CN110526964B - Rapid noninvasive female corpus luteum function monitoring technology - Google Patents
Rapid noninvasive female corpus luteum function monitoring technology Download PDFInfo
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
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Abstract
The invention relates to a non-invasive female fertility monitoring technology, in particular to a full-quantitative or semi-quantitative rapid non-invasive urine detection technology for measuring the cycle production of progesterone metabolites in female urine so as to monitor and evaluate female corpus luteum function, judge different ovulation cycle types and determine whether real ovulation exists or not and whether pregnancy can be performed after ovulation. The first embodiment of the present invention relates to a protein hybridization conjugate for detecting a pregnanediol glucuronide, which comprises a pregnanediol glucuronide and a conjugate protein, and is characterized by further comprising a linker for connecting the pregnanediol glucuronide and the conjugate protein. The hormone protein conjugate provided by the invention is used for detecting PdG by using an unsaturated labeled antibody, the sensitivity can reach 0.1ng/ml, and the linear range can be controlled between 0.1ng/ml and 50 ng/ml.
Description
The application is a divisional application of Chinese patent application with the name of 'female corpus luteum function rapid noninvasive monitoring technology' applied on 17 th 4 th 2015 and the application number of 201510185202.2.
Technical Field
The invention relates to a non-invasive female fertility monitoring technology, in particular to a full-quantitative or semi-quantitative rapid non-invasive urine detection technology for measuring the cycle production of progesterone metabolites in female urine so as to monitor and evaluate female corpus luteum function, judge different ovulation cycle types and determine whether real ovulation exists or not and whether pregnancy can be performed after ovulation.
Background
According to the investigation report of the current situation of infertility in China in 2012, the infertility rate in China is as high as 12.5%, and the reasons for female prescription are more than 50%. The number of people exceeds 5000 ten thousand. The number of patients who failed infertility treatment was 66%, 98.9% of them were caused by lack of timely and accurate detection. Therefore, the invention provides a female corpus luteum function monitoring technology which is noninvasive, low in cost and easy to use in hospitals and families, and is particularly suitable for the requirements of Chinese prenatal and postnatal care.
At the beginning of the menstrual cycle, approximately 15-20 ova begin to mature in each ovary. Follicles filled with ova produce estrogen. Eventually, one follicle predominates and continues to mature, while the remaining follicles begin to collapse. The estrogen level of this dominant follicle rises dramatically several days before ovulation until a critical level is reached. This high estrogen level triggers a surge of another hormone known as Luteinizing Hormone (LH). Triggering ovulation, resulting in the release of ova from the ovaries. Marking the end of the ovulation cycle.
The estrogen and LH peak appearing in sequence in the menstrual cycle have close relation with the ovarian ovulation time. Ovulation usually occurs within the next 12-48 hours once an estrogen or LH surge occurs. Therefore, monitoring of estrogen or LH peak during the menstrual cycle can be used to determine the optimal time to conception, or when contraceptive regimens need to be taken.
The presence of estrogen and LH surges prior to ovulation does not confirm the actual occurrence of ovulation, as estrogen surge and LH surge do not always lead to ovulation. Even with true ovulation, infertility results from insufficient luteal phase or short luteal phase after ova. This ovulation but inability to conceive phenomenon is currently the most common cause of infertility in women.
The commercial inspection in the market is only limited to hormone blood test or LH urine test, the hormone blood test is inconvenient because a large-scale instrument is required, and the inspection result has no unified standard for infertility diagnosis. LH urinalysis (including CN00112779.9 patent of Kunming Yunnan university, CN200610200333 patent of Gansu Lanzhou university, and CN20110072240.9 patent of Wuhan Jinghong Wanfang Tang) is mainly applied to the preliminary estimation of ovulation period. Therefore, at home and abroad, no convenient and easy-to-use commercialized infertility in vitro noninvasive diagnosis product is available, which is used for quantitatively monitoring female corpus luteum function, determining whether ovulation exists or not, whether pregnancy can be realized after ovulation, and related female fertility and hormone infertility.
Disclosure of Invention
The invention aims to provide a cheap and convenient immunoassay method and a detection kit for hospitals, families and individuals to carry out full quantification or semiquantification according to the close relation between the generation amount of hormone progesterone in the urine of women during ovulation and pregnancy to determine the generation amount of progesterone urine metabolite pregnanediol glucuronide PdG after ovulation. The change of corpus luteum function was monitored by quantitatively measuring the concentration of PdG in urine within 6 days after ovulation with the ovulation period as a reference point, thereby verifying the presence or absence of actual ovulation and the ability to conceive after ovulation. (1) When the pregnanediol value was <2.0mg/24h, no ovulation or luteinization did not disrupt the follicle (LUF). (2) If the pregnant glycol value is between 2.0-3.0mg/24h, ovulation is indicated, but the luteal phase after ovulation is insufficient or short, and pregnancy cannot be performed. (3) This cycle is only the ovulation cycle with full fertility if the pregnanediol value >3.0mg/24 h. Therefore, a pregnanediol value of more than 3.0mg/24h within 6 days after ovulation is a criterion for a female to have full fertility and to be able to normally conceive.
If a standard of greater than 3.0mg/24h pregnanediol value is used 6 days post ovulation, the present invention can also use a qualitative immunoassay, such as a colloidal gold semiquantitative immunoassay, to determine whether a female has full fertility for normal conception (>3.0mg/24h) or has infertility (< 3.0mg/24 h).
The present invention monitors the change of the function of corpus luteum by linking pregnanediol glucuronide to conjugate protein through linker to form pregnanediol glucuronide protein hybrid conjugate and measuring the pregnanediol glucuronide protein hybrid conjugate, thereby verifying the presence or absence of real ovulation and whether the pregnanediol can be pregnant after ovulation.
Linkers of the present invention are divided into hydrophobic (e.g., simple non-elastomeric carbon chains) and hydrophilic (e.g., elastomeric carbon chains containing heteroatoms of oxygen, sulfur, nitrogen, etc.) groups having a length of 1 to 100 carbon atoms, preferably 5 to 25 carbon atoms, more preferably 6 to 20 carbon atoms, and most preferably 6 to 18 carbon atoms, wherein one or more carbon atoms may be substituted with a heteroatom selected from oxygen, sulfur, nitrogen, etc. Although the length of the linker and its hydrophilic or hydrophobic properties affect the sensitivity and specificity of the immunoassay in different ways and to different extents. However, the relationship between the two has not been studied systematically so far, and further, how to effectively utilize the length and hydrophilic/hydrophobic properties of the linker to design the immunoassay kit reasonably for commercial applications such as development and application of immunoassay kits has not been reported.
According to the invention, the connection quantity of the pregnanediol glucuronide on the conjugate protein can be controlled by changing the hydrophily and the hydrophobicity of the connector and the length of the chain, so that the density of the pregnanediol glucuronide on a reagent strip detection line is adjusted, namely the higher the density is, the wider the linear range is, the lower the density is, and the better the sensitivity is. Meanwhile, the length and the type of the carbon chain also influence the quality of the combination of the pregnanediol glucuronide-coupled protein hybrid conjugate and the antibody, so that the nonspecific combination with the antibody can be effectively avoided, and the steric hindrance caused by the combination with the antibody can be reduced by the properly long carbon chain.
The first embodiment of the present invention relates to a protein hybridization conjugate for detecting a pregnanediol glucuronide, which comprises a pregnanediol glucuronide and a conjugate protein, and is characterized by further comprising a linker for connecting the pregnanediol glucuronide and the conjugate protein.
A second embodiment of the present invention relates to the conjugate body of the first embodiment, characterized in that the linker is a straight or branched chain group containing 1 to 100 carbon atoms, preferably 5 to 25 carbon atoms, more preferably 6 to 20 carbon atoms, most preferably 6 to 18 carbon atoms.
A third embodiment of the present invention relates to the conjugate body of the second embodiment, wherein one or more carbon atoms of the linker are substituted with a heteroatom.
A fourth embodiment of the present invention relates to the coupling body of the third embodiment, wherein the hetero atom is selected from oxygen, nitrogen and sulfur.
A fifth embodiment of the present invention relates to the conjugate according to any one of the first to fourth embodiments, wherein the conjugate protein is selected from the group consisting of Ovalbumin (OVA), Bovine Serum Albumin (BSA), and hemocyanin (KLH).
A sixth embodiment of the present invention relates to the coupling body according to any one of the first to fourth embodiments, which is selected from the group consisting of:
(1) egg albumin couplet of pregnanediol glucuronide-6 atom carbon chain
(2) Egg albumin couplet of pregnanediol glucuronide-13 atom carbon chain
(3) Egg albumin couplet of pregnanediol glucuronide-20 atom carbon chain
(4) Pregnanediol glucuronide-18 atom carbon chain-ovalbumin couplet.
A seventh embodiment of the present invention relates to the conjugate body according to any one of the first to fourth embodiments, wherein the ratio of pregnanediol glucuronide to linker and conjugate protein is 1:1 to 100: 1.
an eighth embodiment of the present invention relates to a kit for detecting pregnanediol glucuronide, which comprises the protein hybrid conjugate according to any one of the first to seventh embodiments of the present invention, characterized by further comprising an antibody that specifically binds to the protein hybrid conjugate.
A ninth embodiment of the present invention relates to the kit of the eighth embodiment, characterized in that the antibody is a monoclonal antibody or a polyclonal antibody.
A tenth embodiment of the present invention relates to the kit of the ninth embodiment, further comprising a detectable label linked to the antibody.
An eleventh embodiment of the present invention relates to the kit of the ninth embodiment, further comprising a second antibody that specifically binds to the antibody.
A twelfth embodiment of the present invention relates to the kit of the eleventh embodiment, wherein the ratio of the antibody to the second antibody is 1: 2 to 2: 1.
a thirteenth embodiment of the present invention relates to the kit of the eleventh or twelfth embodiment, further comprising a detectable label linked to the second antibody.
A fourteenth embodiment of the present invention is directed to the kit of the tenth or thirteenth embodiment, wherein the detectable label is selected from the group consisting of a colloid, a chromophore, a chemiluminescent group, a fluorophore, an isotope, and an enzyme.
A fifteenth embodiment of the present invention relates to a kit for monitoring corpus luteum function, characterized by comprising the conjugate body or the kit according to any one of the first to fourteenth embodiments of the present invention.
The present invention has an advantageous effect in that, for women with full fertility, it undoubtedly provides accurate information on the ideal time of natural conception and the starting time of pregnancy. For women suffering from infertility and being treated, the invention also provides a doctor and a patient with an ideal tool for judging whether the treatment effect is successful. In the reproductive center, the progesterone measurement provided by the invention is undoubtedly used for determining the optimal time for artificial insemination, monitoring the ovulation induction of gonadotropins and determining the time for ovulation and egg taking in the early development stage of test tube Infants (IVF), so that the success rate of pregnancy is improved, and great help is provided.
It is to be understood that within the scope of the present invention, the various technical features of the present invention described above and those specifically described below (examples) may be combined with each other to constitute new or preferred technical solutions.
Drawings
FIG. 1 shows PdG immune test strip and its fluorescent quantitative analysis principle diagram
As shown in FIG. 1, the test strip of the present invention may be composed of a nitrocellulose membrane, a sample pad, a water absorbent pad, a PVC or PS substrate, and the like. The nitrocellulose membrane was scribed with two lines: one line is a detection line coated by the PdG-protein conjugate, and the other line is an internal standard quality control line coated by an independent secondary antibody. Colloidal gold (20nm-100nm) or various fluorescent reagents can directly mark the PdG monoclonal antibody, and can also mark a second antibody polyclonal antibody firstly, and then is combined with the PdG monoclonal antibody in an immune manner. Preferably, the secondary antibody polyclonal antibody is labeled, and then the optimal ratio of the secondary antibody label to the PdG monoclonal antibody is adjusted and optimized, so as to obtain the most effective immunological binding. The portable quantitative analyzer (TSR3000 Shanghai Jiening Biotech Co., Ltd.) which is suitable for being used with the colloidal gold or the fluorescence quantitative detection test strip can carry out rapid and accurate quantitative analysis on the depth of the colloidal gold red color or the fluorescence intensity value on the reagent strip. The fluorescence intensity value or the depth of colloidal gold red is in inverse relation with the concentration of PdG in urine of women.
The detection principle of the invention is as follows: the female urine diluted according to a certain proportion is dripped on a sample pad (one-step dry method), or is added into a test tube, and is dripped on the sample pad after being fully and uniformly mixed (two-step wet method). The sample pad or the test tube contains a PdG monoclonal antibody mouse antibody and a goat anti-mouse secondary antibody and a goat anti-rabbit secondary antibody (as internal standards) which are respectively marked by fluorescence. In the sample pad or tube, PdG in the urine sample first binds to the fluorescent PdG mab. Through capillary action, the residual fluorescent PdG monoclonal antibody on the sample pad flows to a detection line on the reagent strip again, and is subjected to immunological combination with PdG-protein conjugate coated on the detection line in advance, so that the detection line shows fluorescence. And the goat anti-rabbit secondary antibody on the sample pad fluoresces and flows onto the quality control line to be combined with the coated rabbit antibody, so that the quality control line also shows fluorescence. After the fluorescent test strip is inserted into the portable quantitative analyzer, the fluorescence intensity on the test strip detection line and the quality control line is simultaneously quantitatively analyzed, and the concentration of PdG in urine is determined by applying the ratio of the fluorescence intensity on the detection line and the quality control line. Whether the urine sample contains the hormone PdG or not, the quality control line always shows fluorescence, and if the quality control line does not show fluorescence, the test strip is invalid. It will be appreciated by those skilled in the art that the above-described principles of quantitative immunoassay are equally applicable to quantitative analysis using a colloidal gold immunoassay strip when the secondary antibody is labeled with colloidal gold instead of fluorescence.
FIG. 2 is a graph of the linear range analysis of protein-addition colloidal gold immunoassay
As shown in figure 2, the sensitivity can be as low as 1ng/ml, the linear range can be controlled between 5-100ng/ml (hormone protein couplet 1,2) or 1-100ng/ml (hormone protein couplet 4) (see figure 2), and the colloidal gold quantitative detection test strip can completely meet the quantitative monitoring of PdG (namely corpus luteum function) by hospital and family individuals.
FIG. 3 Linear Range analysis of protein addition fluoroimmunoassay
As shown in figure 3, the sensitivity of the detection of PdG by using the unsaturated labeled antibody can reach 0.1ng/ml, the linear range can be controlled between 0.5 ng/ml and 20ng/ml (hormone protein couplet 1,2) or between 0.1ng/ml and 50ng/ml (hormone protein couplet 4), and the fluorescent quantitative detection test strip can completely meet the quantitative monitoring of PdG (namely corpus luteum function) by hospital and family individuals.
The above summary of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. The following description more particularly exemplifies illustrative embodiments. Throughout the specification, guidance is provided through examples, which examples can be used in various combinations. In each case, the contents of the illustrated examples are merely representative examples and should not be construed as exclusive examples.
Detailed Description
The inventors of the present invention have conducted extensive and intensive studies and have surprisingly found for the first time that the number of pregnanediol glucuronide linked to a conjugate protein can be controlled by changing the hydrophilicity and hydrophobicity of the linker and the length of the chain, so that the measurement sensitivity can reach 0.1ng/ml, and the linear range can be controlled between 0.1ng/ml and 50ng/ml, thereby completing the present invention.
Connecting object
"linker" as used herein, refers to a linker capable of linking a pregnanediol glucuronide to a conjugate protein, including but not limited to the compounds described in US7521425 and US8288349, which are useful as linkers.
Conjugated proteins
As used herein, "conjugated protein" refers to a protein capable of being linked to a pregnanediol glucuronide by a linker of the invention, including but not limited to ovalbumin, bovine serum albumin, hemocyanin.
Coupling body
The term "conjugate" as used herein refers to an adduct in which pregnanediol glucuronide and conjugate protein are linked together by a linker of the invention, including but not limited to:
pregnanediol glucuronide-6 atom carbon chain-ovalbumin couplet (1)
Pregnanediol glucuronide-13 atom carbon chain-ovalbumin couplet (2)
Pregnanediol glucuronide-20 atom carbon chain-ovalbumin couplet (3)
Pregnanediol glucuronide-18 atom polyethylene glycol carbon chain-ovalbumin couplet (4)
Antibodies
The term "antibody" as used herein, refers to an antibody capable of specifically binding to a pregnanediol glucuronide and/or a pregnanediol glucuronide in a conjugate, and the terms "antibody" and "specific antibody" are used interchangeably herein. The invention also includes pregnanediolPolyclonal antibodies and monoclonal antibodies, particularly monoclonal antibodies, specific for glucuronide. As used herein, "specific" means that the antibody is capable of binding to pregnanediol glucuronide, but does not recognize and bind to other unrelated molecules. The invention encompasses not only intact monoclonal or polyclonal antibodies, but also immunologically active antibody fragments, such as Fab' or (Fab)2A fragment; an antibody heavy chain; an antibody light chain; genetically engineered single chain Fv molecules (Ladner et al, U.S. Pat. No.4,946,778); or a chimeric antibody, such as an antibody having murine antibody binding specificity but retaining portions of the antibody from a human. The antibodies of the invention can be prepared by a variety of techniques known to those skilled in the art.
Detectable labels
As used herein, a "detectable label" refers to a substance that can generate a detectable signal, including, but not limited to, a colloid, a chromophore, a chemiluminescent group, a fluorophore, an isotope, or an enzyme.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Examples
It will be understood by those skilled in the art that the following examples are merely illustrative of the synthesis of the conjugate of the invention and that any method that can be used to link the conjugate to the pregnanediol glucuronide and the conjugate protein can be used and that one skilled in the art can control the ratio of the pregnanediol glucuronide to the conjugate protein by adjusting the ratio of the two reactions in accordance with the teachings of the present examples.
Example 1: PdG-Linker-CO2Preparation of H (1)
PdG (0.1mmol) was dissolved in DMF (0.5mL), and a solution of DCC (0.143mmol) and NHS (0.143mmol) in DMF (0.8mL) was added. The reaction mixture was stirred for 7 hours, followed by addition of a solution of 6-aminocaproic acid (0.119mmol) in chloroform (1mL) and triethylamine (0.5 mL). The reaction was stirred overnight and after removal of the solvent, the residue was taken up in dichloromethane: chromatography of methanol (8:2) on silica gel: obtaining a product PdG-Linker-CO2H (1; 0.025 mmol). Yield: 25 percent.
Example 2: preparation of PdG-Linker-OVA couplet (1)
PdG-linker-CO2H (1; 0.05mmol) is put into a small test tube, 0.2ml of DCC/DMF solution is added to dissolve under stirring, then 0.2ml is added, and the reaction is carried out for 3 hours at room temperature until a large amount of precipitate is separated out (urea is a byproduct). NaH2PO4·2H2O (0.1186g) and anhydrous Na2HPO4(0.46g) was dissolved in 20ml of water to prepare a 0.2M phosphate buffer (pH 7.4). OVA (44.34mg, 1. mu. mol) was dissolved in 4ml of the above-mentioned 0.2M phosphate buffer solution at 4 ℃ with stirring; the DCC/NHS PdG solution (0.4ml of DMF) was added dropwise to the OVA solution with stirring. The coupling reaction of PdG-linker-OVA was stirred at 4 ℃ overnight. The conjugate of PdG-Linker-OVA was dialyzed against water for 48 hours 4 times (1.2L each), and then against phosphate buffer (1.2L, pH7.4) for 24 hours. Dialyzing the purified PdG-Linker-OVA couplet (1), collecting, freeze-drying and storing.
Example 3: preparation of PdG-Linker-OVA couplet (2-4)
In a similar manner to example 1 above, three portions of PdG (0.1mmol) were dissolved in DMF (0.5mL), and DCC (0.143mmol) and NHS (0.143mmol) in DMF (0.8mL) were added. The reaction mixture was stirred for 7 hours, then a solution of 7-aza-8-oxo-13-amino-tridecanoic acid, 7, 14-diaza-8, 15-dioxo-20-amino-eicosanoic acid and 4-oxo-5-aza-9, 12, 15-trioxa-18-amino-octadecanoic acid (0.119mmol) in chloroform (1mL) and triethylamine (0.5mL) was added, respectively. The reaction was stirred overnight and after removal of the solvent, the residue was taken up in dichloromethane: chromatography of methanol (8:2) on silica gel: obtaining a product PdG-Linker-CO2H (2-4; 0.023-0.026 mmol). The yields were respectively: 23 to 26 percent.
The PdG-linker-CO was taken out separately by the method of example 22H (2-4; 0.05mmol) is put into a small test tube, 0.2ml of DCC/DMF solution is added for dissolution under stirring, then 0.2ml is added, and the reaction is carried out for 3 hours at room temperature until a large amount of precipitate is separated out (urea is an accessory product). NaH2PO4·2H2O (0.1186g) and anhydrous Na2HPO4(0.46g) was dissolved in 20ml of water to prepare a 0.2M phosphate buffer (pH 7.4). OVA (44.34mg, 1. mu. mol) was dissolved in 4ml of the above-mentioned 0.2M phosphate buffer solution at 4 ℃ with stirring; the DCC/NHS PdG solution (0.4ml of DMF) was added dropwise to the OVA solution with stirring. The coupling reaction of PdG-linker-OVA was stirred at 4 ℃ overnight. The conjugate of PdG-Linker-OVA was dialyzed against water for 48 hours 4 times (1.2L each), and then against phosphate buffer (1.2L, pH7.4) for 24 hours. Dialyzing by the purified PdG-Linker-OVA couplet (2), collecting, freeze-drying and storing.
Example 4: preparation of colloidal gold reagent strip
Labeling of the hormone pregnanediol glucuronide monoclonal antibody (mAb): after the pH value of the colloidal gold solution is adjusted to 7.5-8.5, half of the saturated labeling amount is used for the mAb labeling, and the requirement of sensitivity is met. If a linear range is targeted, the mAb uses saturation labeling. After mAb labeling, blocking with 1% BSA was performed. Finally, centrifugation was performed, and after carefully removing the supernatant, the mAb-gold colloid conjugate was resuspended by adding conventional buffer (10mM PBS, ph7.4, 1% BSA) and stored at 4 °. Overlapping and superposing the absorbent pad and the 8964 material (the sample pad and the coupling body combined pad) with the nitrocellulose membrane for 1-2mm, and then dotting or scratching the membrane. After the secondary antibody was diluted to 1mg/ml with the loading buffer, spotting (0.5-1. mu.l) or streaking (1. mu.l/cm, 0.8mg/ml) was performed on the control line. Using the same principle, PdG-protein conjugates were spotted (1. mu.l) or scored (1. mu.l/cm) on the test line. After natural drying for 1 hour, the test strip was cut into thin strips (4mm wide).
Test (two-step wet process): the PdG antigen standard is diluted with phosphate buffer (PB, 10mM, pH7.2), and the standard blank is the diluent. Aiming at sensitivity, the sample is spotted (10 mu l) on a sample pad after unsaturated labeled colloidal gold (namely, the dosage of saturated labeled half antibody mAb) and working solution are diluted by 1: 1. Targeting the linear range, saturated labeled colloidal gold (saturated labeling of all antibody mabs) was used. After the same 1:1 dilution, spotted (10-12. mu.l per sample pad). Under the premise of controlling the time (7 minutes), the test strip is loaded and inserted into a quantitative analyzer (TSR3000, Shanghai Jiening Biotech Co., Ltd.) to carry out card reader reading, and a quantitative analysis result is obtained.
Detection of PdG with unsaturated labeled antibody: the sensitivity can be as low as 1ng/ml, and the linear range can be controlled between 5-100ng/ml (hormone protein couplet 1,2) or 1-100ng/ml (hormone protein couplet 4). As can be seen from the results of fig. 2: the colloidal gold quantitative detection test strip can completely meet the quantitative monitoring of PDG (corpus luteum function) by hospital and family individuals.
Example 5: preparation of fluorescent reagent strips
PdG-linker-OVA (1-4) was spotted (1. mu.l) or scratched (1. mu.l/cm) on the test line. After natural drying for 1 hour, the test strip is cut into thin strips (4mm wide), and the goat anti-rabbit secondary antibody is spotted or scratched on a control line. Containing 0.1ml of PdG sample and 0.05ml of PdG monoclonal antibody mouse antibody, 0.05ml of fluorescence (such as Cy3) labeled secondary antibody mixture (goat anti-mouse and goat anti-rabbit secondary antibody) is quantitatively spotted on the sample pad for lateral chromatography, and under the premise of controlling time (7 minutes), the test strip is loaded and inserted into a quantitative analyzer for reading by a card reader (the method comprises the steps ofESE-Quant FLUO reader,DCN Inc) And obtaining a quantitative analysis result.
Example 6: data analysis experiments
Hormone protein couplet for analysis of immunoassay kit
[ note ] this method comprises the following steps: the degree of binding of the hormone protein (hapten density), i.e.the average number of hormone molecules attached to each protein molecule, is determined by Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS)，Bruker Daltonics TMAutodlex spectrometer) Is obtained by the measurement of (1).
The inventors have surprisingly found that the magnitude of the high-low and linear ranges of the analytical sensitivity of an immunoassay kit will depend on "Degree of hormone protein binding"and"Water solubility of hormone proteins"two factors, the inventors are not limited to the existingThe principle is that the following brand-new design ideas which are absolutely different from the conventional design ideas are originally provided for the first time, and the excellent technical effects are obtained:
(1) in the synthesis of hormone protein couplets, the degree of hormone protein binding (i.e., hapten density) is simultaneously combined withHormones Hydrophilic properties of molecules and linkersAnd, andmolar ratio of hormone to proteinAre closely related; generally, the better the hydrophilicity of the two is, the higher the molar ratio of hormone molecules to protein molecules is, the higher the binding degree of hormone and protein is; and vice versa. The hydrophilic nature of the linker is particularly important because of the hydrophobic nature of the hormone molecule. Under the premise of controlling the same hormone/protein molecular molar ratio, when the hydrophobic linker is increased from 6 atoms (1) to 13 atoms (2) or 20 atoms (3), the hormone protein binding degree is correspondingly reduced from 4.1 to 2.4. In stark contrast, when hydrophilic 18-atom polyethylene glycol is used as the linker (4), the degree of hormone protein binding is as high as 11, almost 5 times that of the hydrophobic linker (3) with similar chain length.
(2) The introduction of different properties (hydrophilic or hydrophobic) and different lengths of linker between hormone and protein molecules not only results in a great difference in the degree of hormone protein binding beyond that conventionally envisioned, but also directly affects the final product-Laser Water solubility of the protein(ii) a And the water solubility of the protein determines the amount of the hormone protein on the detection line of the immune reagent strip. Thus, the amount of hormone protein on the line and the number of hormones attached per protein molecule determinedDetection of Molecular density of hormone on line. Thereby affecting the sensitivity and linear range of the immunoassay kit assay. When the hydrophobic linker is increased from 6 atoms (1) to 20 atoms (3), not only the hormone protein binding degree is reduced from 4.1 to 2.4, but also the water solubility of the synthesized hormone protein conjugate is sharply reduced, which results in too low density of hormone molecules on the detection line due to the ultra-low hormone protein binding degree and water solubility, and thus, an effective immunoassay kit analysis cannot be performed. When hydrophilic 18-atom polyethylene glycol is used as a linker, not only is the degree of hormone protein binding (11) high, but also the long chain is usedThe good hydrophilicity of the polyethylene glycol linker enables the hormone-polyethylene glycol-protein conjugate to have good water solubility, so that the hormone protein conjugate can be spotted on a detection line at high concentration. The detection line of high density of hormone molecules caused by high protein concentration and high hormone binding degree undoubtedly provides a wider linear range for immunoassay of an immunoassay kit. In addition, the long chain (18 atoms) of the polyethylene glycol linker improves the effective antibody/hormone binding, and the polyethylene glycol carbon chain has the excellent performance of avoiding non-specific antibody binding, so the hormone-polyethylene glycol-protein conjugate (4) has the best sensitivity in immunoassay of an immunoassay kit.
When introducing elements of the present invention or the preferred embodiments of the present invention, the articles "a," "an," "the," and "said" are intended to mean that there are one or more of the elements. The terms "comprising," "including," and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements.
As various changes could be made in the above without departing from the scope of the invention, it is intended that all matter contained in the above description shall be interpreted as illustrative and not in a limiting sense. The scope of the invention is defined by the appended claims and modifications may be made to the above embodiments without departing from the scope of the invention.
Claims (10)
1. Use of a protein hybridization conjugate comprising a pregnanediol glucuronide and a conjugate protein and a linker connecting the two, selected from the group consisting of:
(1) pregnanediol glucuronide-6 atom carbon chain-ovalbumin couplet;
(2) pregnanediol glucuronide-13 atom carbon chain-ovalbumin couplet; and
(3) pregnanediol glucuronide-18 atom carbon chain-ovalbumin couplet.
2. The use according to claim 1, wherein the kit for the determination of pregnanediol glucuronide further comprises an antibody that specifically binds to the protein hybridization conjugate.
3. The use of claim 2, wherein the antibody is a monoclonal or polyclonal antibody.
4. The use according to claim 3, wherein the kit for the determination of pregnanediol glucuronide further comprises a detectable label linked to the antibody.
5. The use of claim 3, wherein the kit for the determination of pregnanediol glucuronide further comprises a second antibody that specifically binds to the antibody.
6. The use of claim 5, wherein the ratio of antibody to second antibody is from 1: 2 to 2: 1.
7. The use of claim 5, wherein the kit for the determination of pregnanediol glucuronide further comprises a detectable label linked to the second antibody.
8. Use according to claim 4 or 7, wherein the detectable label is selected from the group consisting of a colloid, a chromophore, a chemiluminescent group, a fluorophore, an isotope and an enzyme.
9. Use according to any one of claims 1 to 6, wherein the kit is for monitoring luteal function.
10. A method for preparing the conjugate body (1), (2) or (3) as described in claim 1, which comprises a step of DCC/NHS mediated condensation of pregnanediol glucuronide with 6-aminocaproic acid, 7-aza-8-oxo-13-amino-tridecanoic acid or 4-oxo-5-aza-9, 12, 15-trioxa-18-amino-octadecanoic acid, respectively, and subsequently with ovalbumin.
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