CN101086500A - Mid-pregnancy down's syndrome prenatal screening reagent kit - Google Patents

Abstract

The mid-pregnancy complex screening agent box comprises the AFP standard, AFP single crone anti body wrapped plate bar, AFP quality control, AFP enzyme conjugates, beta -HCG standard, beta -HCG single crone antibody wrapped plate bar, beta -HCG quality control, beta -HCG enzyme conjugates, sample dilute solution, dense cleansing solution, light emitting bottom solution, plate seal film, plate frame, inspecting the AFP or beta -HCG of the pregnant woman, using special data analysis treatment software, with accuracy, reliability, free from trauma, with wide promotion value.

Description

The second trimester down's syndrome prenatal screening reagent kit

Technical field

The present invention relates to a kind of kit that is used for the second trimester down's syndrome prenatal screening, belong to medical science and Biological Detection field.

Background technology

Down syndrome claim again mongolism or mongolism (Down ' s Syndrome, DS), it is the modal birth defect that causes the intellectual development obstacle, its incidence is approximately 1/750, its feature mainly shows as serious feeblemindedness, IQ mostly is 20～60, has only normal person's of the same age 1/4～1/2, the microcephaly is arranged, occipitalia is flat, item is thick, palpebral fissure is little, oblique on the outside, endocanthion is dark, eye distance is wide, saddle nose, mouth Chang Bankai, outside normal mouthful of the tongue, stout-fingered, palmmprint has logical passing through, symptom such as curved in the little finger of toe, it has contingency, randomness, incidence can raise with the increase of pregnant woman age, this disease does not have effective methods of treatment at present, prevent that this type of disease from the way of being taked taking place, carry out Prenatal Screening and necessary pre-natal diagnosis at period of gestation exactly, find and take corresponding measure as early as possible, at present, the method of pre-natal diagnosis mainly adopts chorionic villous sampling or amniocentesis to carry out pre-natal diagnosis clinically, these methods detect the Down syndrome fetus for antenatal, certain effect has been played in the birth that prevents this class fetus, but because that amniocentesis has is certain traumatic, can cause miscarriage, and can not directly detect with the method for making a definite diagnosis all pregnant woman on a large scale, also need certain technical equipment condition in addition, spended time is longer, expense is bigger, can not in all pregnant woman, extensively carry out, secondly, even some have the high risk gravida of amniocentesis indication, owing to may cause the feared state of mind of miscarriage to amniocentesis, refusal is accepted amniotic fluid chromosome examination, therefore, the birth number of Down syndrome is still than higher.

Summary of the invention

The purpose of this invention is to provide a kind of result accurately and reliably, non-invasive, safe, highly sensitive second trimester down's syndrome prenatal screening reagent kit.

For achieving the above object, technical scheme of the present invention is: the second trimester down's syndrome prenatal screening reagent kit, it is characterized in that: described kit is by AFP standard items, AFP monoclonal antibody coated slab, AFP quality-control product, AFP enzyme conjugates, β-HCG standard items, β-HCG monoclonal antibody coated slab, β-HCG quality-control product, β-HCG enzyme conjugates, sample diluent, concentrated cleaning solution, luminous substrate liquid, shrouding film, and grillage is formed.

The preparation method of described AFP, β-HCG standard items is: elder generation dissolves AFP, β-HC6 freeze-drying standard items respectively and is diluted to required maximum concentration, adopts coubling dilution to be diluted to desired concn again, obtains AFP, β-HCG standard items.

The preparation of described AFP, β-HCG monoclonal antibody coated slab may further comprise the steps:

(1), adopts monoclonal antibody technique, use AFP or the pure product of β-HCG routinely mouse to be carried out immunity earlier, extracting spleen cell and murine myeloma cell merge, screened pairing, set up AFP and β-HCG monoclonal antibody cell bank, and then preparing ascites through the inoculation mouse peritoneal, specificity is 100% with obtaining behind the ascites purifying again, purity is matched monoclonal antibody greater than 96% AFP or β-HCG;

(2), with the AFP of purifying or β-HCG monoclonal anti body and function carbonate buffer solution dilution, add then in each hole of coated slab, through absorption, washing, sealing, dry back acquisition AFP or β-HCG monoclonal antibody coated slab;

(3), with the special-purpose Aluminium Foil Package pack of packing into of AFP or β-HCG monoclonal antibody coated slab, seal and refrigerate standby.

The preparation of described AFP, β-HCG enzyme conjugates may further comprise the steps:

(1), take by weighing horseradish peroxidase and pour in the brown vial, get the dissolving of 1ml distilled water, get the 200ul sodium periodate solution with sample injector, splash in the bottle of containing horseradish peroxidase room temperature vibration 20min;

(2), horseradish peroxidase solution that vibration is good is taken into bag filter, puts into the acetate buffer solution for preparing, 4 ℃ of refrigerator dialysed overnight;

(3), get antibody to be marked and add in another bag filter, put into the carbonate buffer solution for preparing, place dialysed overnight in 4 ℃ of refrigerators;

(4), next day, take out horseradish peroxidase solution and antibody mixing, put into carbonate buffer solution and stir dialysis 3 hours;

(5), will dialyse good horseradish peroxidase and antibody-solutions, place in the brown vial, add the abundant mixing of sodium borohydride solution, with oscillator vibration 2 hours;

(6), with sephadex G-200 dress post, above-mentioned label is added on the column top, use the eluent wash-out;

(7), receive effluent, detected by good sheep anti mouse lath, get and show the corresponding invisible spectro enzyme conjugates in blue colour table hole with bag in advance with small test tube segmentation successively;

(8), the enzyme conjugates of collecting is added equivalent glycerine mixing, be positioned over-20 ℃ of refrigerators and preserve;

(9), enzyme conjugates is diluted to working concentration, preparation enzyme conjugates working fluid, the reagent bottle of packing into refrigeration is standby.

Described luminous substrate liquid comprises substrate solution A and substrate solution B, and its preparation may further comprise the steps:

(1), the preparation of substrate solution A:

Accurately take by weighing BSA, Tris aequum with electronic balance, add volumetric flask, add an amount of distilled water, jog makes it dissolving fully, with the distilled water constant volume, transfers PH to 9.0 with HCl again, gets H with sample injector ₂O ₂Add in the aforesaid liquid, mixing is measured branch according to the rules with the substrate solution A solution for preparing and is installed in the reagent bottle, and tightening bottle cap, to be placed on 2～8 ℃ of preservations standby;

(2), the preparation of substrate solution B:

Accurately take by weighing BSA, Tris aequum with electronic balance, add volumetric flask, add an amount of distilled water, jog makes it dissolving fully, with the distilled water constant volume, transfer PH to 9.0 with HCl again, again with electronic balance accurately take by weighing luminol, contraposition iodine phenol is poured volumetric flask into, fully dissolving, mixing are measured branch according to the rules with the substrate solution B solution for preparing and are installed in the reagent bottle, tighten bottle cap, it is standby to be placed on 2～8 ℃ of preservations.

Described using method may further comprise the steps:

(1) AFP measures:

The reagent that a, taking-up are stored in cold storage environment reaches the temperature with equilibrium at room temperature;

B, with concentrated cleaning solution by 1: 40 dilution proportion, shake up standby, as using cleansing solution;

C, determine required hole count according to AFP standard items, quality-control product and sample to be measured;

D, AFP standard items, quality-control product are directly got 50ul and are added in the respective aperture, with before shaking up;

E, in specimen hole to be measured, add earlier the 45ul sample diluent respectively;

F, get 5ul serum and add and to have added in the hole of sample dilution, with pipettor piping and druming, liquid becomes blueness by green in the hole;

G, every hole add 50ul AFP enzyme conjugates, vibrate for 30 seconds with oscillator, cover the shrouding film, put 37 ℃ of water-baths 1 hour;

H, wash plate: get rid of liquid in the most plate, fill with each hole, stop and firmly get rid of after 10 seconds, pat dry after repeating 5 times with cleansing solution;

I, add substrate: according to use amount, by 1: 1 abundant mixing, every hole added the luminous substrate 50ul of new preparation, vibrates 20 seconds with oscillator, puts into light-emitting appearance, in 5～20 minutes measurement results with substrate solution A and substrate solution B;

(2) β-HCG measures:

The reagent that a, taking-up are stored in cold storage environment reaches the temperature with equilibrium at room temperature;

B, with concentrated cleaning solution by 1: 40 dilution proportion, shake up standby, as using cleansing solution;

C, determine required hole count according to β-HCG standard items, quality-control product and sample to be measured;

Directly getting 50ul behind d, β-HCG standard items, the quality-control product mixing adds in the respective aperture;

E, in specimen hole to be measured, add earlier the 25ul sample diluent respectively;

F, get 25ul serum and add and to have added in the hole of sample dilution, with pipettor piping and druming several times, liquid becomes blueness by green in the hole;

G, vibrated for 30 seconds, cover the shrouding film, put 37 ℃ of water-baths 1 hour with oscillator;

H, wash plate: get rid of liquid in the most plate, fill with each hole, stop and firmly get rid of after 10 seconds, pat dry after repeating 5 times with cleansing solution;

I, hole add 50ul β-HCG enzyme conjugates, and liquid in the most plate is got rid of in 37 ℃ of water-baths 15 minutes, fills with each hole with cleansing solution, stops and firmly gets rid of after 10 seconds, pat dry after repeating 5 times;

J, add substrate: according to use amount, by 1: 1 abundant mixing, every hole added the luminous substrate 50ul that newly prepares, and vibrates for 20 seconds with oscillator, puts into light-emitting appearance, in 5～20 minutes measurement results with substrate solution A and substrate solution B.

Beneficial effect: the present invention adopts above method, using monoclonal antibody technology, enzyme labeling technology and enzymatic chemiluminescence quantitative measurement technology, in conjunction with chemiluminescence detection by quantitative instrument, AFP in the detection by quantitative pregnancy serum or β-HCG, adopt the exclusive data interpretation software that data are handled then, realize the purpose of second trimester down's syndrome prenatal screening, owing to used the chemiluminescence quantitative measurement technology, the result accurately and reliably, realized non-invasive, safe, highly sensitive Prenatal Screening, the present invention has higher market popularization value.

Points for attention:

1, the reagent of different lot numbers can not be used with;

2, seal off AFP or the β-HCG monoclonal antibody coated elisa plate bar that does not use, must sealing preserve, do not make moist;

3, kit must not be frozen in 2～8 ℃, lucifuge, dry storage.

Embodiment

Embodiment, the second trimester down's syndrome prenatal screening reagent kit, described kit is by AFP standard items, AFP monoclonal antibody coated slab, AFP quality-control product, AFP enzyme conjugates, β-HCG standard items, β-HCG monoclonal antibody coated slab, β-HCG quality-control product, β-HCG enzyme conjugates, sample diluent, concentrated cleaning solution, luminous substrate liquid, shrouding film, and grillage is formed.

Described AFP, β-HCG standard items, its preparation method is: elder generation dissolves AFP, β-HCG freeze-drying standard items respectively and is diluted to required maximum concentration, adopts coubling dilution to be diluted to desired concn again, obtains AFP, β-HCG standard items.

The preparation of described AFP, β-HCG monoclonal antibody coated slab may further comprise the steps:

(1), adopts monoclonal antibody technique, use AFP or the pure product of β-HCG routinely mouse to be carried out immunity earlier, extracting spleen cell and murine myeloma cell merge, screened pairing, set up AFP and β-HCG monoclonal antibody cell bank, and then preparing ascites through the inoculation mouse peritoneal, specificity is 100% with obtaining behind the ascites purifying again, purity is matched monoclonal antibody greater than 96% AFP or β-HCG;

(2), with the AFP of purifying or β-HCG monoclonal anti body and function carbonate buffer solution dilution, add then in each hole of coated slab, through absorption, washing, sealing, dry back acquisition AFP or β-HCG monoclonal antibody coated slab;

(3), with the special-purpose Aluminium Foil Package pack of packing into of AFP or β-HCG monoclonal antibody coated slab, seal and refrigerate standby.

The preparation of described AFP, β-HCG enzyme conjugates may further comprise the steps:

(1), take by weighing horseradish peroxidase and pour in the brown vial, get the dissolving of 1ml distilled water, get the 200ul sodium periodate solution with sample injector, splash in the bottle of containing horseradish peroxidase room temperature vibration 20min;

(2), horseradish peroxidase solution that vibration is good is taken into bag filter, puts into the acetate buffer solution for preparing, 4 ℃ of refrigerator dialysed overnight;

(3), get antibody to be marked and add in another bag filter, put into the carbonate buffer solution for preparing, place dialysed overnight in 4 ℃ of refrigerators;

(4), next day, take out horseradish peroxidase solution and antibody mixing, put into carbonate buffer solution and stir dialysis 3 hours;

(5), will dialyse good horseradish peroxidase and antibody-solutions, place in the brown vial, add the abundant mixing of sodium borohydride solution, with oscillator vibration 2 hours;

(6), with sephadex G-200 dress post, above-mentioned label is added on the column top, use the eluent wash-out;

(7), receive effluent, detected by good sheep anti mouse lath, get and show the corresponding invisible spectro enzyme conjugates in blue colour table hole with bag in advance with small test tube segmentation successively;

(8), the enzyme conjugates of collecting is added equivalent glycerine mixing, be positioned over-20 ℃ of refrigerators and preserve;

(9), enzyme conjugates is diluted to working concentration, preparation enzyme conjugates working fluid, the reagent bottle of packing into refrigeration is standby.

Described luminous substrate liquid comprises substrate solution A and substrate solution B, and its preparation may further comprise the steps:

(1), the preparation of substrate solution A:

Accurately take by weighing BSA, Tris aequum with electronic balance, add volumetric flask, add an amount of distilled water, jog makes it dissolving fully, with the distilled water constant volume, transfers PH to 9.0 with HCl again, gets H with sample injector ₂O ₂Add in the aforesaid liquid, mixing is measured branch according to the rules with the substrate solution A solution for preparing and is installed in the reagent bottle, and tightening bottle cap, to be placed on 2～8 ℃ of preservations standby;

(2), the preparation of substrate solution B:

Accurately take by weighing BSA, Tris aequum with electronic balance, add volumetric flask, add an amount of distilled water, jog makes it dissolving fully, with the distilled water constant volume, transfer PH to 9.0 with HCl again, again with electronic balance accurately take by weighing luminol, contraposition iodine phenol is poured volumetric flask into, fully dissolving, mixing are measured branch according to the rules with the substrate solution B solution for preparing and are installed in the reagent bottle, tighten bottle cap, it is standby to be placed on 2～8 ℃ of preservations.

The using method of described kit may further comprise the steps:

(1) AFP measures:

The reagent that a, taking-up are stored in cold storage environment reaches the temperature with equilibrium at room temperature;

B, with concentrated cleaning solution by 1: 40 dilution proportion, shake up standby, as using cleansing solution;

C, determine required hole count according to AFP standard items, quality-control product and sample to be measured;

D, AFP standard items, quality-control product are directly got 50ul and are added in the respective aperture, with before shaking up;

E, in specimen hole to be measured, add earlier the 45ul sample diluent respectively;

F, get 5ul serum and add and to have added in the hole of sample dilution, with pipettor piping and druming, liquid becomes blueness by green in the hole;

G, every hole add 50ul AFP enzyme conjugates, vibrate for 30 seconds with oscillator, cover the shrouding film, put 37 ℃ of water-baths 1 hour;

H, wash plate: get rid of liquid in the most plate, fill with each hole, stop and firmly get rid of after 10 seconds, pat dry after repeating 5 times with cleansing solution;

I, add substrate: according to use amount, by 1: 1 abundant mixing, every hole added the luminous substrate 50ul of new preparation, vibrates 20 seconds with oscillator, puts into light-emitting appearance, in 5～20 minutes measurement results with substrate solution A and substrate solution B;

(2) β-HCG measures:

The reagent that a, taking-up are stored in cold storage environment reaches the temperature with equilibrium at room temperature;

B, with concentrated cleaning solution by 1: 40 dilution proportion, shake up standby, as using cleansing solution;

C, determine required hole count according to β-HCG standard items, quality-control product and sample to be measured;

Directly getting 50ul behind d, β-HCG standard items, the quality-control product mixing adds in the respective aperture;

E, in specimen hole to be measured, add earlier the 25ul sample diluent respectively;

F, get 25ul serum and add and to have added in the hole of sample dilution, with pipettor piping and druming several times, liquid becomes blueness by green in the hole;

G, vibrated for 30 seconds, cover the shrouding film, put 37 ℃ of water-baths 1 hour with oscillator;

H, wash plate: get rid of liquid in the most plate, fill with each hole, stop and firmly get rid of after 10 seconds, pat dry after repeating 5 times with cleansing solution;

I, hole add 50ul β-HCG enzyme conjugates, and liquid in the most plate is got rid of in 37 ℃ of water-baths 15 minutes, fills with each hole with cleansing solution, stops and firmly gets rid of after 10 seconds, pat dry after repeating 5 times;

J, add substrate: according to use amount, by 1: 1 abundant mixing, every hole added the luminous substrate 50ul that newly prepares, and vibrates for 20 seconds with oscillator, puts into light-emitting appearance, in 5～20 minutes measurement results with substrate solution A and substrate solution B.

Claims (7)

1, second trimester down's syndrome prenatal screening reagent kit, it is characterized in that: described kit is by AFP standard items, AFP monoclonal antibody coated slab, AFP quality-control product, AFP enzyme conjugates, β-HCG standard items, β-HCG monoclonal antibody coated slab, β-HCG quality-control product, β-HCG enzyme conjugates, sample diluent, concentrated cleaning solution, luminous substrate liquid, shrouding film, and grillage is formed.

2, the preparation method of second trimester down's syndrome prenatal screening reagent kit as claimed in claim 1, it is characterized in that: the preparation method of described AFP, β-HCG standard items is: earlier respectively with AFP, β-HCG freeze-drying standard items dissolving and be diluted to required maximum concentration, adopt coubling dilution to be diluted to desired concn again, obtain AFP or β-HCG standard items.

3, the preparation method of second trimester down's syndrome prenatal screening reagent kit as claimed in claim 1 or 2 is characterized in that: the preparation of described AFP, β-HCG monoclonal antibody coated slab may further comprise the steps:

(1), adopts monoclonal antibody technique, use AFP or the pure product of β-HCG routinely mouse to be carried out immunity earlier, extracting spleen cell and murine myeloma cell merge, screened pairing, set up AFP and β-HCG monoclonal antibody cell bank, and then preparing ascites through the inoculation mouse peritoneal, specificity is 100% with obtaining behind the ascites purifying again, purity is matched monoclonal antibody greater than 96% AFP or β-HCG;

(2), with the AFP of purifying or β-HCG monoclonal anti body and function carbonate buffer solution dilution, add then in each hole of coated slab, through absorption, washing, sealing, dry back acquisition AFP or β-HCG monoclonal antibody coated slab;

(3), with the special-purpose Aluminium Foil Package pack of packing into of AFP or β-HCG monoclonal antibody coated slab, seal and refrigerate standby.

4, the preparation method of second trimester down's syndrome prenatal screening reagent kit as claimed in claim 3, it is characterized in that: the preparation method of described AFP, β-HCG quality-control product is: elder generation dissolves AFP, β-HCG freeze-drying standard items respectively and is diluted to required concentration, obtains AFP or β-HCG quality-control product.

5, the preparation method of second trimester down's syndrome prenatal screening reagent kit as claimed in claim 4 is characterized in that: the preparation of described AFP, β-HCG enzyme conjugates may further comprise the steps:

(1), take by weighing horseradish peroxidase and pour in the brown vial, get the dissolving of 1ml distilled water, get the 200ul sodium periodate solution with sample injector, splash in the bottle of containing horseradish peroxidase room temperature vibration 20min;

(2), horseradish peroxidase solution that vibration is good is taken into bag filter, puts into the acetate buffer solution for preparing, 4 ℃ of refrigerator dialysed overnight;

(3), get antibody to be marked and add in another bag filter, put into the carbonate buffer solution for preparing, place dialysed overnight in 4 ℃ of refrigerators;

(4), next day, take out horseradish peroxidase solution and antibody mixing, put into carbonate buffer solution and stir dialysis 3 hours;

(5), will dialyse good horseradish peroxidase and antibody-solutions, place in the brown vial, add the abundant mixing of sodium borohydride solution, with oscillator vibration 2 hours;

(6), with sephadex G-200 dress post, above-mentioned label is added on the column top, use the eluent wash-out;

(7), receive effluent, detected by good sheep anti mouse lath, get and show the corresponding invisible spectro enzyme conjugates in blue colour table hole with bag in advance with small test tube segmentation successively;

(8), the enzyme conjugates of collecting is added equivalent glycerine mixing, be positioned over-20 ℃ of refrigerators and preserve;

(9), enzyme conjugates is diluted to working concentration, preparation enzyme conjugates working fluid, the reagent bottle of packing into refrigeration is standby.

6, each method of the system of second trimester down's syndrome prenatal screening reagent kit as claimed in claim 5 is characterized in that: described luminous substrate liquid comprises substrate solution A and substrate solution B, and its preparation may further comprise the steps:

(1), the preparation of substrate solution A:

Accurately take by weighing BSA, Tris aequum with electronic balance, add volumetric flask, add an amount of distilled water, jog makes it dissolving fully, with the distilled water constant volume, transfers PH to 9.0 with HCl again, getting H2O2 with sample injector adds in the aforesaid liquid, mixing is measured branch according to the rules with the solution for preparing and is installed in the reagent bottle, and tightening bottle cap, to be placed on 2～8 ℃ of preservations standby;

(2), the preparation of substrate solution B:

Accurately take by weighing BSA, Tris aequum with electronic balance, add volumetric flask, add an amount of distilled water, jog makes it dissolving fully, with the distilled water constant volume, transfer PH to 9.0 with HCl again, again with electronic balance accurately take by weighing luminol, contraposition iodine phenol is poured volumetric flask into, fully dissolving, mixing are measured branch according to the rules with the solution for preparing and are installed in the reagent bottle, tighten bottle cap, it is standby to be placed on 2～8 ℃ of preservations.

7, the using method of second trimester down's syndrome prenatal screening reagent kit as claimed in claim 1 is characterized in that: described using method may further comprise the steps:

(1) AFP measures:

The reagent that a, taking-up are stored in cold storage environment reaches the temperature with equilibrium at room temperature;

B, with concentrated cleaning solution by 1: 40 dilution proportion, shake up standby, as using cleansing solution;

C, determine required hole count according to AFP standard items, quality-control product and sample to be measured;

D, AFP standard items, quality-control product are directly got 50ul and are added in the respective aperture, with before shaking up;

E, in specimen hole to be measured, add earlier the 45ul sample diluent respectively;

F, get 5ul serum and add and to have added in the hole of sample dilution, with pipettor piping and druming, liquid becomes blueness by green in the hole;

G, every hole add 50ul AFP enzyme conjugates, vibrate for 30 seconds with oscillator, cover the shrouding film, put 37 ℃ of water-baths 1 hour;

H, wash plate: get rid of liquid in the most plate, fill with each hole, stop and firmly get rid of after 10 seconds, pat dry after repeating 5 times with cleansing solution;

I, add substrate: according to use amount, by 1: 1 abundant mixing, every hole added the luminous substrate 50ul of new preparation, vibrates 20 seconds with oscillator, puts into light-emitting appearance, in 5～20 minutes measurement results with substrate solution A and substrate solution B;

(2) β-HCG measures:

The reagent that a, taking-up are stored in cold storage environment reaches the temperature with equilibrium at room temperature;

B, with concentrated cleaning solution by 1: 40 dilution proportion, shake up standby, as using cleansing solution;

C, determine required hole count according to β-HCG standard items, quality-control product and sample to be measured;

Directly getting 50ul behind d, β-HCG standard items, the quality-control product mixing adds in the respective aperture;

E, in specimen hole to be measured, add earlier the 25ul sample diluent respectively;

F, get 25ul serum and add and to have added in the hole of sample dilution, with pipettor piping and druming several times, liquid becomes blueness by green in the hole;

G, vibrated for 30 seconds, cover the shrouding film, put 37 ℃ of water-baths 1 hour with oscillator;

H, wash plate: get rid of liquid in the most plate, fill with each hole, stop and firmly get rid of after 10 seconds, pat dry after repeating 5 times with cleansing solution;

I, hole add 50ul β-HCG enzyme conjugates, and liquid in the most plate is got rid of in 37 ℃ of water-baths 15 minutes, fills with each hole with cleansing solution, stops and firmly gets rid of after 10 seconds, pat dry after repeating 5 times;

J, add substrate: according to use amount, by 1: 1 abundant mixing, every hole added the luminous substrate 50ul that newly prepares, and vibrates for 20 seconds with oscillator, puts into light-emitting appearance, in 5～20 minutes measurement results with substrate solution A and substrate solution B.