suspension, fluorescent marker, Sample dilution, cleaning solution are respectively provided with
Bottle, (2) separate and concentrate the kit for packing these reagent bottles；Microballon is polystyrene microsphere of uniform size, with different
Fluorescent dye dyes polystyrene microsphere, to obtain the microballon of different coding (such as No. 1-100)；The compounded microbeads
Comprising a variety of detection microbeads, standard microballon, with reference to microballon and blank microballon in suspension, specifically:
The detection microbeads are to be coated with different prenatal and postnatal care pathogen antigens respectively in the bead surface of different coding；
The standard microballon includes at least three kinds microballons for combining various concentration gradient human IgG；
The microballon that anti-human igg is combined with reference to microballon；
The blank microballon is the microballon that any antigen is not added.
Preferably, the diameter of the microballon is 5-7 microns, preferably 5.6 microns.
The prenatal and postnatal care pathogen antigen include: -2 antigen of big and small extracellular antigen, -1 antigen of herpe simplex and herpe simplex,
Rubella antigens and toxoplasma antigen.
The fluorescent marker is goat-anti-human IgG (specific γ of phycoerythrin label as a preferred technical solution,
Chain).
The Sample dilution is phosphatic buffer.Preferably, the composition of each component and contain in Sample dilution
It measures as follows:
The component of the cleaning solution is identical as the Sample dilution, and concentration is its 8-10 times.
The present invention can detect the detection kit of a variety of prenatal and postnatal care pathogen IgG antibodies as a preferred technical solution,
In further include the reagent bottle equipped with quality-control product, the quality-control product includes positive quality control product and negative quality-control product, the positive quality control
Product are people's pooled serum containing antibody corresponding with the prenatal and postnatal care pathogen antigen for including in the detection microbeads, described
Negative quality-control product is people's negative serum to the corresponding negative antibody of prenatal and postnatal care pathogen antigen.The positive quality control product it is dense
Range is spent with specificity between criticizing, i.e., every a collection of reagent has the concentration range of their own.
The preparation method of the compounded microbeads suspension, includes the following steps:
It is coated with different prenatal and postnatal care pathogen antigens respectively using the microballon of different coding, is mixed and made into detection microbeads,
Then with standard microballon, be transferred to Sample dilution together with reference to microballon, blank microballon and mix, it is outstanding to obtain the compounded microbeads
Supernatant liquid.
The detection microbeads are prepared with the following method: microballon activating solution are prepared, then according to including in every milliliter of microballon
Any one prenatal and postnatal care encephalapthy agent antigen is added in the ratio of 10-100ug pathogen antigen, is sufficiently mixed, and coating is different excellent
Raw good child-rearing pathogen antigen, needs the microballon using different coding.
The microballon activating solution is that reaction tube is added in 1000 unit volume microballons, 5-50 unit volume EDC (1- is added
Ethyl-(3- dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate) and 5-50 unit volume Sulfo-NHS (N- hydroxy amber
Amber acid imide) it activates.
The standard microballon is prepared with the following method: microballon activating solution is prepared, then according to including in every milliliter of microballon
Human IgG is added in the ratio of 2-18ug human IgG (immunoglobulin G), is sufficiently mixed.People in three kinds or more standard microballon
The content of IgG is uniform incremented by successively in the range of 2-18ug.For example, when using 3 kinds of standard microballons, the concentration of human IgG
Respectively 2-5ug/ml, 6-10ug/ml, 12-18ug/ml.
It is described to be prepared with the following method with reference to microballon: microballon activating solution to be prepared, then according to including in every milliliter of microballon
Anti-human igg is added in the ratio of 8-15ug anti-human igg, is sufficiently mixed.
The blank microballon is the microballon activating solution that any antigen is not added.
The innovative point that the present invention can detect the detection kit of a variety of prenatal and postnatal care pathogen IgG antibodies is, Ke Yi
The detection for doing multiple pathogen IgG antibodies in same time same hole to people's serum sample, substantially increases related cause of disease
The detection efficiency of body antibody is of great significance to diagnosis and further treatment.
Specific embodiment
The present invention is illustrated below by specific embodiment, but is not intended to limit the present invention.
Embodiment 1:
The specific preparation process that the present invention can detect the detection kit of a variety of prenatal and postnatal care pathogen IgG antibodies is as follows:
1, Sample dilution is prepared
The formula for preparing 8L is as follows:
| Number | Reagent name | Concentration |
| 1 | NaCl | 8.0g/L |
| 2 | Na2HPO4·12H2O | 2.9g/L |
| 3 | KCl | 2.4g/L |
| 4 | KH2PO4 | 2.4g/L |
| 5 | Tris | 0.3g/L |
| 6 | NaN3 | 0.5g/L |
2, the activation of microballon
5.6 microns of 1ml of microballon (polystyrene microsphere of Luminex company of the U.S.) is taken to be added in 1.5ml centrifuge tube,
5-50ul EDC (1- ethyl-(3- dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate) and 5-50 unit volume Sulfo- is added
NHS (N- hydroxy thiosuccinimide) is activated, and microballon activating solution is obtained.The above microballon activating solution makes 10 parts, every part
Microballon coding it is different.
3, the preparation of compounded microbeads suspension
5 portions of microballon activating solutions are taken, a kind of prenatal and postnatal care pathogen antigen 10-100ug is separately added into, is vortexed and mixes 30 seconds,
Ultrasound mixes 30 seconds, and 2-8 DEG C shakes up overnight.Above-mentioned prenatal and postnatal care pathogen antigen difference is as follows: big and small extracellular antigen, simple blister
- 2 antigen of -1 antigen of rash and herpe simplex, rubella antigens, toxoplasma antigen.Detection microbeads are made.
3 portions of microballon activating solutions are taken, standard microballon 1, standard microballon 2 and standard microballon 3 are respectively labeled as, are calibrated in microballon 1
The human IgG of 2-5ug/ml is added, calibrates the human IgG that 6-10ug/ml is added in microballon 2, calibrates in microballon 3 and 12-18ug/ml is added
Human IgG, be vortexed mix 30 seconds, ultrasound mix 30 seconds, 2-8 DEG C shake up overnight.Standard microballon is made.
1 portion of microballon activating solution is taken, the anti-human igg of 8-15ug/ml is added, is vortexed and mixes 30 seconds, ultrasound mixes 30 seconds, 2-8
It DEG C shakes up overnight.It is made with reference to microballon.
1 portion of microballon activating solution is taken, any pathogen antigen is added without as blank microballon, is vortexed and mixes 30 seconds, ultrasound is mixed
Even 30 seconds, 2-8 DEG C shook up overnight.Blank microballon is made.
By above-mentioned each microballon filtering and washing, it is washed after microballon be transferred in Sample dilution, mix, finally use sample
Dilution is settled to 120ml.After the assay was approved, 20 bottles are distributed into, every bottle of 5.5ml.
3, fluorescent marker:
Goat-anti-human IgG (IgG-PE) of phycoerythrin label: initial concentration 1mg/ml.
Working concentration is diluted to Sample dilution.Working concentration is 0.6mg/L.
4, concentrate: component and formula are identical with Sample dilution, and concentration is its 10 times.It is made into 8L originally, now
It is made into 800ml.
5, quality-control product: positive quality control product: it will mix, obtain containing above-mentioned a variety of prenatal and postnatal care pathogen antigen positive serums
People's pooled serum of the analyte positive measures the concentration range of Positive assay object, the concentration range that every a batch reagent has its special.
Negative quality-control product: negative serum is mixed, and obtains negative quality-control product.
6, by above-mentioned prepared compounded microbeads suspension, fluorescent marker, Sample dilution, cleaning solution, quality-control product point
It is not fitted into reagent bottle, is then separated using kit and concentrated and pack these reagent bottles.
Embodiment 2:
The present invention can detect the detection kit of a variety of prenatal and postnatal care pathogen IgG antibodies in human serum sample while examine
Survey the concrete application of multiple prenatal and postnatal care pathogen IgG antibodies.Specific step is as follows:
1. each ingredient takes out from condition of storage, strong light is avoided, is allowed to come to room temperature (20-25 DEG C).
2. determining quality-control product needed for testing and the total quantity of sample.Negative Quality Control is located at the hole A1, and positive quality control is located at the hole B1,
Each Quality Control and sample require a micropore (being shown in Table 1 sample permutations).
A. compounded microbeads suspension should be mixed thoroughly before use to obtain the best reading result time.Most efficient method
It is to vibrate the mixed whirlpool device of microballon suspension 30 seconds, then Ultrasound Instrument is handled 30 seconds.
B. each reagent should be mixed thoroughly when in use.Appropriate method includes being placed on microwell plate on mixed whirlpool device with about
800RPM is mixed 30 seconds, or is inhaled and beaten at least 5 times repeatedly using the reaction liquid that pipettor draws about 1/2 volume.
Table 1
3. diluting the serum of negative quality-control product, positive quality control product and each patient by 1:21 dilution ratio in dilution plate
Sample (such as: by 10ul serum sample+200ul Sample dilution).Note: correctly operation requires: sample and Sample Dilution
Liquid will be uniformly mixed, and can be operated by above-mentioned note 2b.
4. after the number cells needed for determining detection, using Multi-channel liquid transfer device or reusing pipettor in filter plate
50ul compounded microbeads suspension is added in each hole.
5. sample and quality-control product of the 10ul after 1:21 dilutes are added into every hole of filter plate.Correctly operation is wanted
Ask: the substance of addition will be uniformly mixed with the compounded microbeads suspension in hole, can be operated by above-mentioned note 2b.
6. filter plate is set (20-25 DEG C) of room temperature and is incubated 30 minutes.
7. after incubating, being washed according to the following steps to microballon with vacuum filtration pump.
A. filter plate is placed on the pallet of vacuum filtration pump, opens vacuum filtration pump, removes solution in hole, be only left micro-
Pearl is in the bottom of plate.
B. vacuum pump is closed, 1X board-washing liquid 200ul is added into each hole of filter plate.
C. it reuses vacuum pump and removes solution.
D. 7b, 7C step are repeated, needs to be washed three times in total.
8. gently blotting the bottom of filter plate after last time is washed, before next step operates, filter plate is allowed to exist
It is air-dried 3-5 minutes.
9. 150ul fluorescent marker is added in every hole of filter plate, the sequence and rate being added according to sample successively add
Enter into filter plate.Correctly operation requires: the fluorescent marker of addition will be uniformly mixed with the microballon in hole, can be by above-mentioned note
2b operation.Mixture can be transferred to an empty polystyrene when fluorescent marker mixing is added as a kind of selection
In reaction plate.
10. (20-25 DEG C) of filter plate room temperature incubates 30 ± 10 minutes.
11. the detection template of the multi-functional streaming dot matrix instrument of Luminex 200 of U.S. Luminex company is selected to analyze test
As a result.Result can be read from filter plate or reaction plate.
12. filter plate fluorescent marker incubate after the completion of 60 minutes in read result.Filter plate is vibrated before reading
About 15 seconds.Time needed for this selectable step can reduce read plate.
The detection kit that the present invention can detect a variety of prenatal and postnatal care pathogen IgG antibodies realizes same in the same time
The detection of multiple prenatal and postnatal care pathogen IgG antibodies is done in hole to people's serum sample.
The above shows and describes the basic principle, main features and advantages of the invention.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this
The principle of invention, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, these changes
Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its
Equivalent thereof.
Claims (10)
1. a kind of detection kit that can detect a variety of prenatal and postnatal care pathogen IgG antibodies, which is characterized in that the kit packet
It includes: (1) being respectively provided with multiple sealed reagent bottles of compounded microbeads suspension, fluorescent marker, Sample dilution, cleaning solution, (2)
Separate and concentrates the kit for packing these reagent bottles；Microballon is polystyrene microsphere of uniform size, is contaminated with different fluorescence
Material is dyed and is numbered to polystyrene microsphere, to obtain the microballon of different coding；It is wrapped in the compounded microbeads suspension
Containing a variety of detection microbeads, standard microballon, with reference to microballon and blank microballon, specifically:
The detection microbeads are to be coated with different prenatal and postnatal care pathogen antigens respectively in the bead surface of different coding；
The standard microballon includes at least three kinds microballons for combining various concentration gradient human IgG；
The microballon that anti-human igg is combined with reference to microballon；
The blank microballon is the microballon that any antigen is not added.
2. can detect the detection kit of a variety of prenatal and postnatal care pathogen IgG antibodies as described in claim 1, feature exists
In the diameter of the microballon is 5-7 microns.
3. can detect the detection kit of a variety of prenatal and postnatal care pathogen IgG antibodies as described in claim 1, feature exists
In the prenatal and postnatal care pathogen antigen includes: -2 antigen of big and small extracellular antigen, -1 antigen of herpe simplex and herpe simplex, rubeola
Antigen, toxoplasma antigen.
4. can detect the detection kit of a variety of prenatal and postnatal care pathogen IgG antibodies as described in claim 1, feature exists
In the fluorescent marker is goat-anti-human IgG of phycoerythrin label.
5. can detect the detection kit of a variety of prenatal and postnatal care pathogen IgG antibodies as described in claim 1, feature exists
In the composition of each component and content are as follows in the Sample dilution:
6. can detect the detection kit of a variety of prenatal and postnatal care pathogen IgG antibodies as described in claim 1, feature exists
In the component of cleaning solution described in kit is identical as the Sample dilution, and concentration is its 8-10 times.
7. can detect the detection kit of a variety of prenatal and postnatal care pathogen IgG antibodies as described in claim 1, feature exists
In further including the reagent bottle equipped with quality-control product, the quality-control product includes positive quality control product and negative quality-control product, the positive quality control
Product are people's pooled serum containing antibody corresponding with the prenatal and postnatal care pathogen antigen for including in the detection microbeads, described
Negative quality-control product is people's negative serum to the corresponding negative antibody of prenatal and postnatal care pathogen antigen.
8. can detect the detection kit of a variety of prenatal and postnatal care pathogen IgG antibodies as described in claim 1, feature exists
In the preparation method of the compounded microbeads suspension includes the following steps: to be coated with respectively using the microballon of different coding different
Prenatal and postnatal care pathogen antigen is mixed and made into detection microbeads, then shifts together with standard microballon, with reference to microballon, blank microballon
It to Sample dilution and mixes, obtains the compounded microbeads suspension.
9. can detect the detection kit of a variety of prenatal and postnatal care pathogen IgG antibodies as described in claim 1, feature exists
In the detection microbeads are prepared with the following method: prepare microballon activating solution, then according in every milliliter of microballon include 10-
Any one prenatal and postnatal care encephalapthy agent antigen is added in the ratio of 100ug pathogen antigen, is sufficiently mixed, and it is excellent to be coated with different aristogenesis
Pathogen antigen is educated, the microballon using different coding is needed；The microballon activating solution is that 1000 unit volume microballons are added instead
5-50 unit volume 1- ethyl-(3- dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate and 5-50 unit volume is added in Ying Guan
N- hydroxy thiosuccinimide activates.
10. can detect the detection kit of a variety of prenatal and postnatal care pathogen IgG antibodies as described in claim 1, feature exists
In the standard microballon is prepared with the following method: prepare the microballon activating solution, then according in every milliliter of microballon include 2-
Human IgG is added in the ratio of 18ug human IgG, is sufficiently mixed；It is described to be prepared with the following method with reference to microballon: it is living to prepare the microballon
Change liquid, anti-human igg then is added according to the ratio in every milliliter of microballon including 8-15ug anti-human igg, is sufficiently mixed；The blank
Microballon is the microballon activating solution that any antigen is not added；The microballon activating solution is that 1000 unit volume microballons are added to react
5-50 unit volume 1- ethyl-(3- dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate and 5-50 unit volume N- is added in pipe
Hydroxy thiosuccinimide activates.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114113589A (en) * | 2021-10-25 | 2022-03-01 | 江苏纳迪芯生命科技研究院有限公司 | Chlamydia trachomatis and mycoplasma urealyticum antigen detection kit based on magnetic particle luminescence method |
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