AU2020101552A4 - Kit and method for detecting lactoferrin and beta-lactoglobulin and application thereof - Google Patents
Kit and method for detecting lactoferrin and beta-lactoglobulin and application thereof Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/02—Food
- G01N33/04—Dairy products
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/588—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with semiconductor nanocrystal label, e.g. quantum dots
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4713—Plasma globulins, lactoglobulin
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/79—Transferrins, e.g. lactoferrins, ovotransferrins
Abstract
The present invention relates to the technical field of antigen detection, and
particularly relates to a kit and a method for detecting lactoferrin and
p-lactoglobulin and an application thereof. The kit includes a lactoferrin capture
antibody, a lactoferrin detection antibody, a p-lactoglobulin detection antibody and
a p-lactoglobulin standard substance. Quantum dots are labeled on the lactoferrin
detection antibody and the p-lactoglobulin detection antibody; the quantum dots
labeled on the lactoferrin detection antibody are CdTe quantum dots; and the core
of the quantum dots labeled on the p-lactoglobulin detection antibody is CdTe, and
a shell is ZnSe. The established detection method may provide a detection means
that is convenient to operate and capable of performing batch inspection at a time
for quality supervision departments and dairy enterprises.
Drawings of Description
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Description
Drawings of Description
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Description
KIT AND METHOD FOR DETECTING LACTOFERRIN AND p-LACTOGLOBULIN AND APPLICATION THEREOF
Technical Field
The present invention relates to the technical field of antigen detection, and particularly relates to a kit and a method for detecting lactoferrin and p-lactoglobulin and an application thereof.
Background
Infant formula milk powder is an artificial food that is prepared by taking cow milk or other animal milk or other animal/plant components as a basal component and appropriately adding nutrients and may provide nutrition essential for infant growth and development. The infant formula milk powder may serve as a breast milk substitute. In accordance with regulations of Food and Drug Administration (FDA) and China Food and Drug Administration (CFDA), all ingredients in the infant formula milk powder must be generally recognized as safe (GRAS) or may serve as food additives. Lactoferrin (LF) is an iron-binding glycoprotein having a molecular weight of about 80 kD, is one of the most 4 major proteins in human milk, accounts for about % of total proteins of the human milk, belongs to a composition material of an innate immune system, can promote absorption of iron, and has multiple functions of resisting and killing bacteria, resisting viruses and regulating immune response of the body. After birth of a baby, the autoimmune system is not completely sound. The lactoferrin in breast milk is an important material for ensuring healthy growth of the infant. Bovine lactoferrin is added into the infant formula milk powder, so that nutritional ingredients of the milk powder are closer to those in the breast milk. The bovine lactoferrin is extremely important to growth and development of the infant. After the lactoferrin was first added into the infant formula milk powder in
Description
1986, multiple infant foods added with the lactoferrin come out now. However, in case of excessive intake of the lactoferrin, the lactoferrin brings burden to hepatic and renal functions of the infant. Therefore, the use standard of food nutritive fortifier in China (GB14880-2012) stipulates that, an allowed maximum dose of the bovine lactoferrin in infant formula foods is 1 mg/g. Since an efficient and accurate method for detecting bovine lactoferrin in dairy products is lacked at present, bad and good products are mixed together in the market, shoddy goods substitute for quality goods, and the products are intermingled, which is a big challenge for the nascent Chinese lactoferrin product market. Lots of epidemiologic study data show that, when fed with milk proteins, about 20% of infants at an age of 1 may suffer from cow milk allergy. p-lactoglobulin, a-lactalbumin and casein in cow milk are considered as major allergens. The p-lactoglobulin is a major whey protein in ruminant milk. 20% of protein in the cow milk is the whey protein, including the main component of the p-lactoglobulin. The p-lactoglobulin is also the main component of whey protein powder. A -lactoglobulin monomer is composed of 162 amino acid residues, has a stable molecular structure, has effects of resisting gastric acid and resisting protease hydrolysis, can directly pass through the gastrointestinal tract without being absorbed and then enter blood circulation, easily stimulates the immune system to cause a hypersensitivity reaction, and easily causes infant allergy (the digestive system of an infant is not fully developed, and the p-lactoglobulin is easily 'wholly' absorbed; thus, the p-lactoglobulin is judged as pathogeny by the immune system). Therefore, the p-lactoglobulin is considered as the most major allergen in the cow milk, and is the major allergen of the infant. At present, many countries and organizations (including Regulation (EU) No 1169/201 and U.S.FDA-FALCPA 2004) require that, the p-lactoglobulin should be clearly labeled on outer packing of foods. Although there is no legal restriction on the p-lactoglobulin, it is strongly suggested that, the p-lactoglobulin in foods, or even
Description
low-content p-lactoglobulin should be detected by food manufacturers, to prevent consumers from suffering from hypersensitive response and avoiding product recall. Up to now, an effective treatment means on cow milk allergy is lacked, and the unique method is to introduce law and regulation to require food production enterprises to clearly label all allergen components in the foods, thereby preventing sensitization crowds from contacting, inhaling or eating allergens and allergen-containing foods. Therefore, to develop and establish a high-specificity, high-sensitivity, rapid and accurate method for detecting and analyzing cow milk allergens (particularly the p-lactoglobulin) is particularly important. Enzyme Linked Immunoserbent Assay (ELISA) widely applied in the current market has characteristics of high sensitivity, low minimum detection limit and no need of sample purification, but is greatly influenced by environment and operators. Detection results may be influenced by coating time, blocking conditions and antibody dilution, and quantitative repeatability is low. In addition, because most of the used capture antibodies are polyclonal antibodies, a cross reaction and a false positive result are easily caused, leading to deviations from detection authenticity. Since the infant formula milk powder is complicated in component, contains lots of proteins and other trace elements and has relatively low cow lactoferrin content, the detection result has a certain error. Therefore, it is very necessary to seek a method applicable to determination of content of lactoferrin and p-lactoglobulin in the milk powder with advantages of low cost, rapid detection and accuracy. In view of this, the present invention is particularly proposed.
Summary A purpose of the present invention is to establish two different quantum dot-fluorescence immunosorbent assay (QD-FLISA) for detecting a main nutrient
Description
additive lactoferrin and one of the most major allergens, that is, p-lactoglobulin, in infant formula milk powder, optimize the two detection methods and establish a synchronous detection method. The established detection method may provide a detection means that is convenient to operate and capable of performing batch inspection at a time for quality supervision departments and dairy enterprises. By conducting the study, a technical level in detection of the infant milk powder can be increased, and a scientific basis is provided for applications of quantum dots in the field of biological rapid detection. The study is an invention that has great significance and meets market needs and development direction. The present invention relates to a kit for detecting lactoferrin and p-lactoglobulin. The kit includes a lactoferrin capture antibody, a lactoferrin detection antibody, a p-lactoglobulin detection antibody and a p-lactoglobulin standard substance. Quantum dots are labeled on the lactoferrin detection antibody and the p-lactoglobulin detection antibody. The quantum dots labeled on the lactoferrin detection antibody are CdTe quantum dots. The core of the quantum dots labeled on the p-lactoglobulin detection antibody is CdTe, and a shell is ZnSe. According to one aspect of the present invention, the present invention further relates to a method for detecting lactoferrin and p-lactoglobulin by using the above kit. The method includes the following steps: 1) coating the lactoferrin capture antibody and the p-lactoglobulin standard substance on a solid phase carrier; 2) blocking the solid phase carrier with a blocking solution; 3) performing mixed incubation on a to-be-detected sample containing lactoferrin and/or p-lactoglobulin, the lactoferrin detection antibody and the p-lactoglobulin detection antibody so as to obtain a mixed solution;
Description
4) adding the mixed solution into the solid phase carrier to perform incubation developing; and 5) detecting fluorescence intensity of a product in the step 4). According to one aspect of the present invention, the present invention further relates to an application of the above kit and the above method in detection of lactoferrin and/or p-lactoglobulin in infant formula milk powder. Compared with the prior art, the present invention has beneficial effects as follows: According to research results of the quantum dots in China and abroad in recent years, the quantum dot CdTe has obvious advantages that: particles do not agglomerate easily; photochemical stability is obviously increased; and photoquenching is decreased to the lowest degree. The quantum dot CdTe is widely applied to biological labels. In the present invention, by exploring a quantum dot labeled monoclonal antibody technology, after correlated conditions are optimized, study on the QD-FLISA detection technology is conducted based on an antigen/antibody immunology principle. According to the present invention, by synthesizing the quantum dots of different sizes and exploring spectral features of these quantum dot labeled antibodies, a detection technology capable of synchronously detecting multiple proteins is established. Meanwhile, detection objects selected in the present invention, i.e., the lactoferrin and the p-lactoglobulin, have important detection values in the infant formula milk powder. A related report on development of the QD-FLISA detection technology for the proteins is not found in China.
Description of Drawings In order to clearly describe specific embodiments of the present invention or technical solutions in the prior art, drawings to be used in the description of the specific embodiments or the prior art will be simply introduced below. Apparently, the drawings described below are merely some embodiments of the present
Description
invention. Other drawings may be obtained by those ordinary skilled in the art without contributing creative labor according to these drawings. Fig. 1 shows (a) a transmission electron microscope graph of CdTe/ZnSe quantum dots; (b) a high-resolution transmission electron microscope graph; (c) a selected electron diffraction pattern; and (d) particle size analysis; and Fig. 2 shows an absorption spectrum and a fluorescence spectrum of CdTe/ZnSe quantum dots.
Detailed Description
The present invention relates to a kit for detecting lactoferrin and p-lactoglobulin. The kit includes a lactoferrin capture antibody, a lactoferrin detection antibody, a p-lactoglobulin detection antibody and a p-lactoglobulin standard substance. Quantum dots are labeled on the lactoferrin detection antibody and the p-lactoglobulin detection antibody. The quantum dots labeled on the lactoferrin detection antibody are CdTe quantum dots. The core of the quantum dots labeled on the p-lactoglobulin detection antibody is CdTe, and a shell is ZnSe. Preferably, according to the above kit, a particle size of the quantum dots labeled on the lactoferrin detection antibody is 2.6 nm-3 nm; and a particle size of the core of the quantum dots labeled on the p-lactoglobulin detection antibody is 5.2 nm-6.2 nm. Preferably, according to the above kit, the lactoferrin capture antibody, the lactoferrin detection antibody and the p-lactoglobulin detection antibody are all monoclonal antibodies, and amino acid sequences of antigenic epitopes of the three antibodies are shown as SEQ ID NO:1-3 in sequence.
Description
In the present invention, since a to-be-detected material (particularly milk powder) contains a great variety of proteins, requirements for specificity and sensitivity of the antibodies are extremely high. The antigenic epitopes selected in the present invention are high in specificity, are located in a surface loop region of a to-be-detected protein, can be detected without performing protein denaturation, are very suitable for QD-FLISA detection, and may be further developed as test strips and other application scenarios. Because the lactoferrin accounts for a lower proportion (<0.3) in the cow milk protein, the lactoferrin has higher requirement for detection sensitivity. In addition, the lactoferrin has a high molecular weight and contains many antigenic epitopes, so a double-antibody sandwich method is adopted for detecting the lactoferrin in the present invention so as to increase the detection sensitivity and specificity, and the p-lactoglobulin is detected by a competition method. For the lactoferrin, a matching antibody provided in the present invention is relatively far in distance of native conformation of the proteins, is small in steric hindrance and facilitates increase of double-antibody sandwich sensitivity. Preferably, according to the above kit, the kit further includes a solid phase carrier, a blocking solution, a PBST or PBS, and a sample dilution buffer. Preferably, according to the above kit, the blocking solution is 0.4 w/v%-0.6 w/v% OVA. More preferably, the blocking solution is 0.5 w/v% OVA. According to one aspect of the present invention, the present invention further relates to a method for detecting lactoferrin and p-lactoglobulin by using the above kit. The method includes the following steps: 1) the lactoferrin capture antibody and the p-lactoglobulin standard substance were coated on a solid phase carrier; 2) the solid phase carrier was blocked with a blocking solution;
Description
3) mixed incubation was performed on a to-be-detected sample containing lactoferrin and/or p-lactoglobulin, the lactoferrin detection antibody and the p-lactoglobulin detection antibody so as to obtain a mixed solution; 4) the mixed solution was added into the solid phase carrier to perform incubation developing; and 5) fluorescence intensity of a product was detected in the step 4). Preferably, according to the above method, in the step 1), an absorbance value of the lactoferrin capture antibody and the p-lactoglobulin standard substance at 280 nm is 0.8-1.2, and more preferably 1. Preferably, according to the above method, in the step 1), the coating condition is as follows: incubation is performed at 35°C-39°C for 2.5-3.5 hours. More preferably, the coating condition is as follows: incubation is performed at 37°C for 3 hours. Preferably, according to the above method, in the step 4), the incubation developing condition is as follows: incubation is performed at 16°C-20°C for 25-35 minutes. More preferably, the incubation developing condition is as follows: incubation is performed at 18°C for 30 minutes. According to one aspect of the present invention, the present invention further relates to an application of the above kit and the above method in detection of lactoferrin and/or p-lactoglobulin in infant formula milk powder. Implementation solutions of the present invention will be described below in detail in combination with embodiments. However, those skilled in the art will understand that, the embodiments below are only used for describing the present invention, and should not be regarded as a limitation of the scope of the present invention. Embodiments without specific conditions should be performed in accordance with conventional conditions or conditions suggested by manufacturers.
Description
Used reagents or instruments without manufacturers are all conventional products purchased from the market. Embodiment 1 1. Preparation of quantum dots and surface modification of quantum dots with mercaptoacetic acid a) Preparation of quantum dots An aqueous-phase synthesis route was adopted: NaBH 4 and Te powder reacted to produce NaHTe under nitrogen protection; an appropriate pH environment was regulated with stronger ammonia water; CdCl2-2.5H 20was added; magnetic stirring was performed in a water bath at 90°C, and a reflux reaction was carried out; a CdTe-containing aqueous solution sample was taken out after a certain time; and after centrifugal sedimentation, the sample was washed with distilled water for multiple times, thereby obtaining CdTe quantum dots (sizes of the synthesized quantum dots were controlled as 2.6-3 nm and 5.2-6.2 nm, and spectral distribution was in green and red regions). A reaction of dissolving Te with NaBH 4 to produce CdTe is as follows: 4NaBH 4+2Te+7H 20=2NaHTe+Na2B 40 7+14H21 (1) NaHTe+CdCl 2+NH 3-H20= CdTet+NaCl+NH 4C1 (2) The preparation method of the CdTe/ZnSe quantum dots is the known prior art, wherein a molar ratio of CdTe to ZnSe is 1:1. b) Surface modification of quantum dots with mercaptoacetic acid Quantum dot sample powder was respectively dispersed in mercaptoacetic acid; stirring, heating and reflux were performed; after a reaction was carried out at °C for 12 hours, the product was subjected to repeated centrifugal separation and ultrasonic cleaning by taking acetone as cleaning fluid so as to remove mercaptoacetic acid that is not chemically bonded to the quantum dots; and finally, the quantum dots were dispersed in a phosphate buffered solution (PBS). After
Description
surface modification, the QDs can be stably dispersed in the PBS, and a turbid precipitation phenomenon is avoided. 2. Characterization on spectrum, morphology and disperse states of quantum dots Optical properties of the quantum dots before and after antibody labeling were characterized by using a UV-2501PC ultraviolet spectrophotometer (Shimadzu) and an F4600 fluorescence spectrophotometer (Hitachi), and optical property tests were performed at a room temperature. Calculated fluorescence quantum yield (FL, QY) is represented by a relative value, and is a ratio of an area of a quantum dot emission peak to an area of an emission peak of a dye Rhodamine 6G (QY=95%) dissolved into ethanol. In order to prevent multiple absorptions, optical density (OD) of the system was controlled to be less than 0.15. The morphology and disperse states of the sample were characterized by using a JEM2010 transmission electron microscope (under an operating voltage of 120 kV, Japan) and a fluorescence microscope (German Leica). Results are as shown in Fig. 1. 3. Preparation of quantum dot antibodies 3.1 Antibody preparation 3.1.1 Animal immunization Experimental animals were selected from female BALB/c mice at an age of 6-8 weeks, and purchased from Beijing HFK Bio-Technology Co., Ltd. After adapting to the environment within a week, the mice were immunized. For first immunization, synthetic polypeptides shown as SEQ ID NO:1-3 coupled with a virus-like particle adjuvant were taken as antigens and respectively fully mixed with an emulsifier to form emulsion; intraperitoneal injection was performed on the mice; booster immunization was performed after two weeks; the antigen used in booster immunization was the same as the above and had a dose of 50 pg, and after emulsion was formed, intraperitoneal injection was performed on the mice;
Description
booster immunization was performed every 2 weeks according to the same method; totally booster immunization was performed for 3 times; on Day 7 of the last immunization, blood was collected from venous plexus of eye sockets of the mice; serum was subjected to centrifugal separation; an antibody titer was determined by ELISA; high-titer mice were selected for fusion to make hybridoma; within three days before fusion, immunization was performed again; on the day of fusion, spleens were taken in a sterile environment; and single spleen cell suspension was prepared for fusion. 3.1.2 Preparation of hybridoma cells Myeloma cells SP2/0 in logarithmic growth were taken; centrifugation was performed at 1000rpm for 5 minutes; the supernatant was removed; the cells were suspended with an incomplete DMEM culture solution (Gibco, Cat No.11965) and then counted; needed cells were taken; the cells were washed with the incomplete culture solution twice; meanwhile, immune spleen cell suspension was prepared and washed with the incomplete culture solution twice; the myeloma cells and the spleen cells were mixed together according to a ratio of 1:10 or 1:5 and washed in a 50 mL plastic centrifuge tube with the incomplete culture solution once at 1200 rpm for 8 minutes; the supernatant was removed, and residual liquid was absorbed by a dropper; the bottom of the centrifuge tube was slightly touched on the palm, so that precipitated cells were uniformly dispersed; preheating was performed in a water bath at 40°C; ImL of 45% PEG-4000 (pH 8.0, Sigma, Cat No.P7181) preheated to 40°C was added within about 1 minute (preferably, 45 seconds) by a 1 mL sucker; slight stirring was performed while adding (stirring by the sucker); visible particles may appear during observation by naked eyes; 20-30 mL of the incomplete culture preheated to 37°C was added within 90 seconds by a 10 mL sucker so as to terminate PEG effects; standing was performed at 20-37°C for 10 minutes; the supernatant was removed at 1000 rpm for 5 minutes; a 5 mL of HAT culture medium (DMEM+HAT, Sigma, Cat No.1H0262-1OVL) was added; the
Description
precipitated cells were slightly blown and sucked (remember not to hard blowing or beating, so as to prevent the cells fused together from being dispersed); the cells were suspended and uniformly mixed; the HAT culture medium was refilled to -100 mL (until a spleen cell concentration was 1-2x10 6 /nL); the cells were separately charged in 96-well cell culture plates, wherein a volume in each well was 0.1 mL; the cells were separately charged in 24-well plates, wherein a volume in each well was 1.0-1.5 mL; the culture plates were placed at 37°C and cultured in a 6%of CO 2 incubator; totally 6 pieces of 96-well plates were laid; 1/2 culture medium was replaced with the HAT culture medium within 5 days; 7-10 days later, the HAT culture medium was replaced with an HT culture medium (DMEM+HT, Sigma Cat No. H0137-10VL); growth conditions of the hybridoma cells were often observed, and the supernatant was sucked out for antibody detection when the cells grew to 1/10 or more of the well bottom area; and positive cloned cells were subjected to enlarged culture and cryopreserved. 3.1.3 Cloning screening and identification Dot blot preliminary screening was performed on titers of antibodies resistant to lactoferrin and/or p-lactoglobulin; the titers were verified by ELISA experiments; and then antibody pairing experiments were conducted to find out a lactoferrin capture antibody and a lactoferrin detection antibody that can be well paired. 3.2 Quantum dot labeling Both EDC and NHS were prepared with a phosphate buffer having a pH value of 7.4; a concentration of quantum dots was 8 pmol/L; 20 pL of quantum dots were added into 400 pL of a solution containing 2 nmol/L of EDC for performing vortex activation at a room temperature for 5 minutes; the quantum dots were added into 400 pL of a solution containing 0.8 nmol/L of NHS to react for 5 minutes; 40 pL of monoclonal antibody including a lactoferrin monoclonal antibody (an antigenic epitope was shown as SEQ ID NO:2) and a p-lactoglobulin monoclonal antibody (an antigenic epitope was shown as SEQ ID NO:3) was added into the reaction
Description
solution for performing uniform vortex mixing; a light shading reaction was carried out on a 37°C shaker for 2 hours; an optimum labeling effect was achieved by regulating molar ratios of the CdTe quantum dots, the antibodies, the EDC and the NHS in the reaction system; and after coupling was ended, a coupling product was concentrated to 200-300 pL by an ultrafiltration tube having a molecular weight cut-off of 3kDa, and excessive EDC and NHS would be removed with the filtrate. 3.3 Coupling mixture purification (1) Purification was performed by a phosphate buffer solution (PBS) balanced Sephadex-G200 gel column that is 30 cm long; (2) a coupling mixture was loaded after ultrafiltration; elution was performed after sample loading was ended; and an automatic collector was opened for collecting the sample; and (3) after elution was ended, each elution peak protein was collected to perform ultrafiltration and concentration; and functional groups on surfaces of the CdTe quantum dots before and after antibody labeling were analyzed by using an infrared instrument (Shimadzu IRPrestige-21), wherein a wave-number range was 400-4000cm'. 3.4 Fluorescence emission spectrum analysis The quantum dots and the coupling product were diluted with the PBS and added into a cuvette; an emission spectrum was scanned by utilizing a fluorescence spectrophotometer, wherein an excitation wavelength was 365 nm, a slit width was nm, and a detection range of an emission wavelength was 350-700 nm; peak changes of maximum emission peaks of the quantum dots before and after labeling were compared; emission peak position changes may be caused in presence of activators EDC and NHS; carboxyl on surfaces of the quantum dots and amino on surfaces of the antibodies were subjected to covalent coupling, and surface charges of the quantum dots were decreased; thus, dipole-dipole interaction among nano
Description
particles of the quantum dots was decreased, and StokeS shift was decreased. Blue shift of the fluorescence emission peak occurred, thereby further proving the coupling between the quantum dots and the antibodies. Embodiment 2 (1) Lactoferrin capture antibody and/or -lactoglobulin standard substance coating plates were adopted, and incubation was performed under 50mMTris, 200ng/well and at 37°C for 1 hour. A use concentration of the capture antibody/protein was lower; after coating completion, there were many unoccupied voids on the surface of the ELISA plates; in order to avoid antigens or detection antibodies from being nonspecifically adsorbed to these sites in subsequent operation and influencing detection results, these voids should be filled with lots of irrelevant proteins, thereby eliminating readsorption of interfering substances in subsequent steps. 0.4w/v-0.6w/v% OVA was added into the PBS to serve as a blocking solution for blocking the coated antibodies. After 200 pL of the above blocking solution was added into each well, blocking was performed at a room temperature for 1 hour. (2) Standard substance and sample preparation: Standard substance (commercially available p-lactoglobulin standard substance and lactoferrin, analytically pure): 100 ng/nL, subjected to twofold dilution to 0.10 ng/nL. Sample (baby milk): 100 pL of 2.5 N glacial acetic acid/10M urea+ 100 pL of specimen; after 10 minutes, 100 pL of 2.7 N NaOH/1M HEPES was added for neutralization; and 100 pL of PBS containing 1 w/v % of BSA having a pH value of 7.2-7.4 was added for dilution, according to a total dilution ratio of 1:5. The diluted to-be-detected sample and the standard substance were incubated with the lactoferrin detection antibody and the p-lactoglobulin detection antibody; and an incubation condition was as follows: a thermostatic prereaction was carried out at 37°C for 1 hour so as to obtain a mixed solution.
Description
(3) The mixed solution was added into each well, and incubation was performed at 18°C for 30 minutes; (4) PBST washing was performed for 10 minutes x6 times; (5) Quantum dot signals were respectively read at emission peaks 541 nm (green, lactoferrin) and 628 nm (red, p-lactoglobulin). Sensitivity: through multiple experimental verifications, according to the method provided in the present invention, detection sensitivity is about 100 pg/mL while detecting the lactoferrin, and the detection sensitivity is about 150 pg/niL while detecting the p-lactoglobulin. Specificity: the antibodies provided in the present invention have no cross reaction with multiple other common proteins in the milk powder (after subjected to gradient dilution at 20 pg/mL, 10 pg/mL, 1 g/mL, 100 ng/mL, 10 ng/mL and 1 ng/mL, the antigen protein standard substance was subjected to ELISA verification; the antibody concentration was a working concentration in the present invention; and results were as shown in the following table). Protein name SEQ ID NO:1 SEQ ID NO:2 SEQ ID NO:3 Corresponding Corresponding Corresponding monoclonal antibody monoclonal antibody monoclonal antibody detection limit detection limit detection limit
Casein NA NA NA
usi-casein NA NA NA
us2-casein NA NA NA
-casein NA NA NA
k-casein NA NA NA
a-lactoglobulin NA NA 10 tg/mL
P-lactoglobulin NA NA 1 ng/mL
Serum albumin NA NA NA
Lactoferrin 1 ng/mL 1 ng/mL NA
Lactoperoxidase NA 20 tg/mL NA
Immune globulin NA NA NA
Description
Osteopontin NA NA NA
Repetitive experiment: according to a PN value determined in the repetitive experiment, statistical treatment is performed, a value t is equal to 1.70, and P>0.05, which indicates that the difference has no statistical significance. Thus, the experiment has excellent repeatability. Finally it shall be indicated that, the above embodiments are only used for describing the technical solutions of the present invention, rather than limiting the present invention. Although the present invention is described in detail in combination with the above embodiments, those ordinary skilled in the art shall understand that, the technical solutions recorded in the above embodiments may be still modified, or partial or all technical features may be subjected to equivalent replacements. However, these modifications or replacements do not enable the essence of the corresponding technical solutions to deviate from the scope of the technical solutions in various embodiments of the present invention.
Claims (5)
1. A kit for detecting lactoferrin and -lactoglobulin, the kit comprising a lactoferrin capture antibody, a lactoferrin detection antibody, a p-lactoglobulin detection antibody and a p-lactoglobulin standard substance, wherein quantum dots are labeled on the lactoferrin detection antibody and the p-lactoglobulin detection antibody; the quantum dots labeled on the lactoferrin detection antibody are CdTe quantum dots; the core of the quantum dots labeled on the p-lactoglobulin detection antibody is CdTe, and a shell is ZnSe; wherein a particle size of the quantum dots labeled on the lactoferrin detection antibody is 2.6 nm-3 nm; and a particle size of the core of the quantum dots labeled on the p-lactoglobulin detection antibody is 5.2 nm-6.2 nm; the lactoferrin capture antibody, the lactoferrin detection antibody and the p-lactoglobulin detection antibody are all monoclonal antibodies, and amino acid sequences of antigenic epitopes of the three antibodies are shown as SEQ ID NO:1-3 in sequence.
2. The kit according to claim 1, further comprising a solid phase carrier, a blocking solution, a PBST or PBS, and a sample dilution buffer, wherein the blocking solution is 0.4 w/vo-0.6 w/v% OVA.
3. A method for detecting lactoferrin and p-lactoglobulin by using the kit of any one of claims 1-2, comprising: 1) coating the lactoferrin capture antibody and the p-lactoglobulin standard substance on a solid phase carrier; 2) blocking the solid phase carrier with a blocking solution; 3) performing mixed incubation on a to-be-detected sample containing lactoferrin and/or p-lactoglobulin, the lactoferrin detection antibody and the p-lactoglobulin detection antibody so as to obtain a mixed solution;
Claims
4) adding the mixed solution into the solid phase carrier to perform incubation developing, wherein the incubation developing condition is as follows: incubation is performed at 16°C-20°C for 25-35 minutes; and 5) detecting fluorescence intensity of a product in the step 4). 4. The method according to claim 3, wherein in the step 1), an absorbance value of the lactoferrin capture antibody and the p-lactoglobulin standard substance at 280 nm is 0.8-1.2 ; the coating condition is as follows: incubation is performed at 35°C-39°C for 2.5-3.5 hours.
5. An application of the kit of any one of claims 1-2 and the method of any one of claims 3-4 in detection of lactoferrin and/or p-lactoglobulin in infant formula milk powder.
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| AU2020101552A AU2020101552A4 (en) | 2020-07-29 | 2020-07-29 | Kit and method for detecting lactoferrin and beta-lactoglobulin and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112858695A (en) * | 2021-02-08 | 2021-05-28 | 西北农林科技大学 | Preparation method of ELISA (enzyme-Linked immunosorbent assay) detection kit for bovine whey component content in formula goat milk powder |
| CN115141274A (en) * | 2022-05-31 | 2022-10-04 | 南开大学 | Milk allergen beta-lactoglobulin specific nano antibody and application thereof |
| CN115850448A (en) * | 2022-08-10 | 2023-03-28 | 北京博奥森生物技术有限公司 | Lactoferrin polypeptide fragment, preparation method thereof, prepared antibody and application |
| CN116794296A (en) * | 2023-08-15 | 2023-09-22 | 美维仕(北京)健康管理有限公司 | Method and kit for detecting sensitization of hydrolyzed formula food |
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2020
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112858695A (en) * | 2021-02-08 | 2021-05-28 | 西北农林科技大学 | Preparation method of ELISA (enzyme-Linked immunosorbent assay) detection kit for bovine whey component content in formula goat milk powder |
| CN115141274A (en) * | 2022-05-31 | 2022-10-04 | 南开大学 | Milk allergen beta-lactoglobulin specific nano antibody and application thereof |
| CN115141274B (en) * | 2022-05-31 | 2024-02-20 | 南开大学 | Milk allergen beta-lactoglobulin specific nano-antibody and application thereof |
| CN115850448A (en) * | 2022-08-10 | 2023-03-28 | 北京博奥森生物技术有限公司 | Lactoferrin polypeptide fragment, preparation method thereof, prepared antibody and application |
| CN115850448B (en) * | 2022-08-10 | 2024-02-02 | 北京博奥森生物技术有限公司 | Lactoferrin polypeptide fragment, preparation method thereof, prepared antibody and application |
| CN116794296A (en) * | 2023-08-15 | 2023-09-22 | 美维仕(北京)健康管理有限公司 | Method and kit for detecting sensitization of hydrolyzed formula food |
| CN116794296B (en) * | 2023-08-15 | 2024-03-08 | 美维仕(北京)健康管理有限公司 | Method and kit for detecting sensitization of hydrolyzed formula food |
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