CN114609391A

CN114609391A - Human placenta growth factor determination kit and preparation method thereof

Info

Publication number: CN114609391A
Application number: CN202210182409.4A
Authority: CN
Inventors: 高乐乐; 张冉; 郝荣杰; 郭春雨
Original assignee: Beijing Tiger Biotechnology Co ltd
Current assignee: Beijing Tiger Biotechnology Co ltd
Priority date: 2022-02-26
Filing date: 2022-02-26
Publication date: 2022-06-10

Abstract

The invention provides a human placenta growth factor determination kit and a preparation method thereof, relates to the technical field of biological detection, and comprises a detection card and a sample diluent. The detection card comprises a bottom plate, and an integrated pad, a nitrocellulose membrane and absorbent paper which are sequentially connected and fixed on the bottom plate; the integrated pad is fixed with an antibody I marked by a fluorescent microsphere, and the nitrocellulose membrane is provided with a detection line T line and a quality control line C line; the detection line T is coated with an antibody II, and the quality control line C is coated with an antibody III. The fluorescent microsphere-labeled antibody I is arranged at one end close to the nitrocellulose membrane, the antibody I and the antibody II are both mouse anti-placental growth factor antibodies, and the antibody III is goat anti-mouse IgG. The determination method has the advantages of high sensitivity, high accuracy, simple operation and low cost.

Description

Human placenta growth factor determination kit and preparation method thereof

Technical Field

The invention relates to the technical field of biological detection, in particular to a human placenta growth factor determination kit and a preparation method thereof.

Background

Placental dysplasia is likely to cause early-onset preeclampsia and thus premature birth, and promotion of fetal lung maturation and inhibition of uterine contraction are considered before pregnancy for 34 weeks. Currently, the level of human Placental growth factor (PLGF) is assessed quantitatively by: PLGF ≧ 100pg/ml indicates that placenta function is normal; PLGF <100pg/ml suggests placental insufficiency requiring treatment to support placental perfusion. PLGF <12pg/ml suggests severe placental insufficiency, strongly predictive of preterm delivery within 14 days. If therapeutic intervention delays the onset of preterm labor, PLGF levels are accelerated when placental function improves. The effect of placental function improvement following treatment for preterm birth can be assessed by monitoring PLGF levels.

The concentration relation of the placenta growth factor (PLGF) in a blood sample is a reference index for evaluating the growth and development conditions of the placenta, and the concentration relation of the placenta growth factor (PLGF) and soluble fms-like tyrosine kinase-1 (sFlt-1) is closely related to the occurrence and development of the preeclampsia, and researches show that the detection of the blood vessel activity factor sFlt-1 and the PLGF level in the blood serum of a pregnant woman in the middle period of pregnancy has good prediction value on the occurrence of the preeclampsia, so that the detection of the PLGF level can be used as an auxiliary prompt and used as a basis for clinically diagnosing the occurrence of preeclampsia diseases.

At present, the immunological method for detecting the human placenta growth factor mainly comprises the following steps: chemiluminescence methods and electrochemical luminescence methods, etc., wherein the chemiluminescence methods and the electrochemical luminescence methods require large-scale high-precision operating instruments, are expensive, have high requirements on professional level of test operation personnel, and are not beneficial to large-scale detection of the indexes.

Disclosure of Invention

The invention provides a kit for detecting the human placenta growth factor level in blood circulation, which has high sensitivity, high stability, simple and convenient operation and quick application, and a preparation and use method thereof, aiming at overcoming the defects of the prior art, and facilitating the application of the kit for identifying normal pregnancy and preeclampsia before clinical symptoms appear.

In order to solve the technical problems, the invention adopts the following technical scheme.

On one hand, the embodiment of the application provides a human placenta growth factor determination kit, which comprises a detection card and a sample diluent, wherein the detection card comprises a bottom plate, and an integrated pad, a nitrocellulose membrane and absorbent paper which are sequentially connected and fixed on the bottom plate;

an antibody I marked by a fluorescent microsphere is fixed on the integrated pad, and a detection line T line and a quality control line C line are arranged on the nitrocellulose membrane; the detection line T is coated with an antibody II, and the quality control line C is coated with an antibody III;

the fluorescent microsphere labeled antibody I is arranged at one end close to the nitrocellulose membrane, the antibody I and the antibody II are both mouse anti-human placenta growth factor antibodies, and the antibody III is goat anti-mouse IgG.

On the other hand, the invention also provides a preparation method of the human placental growth factor determination kit as a general inventive concept.

The preparation process of the kit comprises the following steps:

preparation of an integral pad: soaking the glass fiber membrane in a surfactant, and drying; preparing the fluorescent microsphere conjugate from the fluorescent microsphere and the antibody I, preparing conjugate working solution, spraying the conjugate working solution on the glass fiber membrane, and drying;

preparation of nitrocellulose membrane: diluting the antibody II, scribing on the detection line T, and drying; diluting the antibody III, scribing on the quality control line C, and drying;

preparation of sample diluent: preparing a phosphate buffer solution, adding tween-20 into the phosphate buffer solution, and uniformly stirring;

assembling the detection card: sequentially adhering the prepared and dried integrated pad, the nitrocellulose membrane and the absorbent paper to a bottom plate, cutting the integrated pad, the nitrocellulose membrane and the absorbent paper into test strips, and then putting the test strips into a card shell to obtain the detection card;

and finally, the prepared detection card and the sample diluent are packed in a box in a matching way.

Compared with the prior art, the embodiment of the invention has at least the following advantages or beneficial effects:

the integrated design on the structure of the detection card greatly reduces the misjudgment of results by human factors; the detection of the concentration by using the fluorescence immunoassay instrument improves the sensitivity and the accuracy of the detection, and compared with the mode of carrying out adhesive assembly on the sample pad and the fluorescent microsphere conjugate pad in the prior art, the structural improvement of the integrated pad of the invention is more convenient for the chromatography of the sample, reduces the adhesive flow in the production, has simple process and saves the time. To sum up, this kit simple structure, it is with low costs, convenient equipment, the simple operation.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a schematic diagram of a test card according to the present invention;

FIG. 2 is a schematic view of the operation of the test card of the present invention;

FIG. 3 is a graph showing a linear correlation obtained by a test of a test card in an experimental example of the present invention.

Icon: 1-integral pad; 2-fluorescent microsphere conjugates; 3-nitrocellulose membrane; 4-absorbent paper; 5-bottom plate.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail below with reference to specific examples.

The invention provides a human placenta growth factor determination kit, which comprises a detection card and a sample diluent, wherein the detection card comprises a bottom plate, and an integrated pad, a nitrocellulose membrane and absorbent paper which are sequentially connected and fixed on the bottom plate;

the integrated pad is fixed with an antibody I marked by fluorescent microspheres, and the nitrocellulose membrane is provided with a detection line T line and a quality control line C line; the detection line T is coated with an antibody II, and the quality control line C is coated with an antibody III.

The fluorescent microsphere labeled antibody I is arranged at one end close to the nitrocellulose membrane, the antibody I and the antibody II are both mouse anti-placental growth factor antibodies, and the antibody III is goat anti-mouse IgG.

The human placenta growth factor determination kit provided by the invention adopts a fluorescence immune double antibody sandwich method to detect the PLGF horizontal concentration of a pregnant woman blood sample, and has high detection efficiency and sensitive reaction.

In some embodiments of the present invention, the integrated pad includes a glass fiber membrane treated and dried by a surfactant, wherein the surfactant is a solution containing 0.1-1 vol% of tween-20, and the addition of the surfactant is very helpful for reducing the surface tension of the liquid sample, and is helpful for the chromatography of the integrated pad.

The particle size of the fluorescent microsphere is 200-400 nm, and the wavelength of the fluorescent microsphere emitted in an excited state is 500-650 nm. The microspheres with different particle size ranges act on different targets in a targeted manner, the fluorescent microspheres with the particle size ranges are helpful for coupling with the antibody I, the particle sizes correspond to the wavelengths of the excited states of the antibodies, and the fluorescent signals can be accurately captured by detecting the corresponding wavelengths through a fluorescence immunoassay analyzer.

In some embodiments of the invention, the working concentration of the antibody II is 0.5-1 mg/ml, and the addition amount on the detection line T is 0.5-1 μ l/cm; the working concentration of the antibody III is 1-2 mg/ml, and the addition amount on the quality control line C is 0.5-1 μ l/cm.

The working concentration obtained by diluting the antibodies II and III corresponds to the scribing amount on the detection line T line and the quality control line C line, and the sufficient amount of the antibodies for interception and combination on each line is ensured.

In addition, the invention also provides a preparation method of the human placenta growth factor determination kit, which comprises the following steps:

The preparation method of the human placenta growth factor determination kit provided by the invention is an integrated and realizable technical scheme, the fluorescent microsphere conjugate integrated pad and the nitrocellulose membrane are prepared and scribed, then the fluorescent microsphere conjugate integrated pad and the nitrocellulose membrane are assembled with the bottom plate and the absorbent paper to form the detection card, the prepared sample diluent is used for diluting a sample, and the diluted sample is detected and analyzed by the detection card.

The preparation method of the fluorescent microsphere conjugate comprises the following steps:

activating the fluorescent microspheres by an activating agent, wherein in some embodiments of the present invention, the mass ratio of the fluorescent microspheres to the activating agent is 1: (0.1-0.5).

The ratio of the fluorescent agent to the activator determines the activation degree, a proper amount of surfactant enables carboxyl of the microsphere to react with 1-ethyl- (3-Dimethylaminopropyl) carbonyl diimine (1- (3-dimethylamino propyl) -3-ethyl carboxyl imide, EDC) and N-hydroxysuccinimide (NHS) to form an amido bond, so that the microsphere is in an excited state and is used for covalent coupling with the antibody, and the excessive surfactant enables the fluorescent microsphere to be completely coated, so that the binding efficiency of the fluorescent microsphere and the antibody is influenced.

Coupling the activated fluorescent microspheres with antibodies, wherein in some embodiments of the present invention, the coupling ratio of the antibody i to the fluorescent microspheres is: 50-200 μ g-Ab/mg-beads.

The coupling ratio of the fluorescent microspheres and the antibody has influence on the preservation of the prepared fluorescent immune microsphere conjugate, so that the agglutination phenomenon occurs before the fluorescent immune microsphere conjugate is sprayed on an integrated pad. When the antibody in the system is insufficient, the surface of the microsphere still has a plurality of reactive groups after crosslinking, and the groups can react with the antibody connected on the microsphere, so that the microsphere is pulled together again, and aggregation is generated. Although this can be solved by adding blocking agents, such as: fetal Bovine Serum Albumin (BSA), but the best way is to match the corresponding amount of antibody to the particle size of the fluorescent microspheres. Generally, the smaller the particle size of the fluorescent microsphere is, the more the amount of the required antibody is, the better the precision and the linearity are, while the selection of the fluorescent microsphere with the larger particle size requires less antibody, the poorer precision and the linearity are, but the sensitivity is good, and the particle size of the fluorescent microsphere provided by the invention is 200-400 nm, so the coupling ratio of the antibody I and the fluorescent microsphere is set as follows: 50-200 μ g-Ab/mg-beads.

Spraying the coupled fluorescent microsphere combination on the glass fiber membrane, wherein in some embodiments of the invention, the spraying amount of the fluorescent microsphere combination on the integrated pad is as follows: 2 to 4. mu.l/cm²。

The spraying amount of the fluorescent microsphere conjugate on the integrated pad reflects the concentration of the antibody, and after the excessive antibody is sprayed on the integrated pad to cause the chromatographic reaction to occur, the antigen and the antibody are combined to form immunityThe immune complex can move forward under the drive of capillary force, and the fluorescent microsphere coupled antibody which is not combined with the antigen can also move forward to be combined with the antibody on the subsequent quality control line C. Therefore, 2 to 4. mu.l/cm ²2 mul/cm in the amount sprayed²The amount of antibody sprayed in the most basic excess antibody spraying amount provided by the present invention also affects the efficiency of chromatography operation, so it is not preferable that the antibody spraying amount is higher, and therefore 4. mu.l/cm is used in the present invention²The highest amount of excess antibody sprayed was used.

In some embodiments of the present invention, the soaking time of the glass fiber membrane in the surfactant is 0.5 to 6 hours. The surfactant can remarkably reduce the surface tension of water and air, the glass fiber film is placed in the surfactant for soaking, sufficient soaking time is helpful for improving the wear resistance, the ductility and the antistatic capability of the glass fiber film, and the soaking time of 0.5h given by the invention is the minimum.

The human placenta growth factor measuring kit is used for collecting samples by a fluorescence immunochromatography principle and processing results by a fluorescence analyzer, and has the advantages of sensitive reaction and high accuracy.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

The embodiment provides a human placenta growth factor determination kit and a preparation method thereof.

The human placenta growth factor determination kit comprises a detection card and a sample diluent, wherein as shown in figure 1, the detection card comprises a bottom plate 5, and an integrated pad 1, a fluorescent microsphere binder 2, a nitrocellulose membrane 3 and absorbent paper 4 which are sequentially connected and fixed on the bottom plate;

an antibody I marked by fluorescent microspheres is fixed on the integrated pad 1, and a detection line T and a quality control line C are arranged on the nitrocellulose membrane 3, wherein the detection line T is coated with an antibody II, and the quality control line C is coated with an antibody III.

The preparation method of the human placenta growth factor determination kit comprises the following steps:

preparation of an integral pad:

1) preparing a surfactant: 2ml of Tween-20 is absorbed by a pipette and added into 1000ml of purified water, and the pipette is blown back and forth and then mixed evenly.

2) Soaking the glass fiber membrane in a surfactant for 0.5h, and fully drying.

3) Preparing fluorescent microsphere conjugate from fluorescent microsphere and mouse anti-PLGF antibody I, and mixing at a ratio of 4 μ l/cm²The dosage of the composite is evenly sprayed on the glass fiber membrane, and the integrated pad is obtained after drying.

The preparation process of the fluorescent microsphere conjugate comprises the following steps:

activating fluorescent microspheres;

and (3) sucking 0.2ml of fluorescent microspheres (containing 2mg of fluorescent microspheres) into a centrifuge tube by using a pipette gun, wherein the fluorescent microspheres are milky in appearance and have the particle size of 200-400 nm. The wavelength emitted by the fluorescent microsphere after being excited is 500 nm-650 nm. Then, 1.8ml of 0.1M MES buffer was added and sufficiently suspended. Add 50. mu.l activation buffer to the above solution and spin-activate on a spin-blender for 30 min. Wherein the activation buffer is 0.1M MES buffer containing 10mg/ml EDC and NHS.

Uniformly mixing and coupling the mouse anti-PLGF antibody I and the activated fluorescent microspheres;

and adding 200 mu g of mouse anti-PLGF antibody I into the activated fluorescent microsphere solution, and uniformly mixing for 60min on a rotary mixer.

And thirdly, centrifuging and retaining the precipitate, and redissolving the precipitate to obtain the fluorescent microsphere conjugate.

And (3) placing the test tube filled with the coupling mixture into an ultracentrifuge, centrifuging at 13000rpm for 15min, then discarding the supernatant, adding 2ml of 0.5M PB buffer solution containing 0.5% BSA and 5% sucrose into the precipitate, and performing ultrasonic mixing to obtain a fluorescent microsphere conjugate solution.

Antibody streaking on nitrocellulose membranes:

diluting the mouse anti-PLGF antibody II to 0.5mg/ml by using 0.02M Tris buffer solution containing 5% trehalose as a working solution for detecting the line T, and diluting the goat anti-mouse IgG to 1.5mg/ml by using 0.02M Tris buffer solution containing 5% trehalose as a working solution for controlling the line C; the nitrocellulose membrane was scratched in an amount of 1. mu.l/cm, and left to dry at 35 ℃ for 24 hours.

Preparation of sample diluent:

preparation: weighing 1.15g of disodium hydrogen phosphate and 0.231g of sodium dihydrogen phosphate, adding 1000ml of purified water for full dissolution, adding 2-5 ml of tween-20, and uniformly stirring.

Subpackaging: subpackaging the prepared sample diluent as follows: 200 to 400. mu.l/tube.

Assembling the detection card:

and (3) sequentially sticking the prepared and dried fluorescent microsphere conjugate integrated pad, the nitrocellulose membrane and the absorbent paper on a bottom plate, cutting the mixture into test strips, and then putting the test strips into a card shell to obtain the detection card.

In addition, the embodiment also provides a use method of the human placental growth factor determination kit.

As shown in figure 2, the collected sample is dripped on the integrated pad 1, and according to the chromatography direction, the sample is absorbed by the integrated pad 1, then passes through the attachment point of the fluorescent immune microsphere conjugate 2 to reach the nitrocellulose membrane 3, and then passes through the detection line T line, then passes through the quality control line C line, and finally reaches the absorbent paper 4 to be absorbed.

The working principle is as follows: fluorescence immune double antibody sandwich principle. The sample is dragged by capillary force on the integrated pad 1, when passing through the attachment point of the fluorescent immune microsphere conjugate, the PLGF antigen is combined with the mouse anti-PLGF antibody I coupled with the fluorescent microsphere to form a fluorescent microsphere coupled antibody-PLGF antigen complex, then the complex is continuously diffused to the nitrocellulose membrane 3 along with the sample, the complex is intercepted by the mouse anti-PLGF antibody II coated on the detection line T line to form an immune complex of the fluorescent microsphere coupled antibody-PLGF antigen-mouse anti-PLGF antibody II, and the mouse anti-PLGF antibody I not coupled with the fluorescent microsphere coupled with the PLGF antigen is continuously forwards along the nitrocellulose membrane, moves to the quality control line C line and then reacts with the goat anti-mouse IgG coated antibody again to form a fluorescent microsphere coupled mouse anti-PLGF antibody I-goat anti-mouse IgG antibody complex.

The detection line T containing the immune complex of the fluorescent microsphere coupled antibody-PLGF antigen-mouse anti-PLGF antibody II and the quality control line C containing the immune complex of the fluorescent microsphere coupled mouse anti-PLGF antibody I-goat anti-mouse IgG antibody are collected to fluorescent signals in a fluorescence immunoassay analyzer.

And (3) calculating the concentration value of the sample according to the relative fluorescence signal processing value (T/C: T is the fluorescence signal of the detection line, C is the fluorescence signal of the quality control line) of the sample and the self-carried calibration curve of the reagent, and then completing the determination of the human placenta growth factor in the serum and plasma samples to be detected.

Example 2

This example is essentially the same as example 1, except that an integral pad was prepared:

1) preparing a surfactant: and (3) sucking 1ml of Tween-20 by using a pipette gun, adding into 1000ml of purified water, blowing and sucking the pipette gun back and forth, and mixing uniformly.

2) And soaking the glass fiber membrane in a surfactant for 4 hours and then fully drying.

Example 3

1) preparing a surfactant: 10ml of Tween-20 is sucked by a pipette gun and added into 1000ml of purified water, and the pipette gun is blown back and forth and then mixed evenly.

2) And soaking the glass fiber membrane in a surfactant for 6h, and fully drying.

Example 4

This example is essentially the same as example 1, except that the activation of the fluorescent microspheres:

and (3) sucking 0.2ml of fluorescent microspheres (containing 2mg of fluorescent microspheres) into a centrifuge tube by using a pipette gun, wherein the fluorescent microspheres are milky in appearance and have the particle size of 200-400 nm. The wavelength emitted by the fluorescent microsphere after being excited is 500 nm-650 nm. Then, 1.8ml of 0.1M MES buffer was added and sufficiently suspended. Add 20. mu.l of activation buffer to the above solution and spin-activate on a spin-blender for 30 min. Wherein the activation buffer is 0.1M MES buffer containing 10mg/ml EDC and NHS.

Example 5

and (3) sucking 0.2ml of fluorescent microspheres (containing 2mg of fluorescent microspheres) into a centrifuge tube by using a pipette gun, wherein the fluorescent microspheres are milky in appearance and have the particle size of 200-400 nm. The wavelength emitted by the fluorescent microsphere after being excited is 500 nm-650 nm. Then, 1.8ml of 0.1M MES buffer was added and sufficiently suspended. To the above solution, 100. mu.l of activation buffer was added, and the mixture was rotary-activated for 30min on a rotary homogenizer. Wherein the activation buffer is 0.1M MES buffer containing 10mg/ml EDC and NHS.

Example 6

This example is essentially the same as example 1, except that the murine anti-PLGF antibody I was coupled to activated fluorescent microspheres:

and adding 100 mu g of mouse anti-PLGF antibody I into the activated fluorescent microsphere solution, and uniformly mixing for 60min on a rotary mixer.

Example 7

and adding 400 mu g of mouse anti-PLGF antibody I into the activated fluorescent microsphere solution, and uniformly mixing for 60min on a rotary mixer.

Example 8

This example is substantially the same as example 1, except that:

preparing fluorescent microsphere conjugate from fluorescent microsphere and mouse anti-PLGF antibody I, and mixing at a ratio of 2 μ l/cm²The dosage of the composite is evenly sprayed on the glass fiber membrane, and the integrated pad is obtained after drying.

Example 9

This example is substantially the same as example 1, except that:

preparing fluorescent microsphere conjugate from fluorescent microsphere and mouse anti-PLGF antibody I, and mixing at a ratio of 3 μ l/cm²The dosage of the composite is evenly sprayed on the glass fiber membrane, and the integrated pad is obtained after drying.

Example 10

This example is substantially the same as example 1, except that the amount of antibody streaking on the nitrocellulose membrane:

diluting the mouse anti-PLGF antibody II to 0.8mg/ml by using 0.02M Tris buffer solution containing 5% of trehalose as a working solution for detecting the line T, and diluting the goat anti-mouse IgG to 1mg/ml by using 0.02M Tris buffer solution containing 5% of trehalose as a working solution for controlling the line C; the nitrocellulose membrane was scratched in an amount of 1. mu.l/cm and left to dry at 38 ℃ for 24 hours.

Example 11

diluting the mouse anti-PLGF antibody II to 1mg/ml by using 0.02M Tris buffer solution containing 5% trehalose as a working solution for detecting the line T, and diluting the goat anti-mouse IgG to 2mg/ml by using 0.02M Tris buffer solution containing 5% trehalose as a working solution for detecting the line C; the nitrocellulose membrane was scratched in an amount of 1. mu.l/cm, and left to dry at 40 ℃ for 24 hours.

Test example 1

In this test example, the detection cards prepared in examples 1 to 11 were used to detect the human placenta growth factor internal control, and performance analysis was performed on the blank limit, the linear correlation coefficient R, the repeatability CV, and the accuracy thereof, respectively.

It should be noted that, the internal control used in the detection of the present invention is a clinical sample (serum sample selected in this experiment) collected by a hospital and having a corresponding clinical background value, for the measurement of the linear range, the clinical serum sample close to the linear high value (3500pg/mL) is prepared into clinical serum with theoretical concentrations of 2800, 2000, 1500, 700, 350 and 15 (unit: pg/mL) according to a certain proportion, and the internal control for repeatability and accuracy is selected from clinical serum with a corresponding theoretical concentration for detection.

(1) Detection of white space limit

Using 20 detection cards randomly selected from examples 1-11 to perform 80-120 μ L sample application detection on the sample diluent, and calculating the mean value of the detection values

And a standard deviation SD. The measurement results of the blank limit are shown in Table 1:

TABLE 1 measurement of blank limits

As can be seen from Table 1, the blank limit is less than 0.08ng/mL, which indicates that the detection card provided by the invention has good background and the sensitivity meets the requirements.

(2) Detection of linear range

Mixing 3500, 2800, 2000, 1500, 700, 350 and 15 (unit: pg/mL) and sample diluent, adding 80-120 μ L of sample, detecting, and calculating average value (pg/mL)

And its coefficient of correlation R with the theoretical concentration (pg/mL). The results of the linear range measurement are shown in table 2 and fig. 3:

TABLE 2 Linear Range measurements

As can be seen from Table 2 and FIG. 3, when the linear range of the detection card provided by the present invention is (15-35) pg/mL, the correlation coefficient R of the dose-response curve is 0.998, which is greater than the standard value of 0.990, the linearity is good, and the requirement is satisfied.

(3) Detection of coefficient of variation of repeatability

And respectively measuring 10 detection cards aiming at 2000pg/mL and 50pg/mL by 60 detection cards of 3 lot numbers in the detection cards, and calculating the mean value x and standard deviation SD of the detection values, thereby calculating the CV in the lot and among the lots. The results of the measurement of the coefficient of variation with respect to reproducibility are shown in Table 3:

TABLE 3 measurement results of repetitive coefficient of variation

As can be seen from Table 3, the 3 lot numbers have intra-lot CV values within 10% and inter-lot CV values within 10% for both 2000pg/mL and 50pg/mL, and have good repeatability and meet the requirements.

(4) Detection of accuracy

Taking 6 detection cards, respectively adding a human placental growth factor (PLGF) sample (A) with the concentration of about 2000pg/ml (the concentration deviation is allowed to be +/-20%) into a serum or other corresponding matrix sample B, wherein the volume of the added A is 10% of the total volume (A + B), measuring 3 cards respectively for the recovered sample and the low-value sample, respectively calculating the average value, and calculating the recovery rate R according to a formula.

In the formula:

r-recovery rate;

v-volume of sample A;

V₀-the volume of sample B;

c, the detection concentration of the sample A after the sample A is added into the sample B;

C₀-the concentration of sample B;

C_s-concentration of sample a.

The accuracy measurement results are shown in table 4:

TABLE 4 determination of accuracy

As can be seen from Table 4, the recovery rate of the detection card of the present invention is 100.99%, the recovery rate is within the range of standard 85% -115%, and the accuracy meets the requirement.

Test example 2

The experimental example combines a soluble fms-like tyrosine kinase-1 determination kit to provide clinical application of a determination reagent for preeclampsia. All samples in this experimental example were clinical samples.

The results are judged according to the reference intervals of different gestational periods of the kit, the reference interval of the kit is generally used as the reference interval value by 5-95% of the measured value of blood samples of normal persons or other samples, the reference intervals of different gestational periods are shown in a table 5, and the detection results of clinical samples are shown in a table 6:

TABLE 5 preeclampsia assay kit reference interval

TABLE 6 clinical specimen test results

In Table 6, + represents positive, -represents negative, - ↓ represents higher than the upper limit of the reference range, and ↓ represents lower than the lower limit of the reference range. As can be seen from tables 5 and 6, the clinical diagnosis of 11 positive samples and 14 negative samples, the kit detected 10 positive samples, the sensitivity was up to 90%, and 12 negative samples were consistent with the clinical results. In addition, the kit detects positive in two cases of clinical negative samples G8 and G24, and more clinical observation and detection are needed.

The human placenta growth factor determination kit provided by the invention adopts a fluorescence immune double antibody sandwich method to detect the PLGF horizontal concentration of a pregnant woman blood sample, and the determination of the concentration of the human placenta growth factor (PLGF) in a sample to be detected can be completed by determining through a fluorescence immune analyzer and contrasting with a calibration curve.

In conclusion, the detection card of the human placenta growth factor determination kit provided by the invention is structurally integrated, so that misjudgment of results by human factors is greatly reduced; the fluorescence immunoassay analyzer is used for detecting the concentration, so that the sensitivity and the accuracy of detection are improved; in addition, the reagent materials required by the preparation method of the human placenta growth factor determination reagent kit are easy to obtain, the process is simple, the operation is easy, and the performance of the prepared reagent kit is good; the determination method also has the advantages of high sensitivity, high accuracy, simple operation and low cost.

The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Claims

1. Human placenta growth factor determination kit, its characterized in that: the kit comprises a detection card and sample diluent, wherein the detection card comprises a bottom plate, and an integrated pad, a nitrocellulose membrane and absorbent paper which are sequentially connected and fixed on the bottom plate;

the fluorescent microsphere-labeled antibody I is arranged at one end close to the nitrocellulose membrane, the antibody I and the antibody II are both mouse anti-human placental growth factor antibodies, and the antibody III is goat anti-mouse IgG.

2. The human placental growth factor assay kit of claim 1, wherein the integral pad comprises a dried glass fiber membrane treated with a surfactant, wherein the surfactant is a solution comprising 0.1 to 1% tween-20 by volume.

3. The human placental growth factor assay kit according to claim 1, wherein the particle size of the fluorescent microspheres is 200 to 400nm, and the wavelength of the fluorescent microspheres emitted in an excited state is 500 to 650 nm.

4. The human placental growth factor assay kit according to claim 1, wherein the working concentration of antibody ii is 0.5-1 mg/ml, and the amount added on the detection line T is 0.5-1 μ l/cm; the working concentration of the antibody III is 1-2 mg/ml, and the adding amount on the quality control line C line is 0.5-1 mu l/cm.

5. The human placental growth factor assay kit of claim 1, wherein the sample diluent comprises a phosphate buffer.

6. A method for preparing the human placental growth factor assay kit according to any one of claims 1 to 5, comprising the steps of:

preparation of an integral pad: soaking the glass fiber membrane in a surfactant, and drying; preparing the fluorescent microsphere and the antibody I into the fluorescent microsphere conjugate, preparing a conjugate working solution, spraying the conjugate working solution onto the glass fiber membrane, and drying;

preparation of nitrocellulose membrane: diluting the antibody II, scribing lines on the detection line T, and drying; diluting the antibody III, scribing a line on the quality control line C, and drying;

preparation of sample diluent: preparing a phosphate buffer solution, adding tween-20 into the phosphate buffer solution, and stirring;

7. The human placental growth factor assay kit according to claim 6, wherein the preparation of said fluorescent microsphere conjugate comprises the steps of:

activating fluorescent microspheres by an activating agent, wherein the mass ratio of the fluorescent microspheres to the activating agent is 1: (0.1 to 0.5);

coupling the activated fluorescent microspheres with antibodies, wherein the coupling ratio of the antibodies I to the fluorescent microspheres is 50-200 mug-Ab/mg-beads.

8. The method for preparing a human placental growth factor assay kit according to claim 6, wherein the amount of the fluorescent microsphere conjugate sprayed on the glass fiber membrane is 2 to 4 μ l/cm²。

9. The method of claim 7, wherein said activator is 0.1M MES buffer.

10. The method for preparing the human placental growth factor assay kit according to claim 6, wherein the time for soaking the glass fiber membrane in the surfactant is 0.5 to 6 hours.