CN111537744A - ELISA quantitative detection method of milk agglutinin or mucin 1 based on milk fat globule membrane characteristics - Google Patents

Abstract

The invention relates to an ELISA quantitative detection method of milk agglutinin or mucin 1(MUC1) based on milk fat globule membrane characteristics, in particular to an enzyme-linked immunosorbent assay (ELISA) method, which comprises the following steps: collection of Breast milk sample → use

lyses buffer pre-treated the breast milk sample and placed on ice lysis → Western Blot quantitation → ELISA quantitation. The invention also includes a kit for quantitatively detecting the lactadherin and MUC1 protein in a breast milk sample, which comprises

lysis buffer reagent and instructions. The invention has the advantages that: the method is used for quantitatively detecting the milk agglutinin and the MUC1 protein in the breast milk of the puerpera in different periods, has high flux, high accuracy, simplified detection steps and low cost, and provides science for improving the formula milk powderAccording to the method.

Description

ELISA quantitative detection method of milk agglutinin or mucin 1 based on milk fat globule membrane characteristics

Technical Field

The invention relates to the technical field of biological detection, in particular to an ELISA quantitative detection method of milk agglutinin or mucin 1 based on milk fat globule membrane characteristics.

Background

Breast milk is the best food for infants, and is the only, safest and most complete natural food required for the growth and development of infants. Breast feeding can not only meet the physique growth and development of infants, but also promote intelligence development and is beneficial to psychological growth; the breast-fed infants have good immunity development, strong disease resistance and low incidence of three-high diseases (hypertension, hyperlipidemia and hyperglycemia) after adult; in addition, breast feeding may reduce the incidence of breast cancer in mothers. Pure breast feeding of newborn and small infants is a scientific infant-raising method advocated by the world health organization and the foundation of children of the United nations, and is the most basic measure for ensuring the healthy growth of children. The WHO advocates breast feeding of infants for at least 4-6 months after birth to ensure the normal growth and development of infants, improve the immunity of the organism and prevent the occurrence of infectious diseases. However, due to the work status, occupation, disease, etc., breast feeding is not optimistic and lack of breast milk is very common. In recent years, the breast-feeding rate of China is continuously reduced, and the pure breast-feeding rate of infants in 0-6 months in China is only 40-50%, which is obviously reduced compared with the 90 s in the last century. Therefore, the infant formula becomes the preferred formula. However, although the infant formula is nutritious, it still differs greatly from breast milk in functionality. In the later stage of the 20 th century, along with the continuous and deep research on breast milk components, a series of infant formula milk powder added with taurine, nucleotide, glycan, linoleic acid, linolenic acid and other active components appears, so that the breast milk emulsification degree is further improved. Although the current commercial infant formula milk powder is close to the breast milk in the content of macro nutrient components, functional components in the breast milk are not accurately copied and embodied in the infant formula milk powder. In addition, most of the previous research based on breast milk is foreign data, and the data of the domestic research based on the breast milk of Chinese population is less. In fact, the characteristic data of the breast milk of western people is not completely suitable for the Chinese population due to the difference of regions, race, eating habits, and the like. Therefore, the determination of the components of the human milk of Chinese people and the research work of the health protection effect of the human milk on the mother and the infant are very important.

The fat component in the breast milk exists in the form of fat globules, and forms a special structure with a triple membrane system surrounding a triglyceride coreThe complex structure of triple Membrane encasing the fat droplet is called Milk Fat Globule Membrane (MFGM). MFGM is mainly composed of cholesterol, glycerophospholipid, sphingolipid and protein, wherein the protein accounts for 1-4% of total milk protein content and 1% of total globule mass⁽¹⁾. The various protein components in MFGM play an important role in the growth and development of infants, infection resistance, immune development and the like, and attract extensive attention. Wherein, Lactadherin (Lactadherin) and mucin 1(mucins is abbreviated as MUC1) are two main protein components in MFGM protein. Milk agglutinin, also known as Milk Fat Globule epidermal growth Factor 8(Milk Fat Globule EGF Factor 8, MFGE8), is a glycosylated membrane protein with a molecular weight of 46kDa⁽²⁾The N-terminal domain of the amino acid sequence of lactadherin contains two EGF domains, wherein the second EGF domain contains an RGD (Arg-Gly-Asp) motif that binds to integrin α v β 3/5 to promote cell adhesion and induce integrin-mediated signal transduction, and the C-terminal domain contains two dishonest domains that bind to apoptotic cell surface phosphatidylserine residues and mediate phagocyte function^(3,4)Because the C-terminal domain has homology with the coagulation factors V and VIII, the lectin is also involved in the coagulation process, and can inhibit prothrombinase, X factor, VIIa and the like^(5,6). Research shows that the milk agglutinin may participate in the formation of new blood vessel and regulate the function of cell adhesion protein in smooth muscle and artery fiber⁽⁷⁾Lactadherin is involved in promoting dietary triglyceride absorption and fatty acid uptake by cells, and regulates the hydrolysis of cytoplasmic lipid droplets in intestinal epithelial cells by promoting intestinal epithelial triglyceride hydrolase activity following interaction with integrins α v β 3 and α v β 5^(8,9)Milk agglutinin combined with RGD binding integrin α 8 β 1 can control intestinal muscle contraction frequency, reduce intestinal peristalsis to promote nutrient absorption^(10,11). MFGM protein mainly containing lactadherin promotes C2C12 cell proliferation through PI3K/Akt/mTOR/P70S6K signaling pathway⁽¹²⁾. These studies suggest that our intake of milk lectin through breast milk may be of great significance for gastrointestinal development and nutrient absorption in infants and young children. In addition, in vitro and in vivo studiesIt was shown that milk lectin inhibits rotavirus binding and protects breast-fed children from symptomatic rotavirus infection⁽¹³⁾. Lachesins also help to establish an intestinal barrier in neonates, preventing Necrotizing Enterocolitis (NEC) in neonates⁽¹⁴⁾. Milk agglutinin has also been shown to be associated with systemic lupus erythematosus, an autoimmune disease⁽¹⁵⁾And may help to reduce neonatal sepsis and lung injury⁽¹⁶⁾. MUC1 is a high-content glycoprotein, has a complex structure, and is composed of a homocardia state and a sugar chain, wherein the sugar chain accounts for 80%, and the sugar chain and the homocardia are linked by O-shaped glycosidic bonds. At the carboxy terminus of MUC1, three parts were included, an intracellular region consisting of 72 amino acids, a transmembrane region consisting of 28 amino acids, and an extracellular region consisting of 58 amino acids. The structures of the intracellular segment and the transmembrane segment in different species have high conservation types, which indicates the important biological functions of the intracellular segment and the transmembrane segment. The extracellular domain contains a tandem repeat sequence consisting of 20 amino acids, which determines the spatial structure specificity and immunogenicity of MUC 1. The main biological functions of MUC1 include lubrication, protection of cells, maintenance of viscosity, recognition of cells, immune regulation, etc.; in addition, the MUC1 plays an important role in the aspects of tumor generation, metastasis and the like.

However, since both lactadherin and MUC1 are present in MFGM and are not easily separated, their fat content greatly interferes with their quantitative determination, thus making the measurement of their content a difficult problem. Previously, MFGM proteins were quantified mostly by SDS-PAGE techniques, such as SDS-PAGE and subsequent Coomassie Brilliant Blue (CBB) staining, but such methods suffer from the influence of the degree of binding of the protein to the dye, e.g., glycoprotein inhibition and CBB binding, resulting in poor staining, and such methods have difficulty in absolute quantification of the protein⁽¹⁷⁾. Improvements to research techniques have since emerged, such as fluorescence measurements based on correlation with electrophoretically separated protein bands and their tryptophan content (18). In addition, absolute quantification techniques based on proteomics techniques are also used for detection and quantification of MFGM. However, the above techniques have drawbacks of being difficult to popularize and expensive for the detection of MFGM. In general, the above techniques are commonly used to evaluate MFGM proteinsThe relative composition of the groups is not suitable for large-scale sample detection of single proteins.

In addition, although enzyme-linked immunosorbent assays have been widely used in the prior art for quantifying various breast milk proteins, there has been no study on their use in the quantitative analysis of MFGM proteins. The main reasons are as follows:

(1) the MFGM protein may be partially or fully embedded in the membrane, which may cause difficulties for immune recognition of the protein.

(2) The process of treating the sample may result in the loss of MFGM protein, and this error is not stable.

(3) The binding ability of MFGM to solid support is difficult to assess and proteins on the same MFGM may compete with each other.

(4) MFGM is a special structure for wrapping fat globules, and the existence of lipid can also have certain influence on immunoassay.

In summary, quantitative analysis of the protein in MFGM is still the blank of research, and the quantitative analysis of the protein in MFGM helps people to understand the functions of the components of breast milk and the relationship between breast milk and infant growth and development; meanwhile, scientific basis is provided for the preparation of formula milk powder and various functional foods.

Accordingly, it is an object of the present invention to develop an enzyme-linked immunosorbent assay (ELISA) method for determining the concentration of milk agglutinin or MUC1 in breast milk. This is of great importance for the intensive study of breast milk.

The ELISA quantitative detection method of the milk agglutinin or mucin 1 based on the milk fat globule membrane characteristic is not reported at present.

Disclosure of Invention

The first purpose of the invention is to provide an ELISA quantitative detection method of milk agglutinin or MUC1 protein based on milk fat globule membrane characteristics aiming at the defects of the prior art.

A second object of the present invention is to address the deficiencies of the prior art by providing the use of a method as described above.

The third purpose of the invention is to provide a kit for quantitatively detecting the lactadherin or MUC1 protein in the breast milk aiming at the defects of the prior art.

In order to achieve the first purpose, the invention adopts the technical scheme that:

an ELISA quantitative detection method of milk agglutinin based on milk fat globule membrane characteristics, the detection method is an enzyme-linked immunosorbent assay, and comprises the following steps:

(1) collecting a breast milk sample;

(2) pretreating the collected breast milk sample: use of

Pretreating a breast milk sample by using lysine buffer, and putting the breast milk sample on ice for cracking to obtain a full-emulsion lysate;

(3) homogenizing the obtained whole milk lysate, diluting, and then quantitatively detecting the milk agglutinin in a breast milk sample by using an ELISA kit;

(4) taking a standard substance solution with gradient concentration to obtain a standard curve of the concentration of the standard substance solution;

(5) fitting a standard curve according to a linear regression method, wherein the fitting function equation is Y ═ a + bX, and calculating the concentration of milk agglutinin in the breast milk sample according to the standard curve;

the test method does not include centrifuging the breast milk sample.

Preferably, the method further comprises performing Western Blot on the breast milk sample to detect the expression level of the lectin in the sample.

Preferably, the sample of breast milk in step (1) comprises mature milk and colostrum.

Preferably, the cracking time on ice in step (2) is 20-40 min; in a volume ratio of

lysine buffer: breast milk samples were 9: 1.

Preferably, breast milk samples are cooked with 5 x SDS loading buffer prior to Western Blot detection and treated with a metal bath at 90-110 ℃ for 5-10 minutes.

Preferably, the gradient concentration in step (4) is: 4000pg/ml, 2000pg/ml, 1000pg/ml, 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml, 0 pg/ml.

An ELISA quantitative detection method of MUC1 protein based on milk fat globule membrane characteristics is disclosed, and the method is the same as the above method.

Preferably, the gradient concentration in step (4) of the ELISA quantitative detection method for MUC1 protein based on milk fat globule membrane characteristics is as follows: 1000mU/ml, 400mU/ml, 160mU/ml, 64mU/ml, 25.6mU/ml, 10.24mU/ml, 4.10mU/ml, 0 mU/ml.

The ELISA reagents used for the determination of lactadherin and MUC1 were different, pg/ml being the mass concentration and pg representing the mass; mU/ml refers to the activity concentration. The two protein concentration determinations used different kits, one mass concentration and one activity concentration.

In order to achieve the second object, the invention adopts the technical scheme that:

the method is applied to the formula research of the milk powder.

The method is applied to the preparation of a kit for quantitatively detecting the lactadherin or the MUC1 protein in the breast milk.

In order to achieve the third object, the invention adopts the technical scheme that:

a kit for quantitatively detecting lactadherin or MUC1 protein in breast milk comprises

lyses buffer reagent, ELISA detection reagent and kit instructions, the kit instructions for use describe the method.

It should be noted that the only differences in the kit for quantitatively detecting lactadherin or MUC1 protein in breast milk are: the ELISA detection reagents used are different, namely the antibodies on the used ELISA plates are different, the kit used in the invention is a commercial kit, wherein, the kit of boster company is used for the lectin, and the product number is as follows: EK 1201; MUC1 used was Thermo fisher corporation, MUCIN 1 kit, cat no: CA 15-3.

Preferably, the ELISA reagent comprises (1)1X Assay Diluent B: 2ml stock solution +8ml deionized water;

(2)1X Wash Buffer: 20mL of 20X Wash Buffer +380mL of deionized water;

(3)1X biotinylated antibody concentrate: centrifuging a Biotinylated antibody reagent vial, adding 100. mu.L of 1XAssay Diluent B, gently mixing up and down by a pipette (the concentrate can be stored at 4 ℃ for 5 days), and diluting with 1XAssay Diluent B by 80 times according to the amount to obtain 1X antibody;

(4)1X Streptavidin-HRP Reagent: Streptavidin-HRP Reagent was centrifuged briefly and mixed gently with an upper and lower pipette before use, diluted 200-fold with 1XAssay Diluent B depending on the amount used. (50ul +10ml ready to use).

Quantitative detection of milk agglutinin or MUC1 protein in breast milk can be achieved by the above method.

Preferably, the ELISA reagent of MFGE8 comprises (1) MFGE8 antibody working solution: preparing according to the requirement of 110ul (more) of each hole, and adding 99ul of antibody diluent into 1ul of antibody stock solution (2 h before use);

(2) ABC working solution: preparing according to the requirement of 110ul (more) of each hole, and adding 99ul of antibody diluent into 1ul of antibody stock solution (1 h before use);

(3)1X Wash Buffer: 20mL of 20 × Wash Buffer +380mL of deionized water.

The invention has the advantages that:

1. the method is used for quantitatively detecting the milk agglutinin and the MUC1 protein in the breast milk of the puerpera in different periods, has high flux and high accuracy, particularly simplifies the detection steps, has low cost, overcomes the defects that the MFGM protein can be partially or completely embedded in a membrane, possibly causes difficulty for the immune recognition of the protein and can cause loss of the MFGM protein, reduces errors, pretreats the sample in advance for cracking, and eliminates the influence on the immune detection caused by the existence of substances such as MFGM lipid and the like.

2. The method simplifies the experimental steps, the experimental results show that the results are not affected on the premise of simplifying the experimental steps, the method is low in cost, does not need expensive equipment and has the same accuracy rate compared with methods for carrying out related fluorescence measurement on the protein bands separated by electrophoresis and the tryptophan content thereof and the like, and the method can be well applied to clinic and provides scientific basis for the formula research of the milk powder.

Drawings

FIG. 1 is a Western Blot to characterize the expression of lectins in different treatment groups. In FIGS. A and B, 1 represents whole milk lysate: (

lysine of white Breast milk, NLWB); 2 represents the supernatant of the whole emulsion lysate (Supernatantiof NLWB, SNLWB); 3 represents the Whole milk control group (white Breast milk, WB); 4 represents Whey control (Whey, Why); 5 represents Precipitation group (Precipitation of NLWB, PNLWB). P represents a sample of breast milk from different women. As seen in Panel A, 2 major bands with molecular weights between 40-55kDa, corresponding to the bands of the lactoagglutinin glycosylated variants, can be identified based on molecular mass. The gray values of the 1-4 groups are compared by image J as can be seen in graph B.

FIG. 2 is a comparison of the concentrations of milk agglutinin in various groups of breast milk. A. Comparing the milk agglutinin content of whole milk and whey; use of B

Whether insoluble material was removed after lysis buffer affects the lectin ELISA assay.

FIG. 3 is a schematic representation of the use

Effect of lysine buffer on the detection of milk agglutinin in breast milk.

FIG. 4 shows the expression of MUC1 in different treatment groups characterized by Western Blot. 1 in FIGS. A and B represents WB; 2 represents Why; 3 represents NLWB; 4 represents SNLWB; and 5 represents PNLWB. P represents a sample of breast milk from different women. The gray values of the 1-4 groups are compared by image J as can be seen in graph B.

FIG. 5 is a comparison of the concentration of MUC1 in each group of breast milk, the difference in MUC1 in A, whole milk and whey; B. use of

Whether to remove the effect of insoluble matter on MUC1 after lysis buffer.

FIG. 6 is a schematic representation of the use

Effect of lysine buffer on MUC1 detection.

Detailed Description

The invention will be further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, it should be understood that various changes and modifications can be made by those skilled in the art after reading the disclosure of the present invention, and equivalents fall within the scope of the appended claims.

Example 1 quantitative determination of milk agglutinin

1 materials and methods

1.1 Collection and Split charging of Breast milk

40 breast milk of pregnant women who are born in the same time or by caesarean delivery in the south of Punan Hospital, Shanghai are randomly selected, all the selected pregnant women are healthy lying-in women, and the history of infectious diseases or chronic diseases such as hypertension, heart diseases and the like and the history of diseases in the gestational period do not exist. All enrolled parturients signed informed consent and voluntarily participated in the study. After obtaining maternal breast milk in Shanghai Mikan, the mother's milk was transported to the laboratory in an ice box within 2h, and a portion of the breast milk was immediately selected and centrifuged (4 ℃, 3000g, 10min) to obtain the whey fraction. The whole milk and whey are separately packaged and stored at-80 ℃ until use, and the homogenization of the sample is ensured and repeated freeze thawing is avoided in the process.

1.2 Primary reagents

lysine buffer (ab 156035; abcam), lactadherin ELISA Kit (Boster BioEK1201), MFGE8 antibody (MAB2767-SP, R)&D system)。

1.3 treatment of the samples

Corresponding portions of milk were taken, each portion comprising 3 parts of 100. mu.L whole milk and 1 part of 100. mu.L whey. Each breast milk was treated according to the following five groups:

1.3.1 Whole emulsion lysate (NLWB) group: adding 900 μ L of whole milk sample

lysis buffer (ab 156035; abcam), lysed on ice for 30 min;

1.3.2 Whole emulsion lysate Supernatant (SNLWB) group: adding 900 μ L of nasal lysis buffer into one whole milk sample, standing on ice for 30min for fully cracking, centrifuging at 12000r for 30min at 4 deg.C with refrigerated centrifuge, and separating supernatant of cracked whole milk with needle cylinder;

1.3.3 whole milk control group (WB): adding 900 mu L of PBS solution into one part of whole milk;

1.3.4 whey control (Why): adding 900 mu L of PBS solution into the whey sample;

1.3.5 precipitation group (PNLWB): the remaining fractions after removing the clarified fraction in the above SNLWB group were washed 2 times with 1mL of PBS solution. The wash solution was removed as much as possible to retain the upper layer with the precipitated insoluble material.

1.4 Western Blot to determine the expression level of lactadherin in breast milk

Expression levels of collectin were analyzed in 5 groups from 8 breast milk samples using Western Blot. 40 μ L samples from groups 1 to 4 (NLWB, SNLWB, WB, Why) were mixed with 10 μ L of 5 x SDS loading buffer and treated with a metal bath at 100 ℃ for 5 minutes. In group 5, an equal amount of PNLWB was added to SDS loading buffer and mixed well, followed by treatment at 100 ℃ for 5 minutes. The gel plates were prepared using a pre-formed polyacrylamide gel from Invitrogen NuPAGE and run for 60min using an electrophoresis apparatus at constant pressure 120V after loading. After electrophoresis, the membrane is switched at 100V for 60min by a sandwich structure, and after sealing, the membrane is used for 1: a 1000 concentration dilution of MFGE8 antibody (MAB2767-SP, R & D system) was incubated overnight at 4 ℃ with three PBS washes for 5 minutes each; after addition of secondary antibody, incubation was carried out for two hours at room temperature, and ECL chemiluminescence and exposure development were carried out after three PBS washes.

1.5 ELISA detection

Diluting after homogenizing NLWB, SNLWB, WB, Why; the dilution is 1000 times, namely the total dilution is 10000 times. The lactadherin ELISA kit (Boster Bio EK1201) was used. Prior to use, the ABC working solution and TMB should be equilibrated at 37 ℃ for at least 30 min. Adding 100 mu L of standard solution or diluted sample into each hole, covering by a coating film, and then incubating for 1.5h at 37 ℃; discarding the solution without washing; then, 100. mu.L of the prepared MFGE8 antibody working solution was added to each well, and incubated at 37 ℃ for 1 hour after covering with a cover film; washing with lotion for 3 times, and soaking for 1min each time; add 100. mu. LABC working solution to each well; incubating for 30min at 37 ℃ after covering with the covering film; washing was repeated 5 times; add 90. mu.L TMB substrate to each well, incubate 20-25 minutes at 37 ℃ in the dark; the detection should be performed within 30 minutes after adding 100. mu.L of the stop solution per well. Absorbance was measured at 450nm and 550nm using a microplate reader, and the 550nm value was subtracted from the 450nm value to correct for optical defects in the microplate.

1.6 statistical analysis

Statistical significance of the data was analyzed using GraphPad Prism6 software analysis mapping and IBM SPSS software 19.0(IBM Corp., Armonk, NY). Paired t-tests were used to assess the difference between the means between the two groups; analysis of variance assesses the differences between three and more groups. When P <0.05, the data were considered to be significantly different.

2 results

2.1Western Blot characterization of the milk lectin component of the samples

Characterization was performed by Western Blot (fig. 1). Milk agglutinin is present in breast milk as MFGM protein. Use of

After lysis of lysine buffer, the milk agglutinin may fall off from the membrane and dissolve in whey; may still be present on the membrane as part of the insoluble material. By processing different groups, NLWB should contain all milk coagulation; SNLWB removes the milk agglutinin contained in the insoluble matter; WB group and Why group were control groups of whole milk and whey, respectively. PNLWB stands forCollectin contained in insoluble substances was removed.

As shown in FIG. 1A, the PNLWB band was hardly visible, indicating that the milk agglutinin contained in the insoluble matter was almost absent, suggesting that

lysine buffer can better crack the protein in MFGM. Whey was also able to detect the presence of lactadherin, suggesting that some of the lactadherin was removed from the MFGM and dissolved in the whey.

The results of Western Blot were subjected to grey scale analysis as in FIG. 1B and variance analysis to examine the differences of each group; and the differences between each two groups were examined by post-hoc pairwise comparison (LSD). Analysis of variance suggests that the four groups of gray values are not all identical (P < 0.05). Wherein the bands of the NLWB and SNLWB groups have no significant difference (P is 0.959); the gray values of the NLWB group and the SNLWB group are different from that of the WB group (P is 0.032; P is 0.036); why group was significantly different from WB group (P < 0.024). The results show that better results are obtained with both lysate and whole milk compared to whey. Therefore, if the MFGM component is directly removed, it will cause a loss of milk agglutinin in breast milk. The NLWB and SNLWB groups using the lysate gave better detection results, suggesting that the complex structure of MFGM may interfere with the quantitative analysis of its protein components. But NLWB and SNLWB bands did not differ significantly (P >0.99), but Western Blot was erroneous due to the absence of internal controls in breast milk, and this study was further validated by ELISA.

2.2 concentration of milk agglutinin in Breast milk

Next, we further verified the lectin in WB and Why by ELISA. The results showed that, as with Western blot, the level of collectin in whole milk was significantly higher than that of whey (t 4.589, P <0.05), suggesting that the use of whey to detect collectin does not represent the actual concentration of collectin.

Then, we performed comparative detection of milk agglutinin of NLWB and SNLWB, and found no significant difference in milk agglutinin detection between the two (t-0.2196, P-0.8324). In the process of adding

After lysis buffer, there was no significant difference in the concentration detected by MFGE8 between the SNLWB group from which lysed insoluble material was removed by centrifugation and the NLWB group without centrifugation step. It was found that the removal of the lysed insoluble material in the required amount for ELISA did not affect the detection of lactadherin. As shown in fig. 2.

2.3 influence of nasal lysine Buffer on the detection of milk lectin

2.2 the results demonstrate that ELISA assays must use whole milk samples, and add

After lysis buffer, whether insoluble material was removed or not had no effect on ELISA. Therefore, to reduce the loss of milk agglutinin by excessive sample handling steps, we chose not to perform the separation step after lysis.

But do not

The effect of lysine buffer on the specific structure of milk fat globule membrane and whether it helps in the detection of ELISA results still requires further testing. Therefore, we performed by using 40 portions of breast milk

One half hour after the action of lysbuffer (NLWB) or PBS solution (WB) on ice, further investigations were carried out

Effect of lysine buffer on ELISA detection of milk agglutinin in breast milk. As shown in fig. 3. Results demonstrate use

lysine buffer increased the concentration of lectin detection using ELISA (t. 7.065, P)<0.05)。

Discussion of 3

ELISA is a protein detection method with simple operation, high speed, high sensitivity and strong specificity. Although Ultra High Performance Liquid Chromatography (UHPLC) in combination with tandem mass spectrometry (MS/MS) is a very accurate technique that can replace e.g. ELISA for quantification, the use of ELISA is still advantageous: ELISA is a high throughput method and the samples do not require any special preparation. Furthermore, the equipment used for UHPLC-MS/MS and the internal standards and reagents make this technique much more expensive than ELISA. In contrast, since only an microplate reader and basic laboratory materials are required, ELISA detection can be performed in most laboratories. However, the specific structure of MFGM makes it difficult to detect the protein in MFGM by ELISA.

In order to make ELISA assays possible, the effects of MFGM structure and lipid components must be addressed. Whey-soluble lactadherin can be obtained using a lysate, but the use of a different lysate can cause problems with protein solubility and detergent. We have selected one

lysine buffer (ab 156035; abcam). Such as

lysis buffer uses mild detergent to enable its use in ELISA detection. To prove that

The lysis efficiency of lysine buffer to the lactadherin protein is found by the result of Western Blot, and the content of the lactadherin in the insoluble substance after lysis is extremely low, which indicates that

lysine buffer can effectively crack the milk agglutinin in MFGM. This is probably because the loose attachment of milk agglutinin on MFGM membranes makes it easy to shed from the membrane.

It is secondly considered that the lysed breast milk leads to lipid overflow from the membrane, and that the presence of both lipids and insoluble material produced after the rupture may affect the accuracy of the ELISA assay. Accordingly, we formed the lysed breast milk into NLWB and SNLWB control groups by centrifugation, and the results demonstrated that the presence of insoluble materials and lipids therein had no effect on the ELISA detection. This is probably due to the large dilution ratio of the lysate and ELISA kit together on the original sample to fit the ELISA measurable concentration range, so that the effect of the actual lipids and insoluble material is negligible.

Finally, to verify

Actual role of lysine buffer for the detection of milk agglutinin. We compared the ELISA detection effect before and after breast milk lysis. The results show that after lysis ELISA detects higher concentrations of lactadherin. This means that

Cleavage of lysis buffer was effective for ELISA quantification of lactadherin.

Combines double verification of Western Blot and ELISA to prove that the method is based on

The lectin ELISA assay of lysine buffer was of interest.

By the ELISA protocol developed by this study, we can calculate widely the concentrations of milk agglutinin of different puerperae at different times. The amount of samples needed by the research is small, so the method has wide application significance.

As an important immune component in breast milk, milk agglutinin directly links the breast milk status with the newborn development. Our studies have driven interest in both clinical transformation of milk agglutinin and the study of other proteins of MFGM.

Example 2 quantitative detection of MUC1 protein

1 Primary reagent

MUC1 ELISA kit (Thermo EHMUC 1); native lysine buffer (ABCAM ab 156035); TBST; 5 SDS loading buffer; antibody diluent; running buffer; skimmed milk powder; primary anti (Ab 1): anti-MUC 1 antibody (D908K, Cell Signaling).

2 major consumables

A disposable Tip; absorbent paper; 1.5/2ml EP tube; shaking table, incubation box, pipette gun, filter paper, Nitrocellulose (NC) membrane.

3 Experimental methods

3.1 Collection and storage of Breast milk samples

3.1.1 Collection of Breast milk samples

(1) Professional medical staff carry out propaganda and education work and sign informed consent; 1 trained medical staff carries out breast milk collection work;

(2) the pump is used for collecting breast milk once after the parturient is born and in the follow-up visit of the parturient for 42 days after the parturition. The teats were rinsed with sterile water and all milk from one breast was collected.

(3) Collected breast milk is placed in a sterile centrifuge tube and immediately placed in an ice box for transportation to a laboratory.

3.1.2 isolation and storage of Breast milk samples

(1) Partial breast milk is subpackaged in a 1.5ml EP tube, and the subpackaged breast milk is marked with the number, name and time and is placed in a refrigerator at minus 80 ℃ for storage;

(2) centrifuging the other part of breast milk at 4 deg.C for 10min at 3000 g;

(3) sucking the upper layer fat and the whey layer into a 1.5ml EP tube, and separating the lower layer precipitate;

(4) adding 1ml of PBS into the lower layer precipitate, centrifuging for 14000r for 5 min; and washed 2 times with PBS;

(5) counting cells according to the volume of the source breast milk and recording in units of cell/ml;

(6) adding 1ml of TRIzol into the precipitate, dissolving the precipitate completely, standing for 5min, and storing in a refrigerator at-80 deg.C;

(7) centrifuging the tube filled with fat and whey again at 4 deg.C for 10min at 3000 g;

(8) carefully sucking the whey layer by using a syringe, subpackaging and storing the whey at-80 ℃ for later use;

(9) and sucking the residual liquid under the fat layer as clean as possible by using a syringe, and storing at-80 ℃ for later use.

3.1.3 storage and use of Breast milk samples

All breast milk is preserved at-80 ℃ and reasonably subpackaged to ensure the homogenization of the sample. One sample was taken for each test to avoid repeated freeze-thawing.

3.2 methodological Studies of ELISA detection of MFGM protein

3.2.1 treatment of the samples

(1) Materials: each portion of breast milk comprises 3 portions of 100ul of whole milk and 1 portion of 100ul of whey sample; and 5 different comparison groups were established:

whole emulsion lysate (NLWB) group: adding 900ul of whole milk sample

lysis in ice for 30 min;

whole emulsion lysate Supernatant (SNLWB) group: adding 900ul of negative lysis buffer into one whole milk sample, standing on ice for 30min for fully cracking, centrifuging at 12000r for 30min at 4 deg.C with a refrigerated centrifuge, and separating whey component of cracked whole milk with a syringe;

whole milk control group (WB): 900ul of PBS solution was added to one portion of whole milk;

whey control group (Why): to the whey samples, 900ul of PBS solution was added:

pellet group (PNLWB): the remaining fractions after removing the clarified fraction in the above SNLWB group were washed 2 times with 1ml of PBS solution. The wash solution was removed as much as possible to retain the upper layer with the precipitated insoluble material.

3.2.2 Western Blot to examine the protein expression level of MUC1 in breast milk

(1) Preparation before sample loading

8 random samples were selected and divided into 5 groups according to the above requirements.

40 μ L samples from groups 1 to 4 (NLWB, SNLWB, WB, Why) were mixed with 10 μ L of 5 x SDS loading buffer and treated with a metal bath at 100 ℃ for 5 minutes. In group 5, an equal amount of PNLWB was added to SDS loading buffer and mixed well, followed by treatment at 100 ℃ for 5 minutes.

(2) Western Blot to detect protein level of MUC1

Electrophoresis was performed for 60min at 120V constant voltage using an electrophoresis apparatus. After electrophoresis, the membrane is switched at 200A and 3h by a sandwich structure, and after sealing, the membrane is used for 1: incubation was performed with 1000 concentration diluted MUC1 antibody overnight at 4 ℃, and the washed NC membrane was added to the secondary antibody, incubated for two hours at room temperature, washed and developed by ECL chemiluminescence and exposure.

(3) Image J Gray value analysis

3.2.3 ELISA detection of the concentration of MUC1 in Breast milk

Diluting after homogenizing NLWB, SNLWB, WB, Why; an ELISA test of MUC1 was performed. The dilution factor and ELISA detection method are as follows.

3.3 ELISA detection of MUC1

3.3.1 reagent preparation

(1)1X Assay dilution B: 2ml stock solution +8ml deionized water.

(2)1X Wash Buffer: 20mL of 20 × Wash Buffer +380mL of deionized water.

(3)1X biotinylated antibody concentrate: after centrifuging a Biotinylated antibody reagent vial, 100. mu.L of 1X Assay reagent B was added, gently mixed up and down by a pipette (the concentrate may be stored at 4 ℃ for 5 days), and then diluted 80-fold with 1X Assay reagent B according to the amount to obtain 1X antibody.

(4)1X Streptavidin-HRP Reagent: Streptavidin-HRP Reagent was centrifuged briefly and mixed gently with an upper and lower pipette before use, diluted 200-fold with 1X Assay Diluent B depending on the amount used. (50ul +10ml ready-to-use)

3.3.2 sample treatment

(1) In a volume ratio of

lysine buffer: whole milk samples-9: 1 breast milk was diluted 10-fold and left to lyse on ice for half an hour. Homogenizing and diluting; breast milk is diluted 10^4 times.

(2) Standard curve: a vial of lyophilized standard was briefly centrifuged. Add 400. mu.L of Assay Diluent A (for serum/plasma samples) to the lyophilized standard vials to prepare 100U/mL standards. The powder was dissolved thoroughly with gentle mixing. Add 5. mu.L of CA15-3 standard (100U/mL) from the sample vial. The lyophilized standard was put into a test tube containing 495. mu.L of Assay Diluent A to prepare a standard solution of 1, 000 mU/mL. Pipette 300. mu.L of Assay Diluent A was added to each tube. Double dilutions were made using 1, 000mU/mL standard solution. Standard curves of 1000mU/ml, 400mU/ml, 160mU/ml, 64mU/ml, 25.6mU/ml, 10.24mU/ml, 4.10mU/ml, 0mU/ml were formed.

3.3.3 Experimental procedures

(1) All reagents and samples were left at room temperature (18-25 ℃) prior to use.

(2) Add 100. mu.L of standard solution or diluted sample per well; the membrane was covered and incubated at room temperature for 2.5 hours.

(3) The solution was discarded and washed 4 times with 1 × Wash Buffer (300 μ L). And using clean absorbent paper to control the moisture.

(4) Add 100. mu.L of prepared 1 × biotinylated antibody concentrate to each well; incubate at room temperature for 1 hour with gentle shaking.

(5) The washing was repeated as per step 3.

(6) Add 100. mu.L of 1X Streptavidin-HRP Reagent to each well; incubate for 45 minutes and shake gently at room temperature.

(7) The washing was repeated as per step 3.

(8) To each well 100. mu.L of TMB substrate was added. Protected from light and incubated for 30 minutes at room temperature with gentle shaking.

(9) 100ul of stop solution was added to each well.

(10) The test must be performed within 30 minutes after the reaction is stopped. Absorbance was measured at 450nm and 550nm using a microplate reader, and the 550nm value was subtracted from the 450nm value to correct for optical defects in the microplate. If 550nm is not present, the absorbance is measured only at 450 nm.

3.3.4 calculating sample concentration

And (3) fitting a standard curve according to a linear regression method, wherein the fitting function equation is Y ═ a + bX, and calculating the concentration of the breast milk sample according to the standard curve.

3.3.5 statistical analysis

The analysis was performed using SPSS19.0, with α ═ 0.05 as the test standard. Analysis of significant differences between the two groups was performed using Student's t-test and differences between mean of the samples of the multiple groups were tested using analysis of variance. Mapping was performed using GraphPadPrism 6.

4 results

4.1 characterization of MUC1 in Breast milk by Western Blot

The bands of the PNLWB (band 5) group were barely visible as shown in FIG. 4A, indicating that MUC1 contained in the insoluble matter was barely visible, suggesting that

lysine buffer can better crack the protein in MFGM. Whey (lane 4) also detected the presence of MUC1, suggesting that some MUC1 was separated from MFGM and dissolved in whey.

Carrying out grey value analysis on the result of the Western Blot, and testing the difference of each group by variance analysis; and the differences between each two groups were examined by post-hoc pairwise comparison (LSD). Analysis of variance suggests that the four groups of gray values are not all identical (P < 0.05). Wherein the bands of the NLWB and SNLWB groups have no significant difference (P ═ 0.775); the gray values of the NLWB group and the SNLWB group are different from that of the WB group (P < 0.001; P < 0.001); why group was significantly different from WB group (P < 0.001). The results show that better results were obtained with both lysate and whole milk compared to whey, while better detection results were obtained with the lysate for the NLWB and SNLWB groups, suggesting that the complex structure of MFGM may interfere with the quantitative analysis of its protein components. But NLWB and SNLWB bands did not differ significantly (P >0.99), but Western Blot was erroneous due to the absence of internal controls in breast milk, and this study was further validated by ELISA.

4.2 ELISA to detect the concentration of MUC1 in different Breast milk fractions

4.2.1 ELISA for detection of MUC1

The WB group MUC1 was found to be significantly higher than Why (t 2.763, P <0.05) by ELISA; the concentration of MUC1 in whole milk is higher than the concentration of MUC1 detected with whey.

No significant difference in MUC1 concentrations for NLWB and SNLWB (t 1.637, P0.1457); in the process of adding

After lysis buffer, no significant difference in MUC1 concentration was detected between the SNLWB group from which lysed insoluble material was removed by centrifugation and the NLWB group without centrifugation. Indicating that the presence of insoluble material did not adversely affect the ELISA assay, see FIG. 4.

4.2.2 Large sample validation of influence of Naive lysine Buffer on ELISA detection of MUC1

Based on the above results, we eliminated the step of centrifugation after lysis. For 40 parts of breast milk

One half hour after the action of lysbuffer (NLWB) or PBS solution (WB) on ice, further investigations were carried out

The effect of lysine buffer on ELISA detection of MUC1 in breast milk. As shown in fig. 5, the concentration of MUC1 detected by the NLWB group ELISA was higher than that detected by the WB group (t 2.061, P)<0.05). Description of the use

After lysis buffer, ELISA can detect MUC1 with higher concentration than the direct use of whole milk,

lysine buffer can better crack MUC1 in MFGM.

Discussion of 5

Quantitative determination is a difficult problem due to the presence of MUC1 in breast milk MFGM. Therefore, a new detection mode is developed to quantitatively analyze MUC1 in whole milk.

In order to make ELISA assays possible, the effects of MFGM structure and lipid components must be addressed. The use of lysates may render MFGM proteins detectable, but lysates also pose problems with protein solubility and detergent. Selected by us

lysine buffer makesMild detergents were used to enable ELISA detection.

For further demonstration

Cleavage Effect of lysine buffer on MFGM. We have shown by Western Blot that MFGE8 has better cleavage effect than MUC 1. This is probably because the loose attachment of MFGE8 on MFGM makes it easy to peel off from the film. To confirm further, we used ELISA to actually test

lysis recovery of lysine buffer. It is considered that the presence of lipids in the lysed breast milk, as well as insoluble material produced after the rupture, may affect the accuracy of the ELISA assay. Accordingly, we added a centrifugation step to the lysed breast milk, and the results demonstrated that no matter whether insoluble materials and lipids were removed by centrifugation had any effect on the ELISA assay. This is probably due to the fact that high-fold dilution of the original samples allowed the samples to fit within the range of ELISA measurable concentrations, so that the effect of actual lipids and insoluble matter could be neglected.

Finally, to verify

Actual lysis Effect of lysine buffer. We compared the ELISA detection before and after lysis. The results show that higher concentrations of MFGE8 and MUC1 were detected by ELISA after lysis. This means that

Premature cleavage of lysine buffer is of interest for MFGM proteins. The detection method adopted in the research is proved to be effective by combining double verification of Western Blot and ELISA.

By the ELISA protocol developed by this study, we can calculate widely the concentrations of milk agglutinin of different puerperae at different times. This allows data to be obtained with a smaller sample size. Has wide application significance. To date, few studies have estimated the concentration of MFGM protein in breast milk, and our approach to the study has removed the effects of milk fat globule membranes. Milk fat has been poorly studied for this protocol because of the dilution required for lectin detection using ELISA.

The research detects the MFGM protein in human breast milk for the first time, and the obtained result has high referential property.

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the foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and additions can be made without departing from the principle of the present invention, and these should also be considered as the protection scope of the present invention.

Claims (10)

1. An ELISA quantitative detection method of milk agglutinin based on milk fat globule membrane characteristics is characterized in that the detection method is an enzyme-linked immunosorbent assay method, and comprises the following steps:

(1) collecting a breast milk sample;

(2) pretreating the collected breast milk sample: use of

Pretreating a breast milk sample by using lysine buffer, and putting the breast milk sample on ice for cracking to obtain a full-emulsion lysate;

(3) diluting the obtained whole milk lysate after liquefaction, and then quantitatively detecting the milk agglutinin in a breast milk sample by using an ELISA kit;

(4) taking a collectin standard substance solution with gradient concentration to obtain a standard curve of the concentration of the standard substance solution;

(5) calculating the concentration of the milk agglutinin in the breast milk sample according to the standard curve;

the test method does not include centrifuging the breast milk sample.

2. The method of claim 1, further comprising western blot of a sample of breast milk for measuring the expression level of lectin in the sample.

3. The method of claim 1, wherein the sample of breast milk in step (1) comprises mature milk and colostrum.

4. The method according to claim 1, wherein the cracking time on ice in step (2) is 20-40 min; in a volume ratio of

lysine buffer: breast milk samples were 9: 1.

5. The method of claim 2, wherein the breast milk sample is cooked with 5 x SDS loading buffer prior to Western Blot detection and treated with a metal bath at 90-110 ℃ for 5-10 minutes.

6. The method of claim 1, wherein the gradient concentration in step (4) is: 4000pg/ml, 2000pg/ml, 1000pg/ml, 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml, 0 pg/ml.

7. An ELISA quantitative detection method of MUC1 protein based on milk fat globule membrane characteristics, characterized in that the method is the method of any one of claims 1-5. Characterized in that, the gradient concentration in the step (4) is as follows: 1000mU/ml, 400mU/ml, 160mU/ml, 64mU/ml, 25.6mU/ml, 10.24mU/ml, 4.10mU/ml, 0 mU/ml.

8. Use of the method of any one of claims 1 to 7 in the study of milk powder formulations.

9. Use of the method of any one of claims 1 to 7 for the preparation of a kit for the quantitative detection of lactadherin or MUC1 protein in breast milk.

10. A kit for quantitatively detecting lactadherin or MUC1 protein in breast milk is characterized by comprising

lyses buffer reagent, ELISA reagent, and kit instructions for use, the kit instructions comprising the method of any one of claims 1-7.

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