Introduction to AMH structure and immunoactive materials

1、 Background

All members of this family are dimeric glycoproteins, which play different regulatory roles in tissue growth and cell differentiation. Like other members of the family, AMH protein is first synthesized as a large precursor, containing 18 signal amino acid sequences, and then processed into prohormone, finally forming homodimer. Before secretion, mature hormones undergo glycosylation and dimerization to form a 140 kD dimer. The dimer is formed by two identical 70kD monomer subunits linked by disulfide bonds. Each monomer is composed of N end (i.e. "pro" area) and C end (i.e. "math" area). In the process of cytoplasmic transport, the single receptor of 70kD AMH hydrolyzes at specific sites in the "pro" region and the "maturity" region to form two polypeptides: the "pro" region of 58kD and the "maturity" region of 12kD. After hydrolysis, the two peptides will still bind in a non covalent form.

2、 Gene and protein composition

The gene encoding AMH is located on the short arm of No. 19 staining. The amino acid sequence of AMH is shown in the figure below

3、 Reagents for detecting early AMH

Early foreign AMH detection reagents (such as DSL/IOT/Beckman Gen II) have different affinities to different forms of AMH (such as natural, recombinant (processed or processed)). Antibodies of these reagents can bind either monomer AMH or dimer AMH, so they cannot be effectively identified. DSL/IOT two reagents, because different antibodies and different calibrators are used, different test results will be obtained when determining the same sample; Research shows that the test results of Gen II are 20-40% lower than those of DSL; However, the declaration data of Gen II shows that the test result of Gen II will be 40% higher than that of DSL. These data indicate that AMH exists in an unstable form under certain conditions, and the test lacks recognized detection standards. This has limited the application of AMH in reproductive evaluation and scientific research transformation for a certain period of time.

At present, domestic AMH detection is mainly based on the ELISA results of Beckman Gen II, for example, Roche cobas AMH will trace the detection results here.

4、 Structure of AMH

AMH is formed by two identical 70kD monomer subunits linked by disulfide bonds. Each monomer is composed of 560 amino acids. Generally, the subunits of homodimers or other oligomers are arranged symmetrically. Although some examples show that symmetry in dimer can be broken. In this case, the site on one monomer that can be recognized by the antibody may not be recognized on the other monomer. At present, no theory or mechanism shows that AMH is a dimer with broken symmetry. Therefore, the antibody that recognizes the upper position of one monomer can also recognize the position of another monomer。

After reductant treatment, the disulfide bond of dimer AMH is opened, and AMH will form two identical monomers。

There is a hydrolysis site at 451 amino acids, namely - RAQR ｜ SAGA. After protease treatment, the full length AMH will be divided into two polypeptides, 1-451 amino acids are the "pro" region and 452-560 amino acids are the "nature" region。

After hydrolysis, AMH spontaneously polymerizes to form non covalently bound hydrolysis recombined AMH。

Remove the signal peptide (1-18) and seven prepreptides, and AMH will produce two fragments, one of which has 26-451 amino acids; The other has 452-560 amino acids. The latter is the texture fragment of AMH. 26-451 can also be further processed into two fragments, one is 26-229, which is called N-ter pro region here, and the other 230-451 is called midpro region here。

Although the form of AMH in clinically relevant samples is not clear, it is known that there are different forms of AMH protein in mammalian metabolic cycle, and different forms of AMH protein can also be confirmed in a variety of tissues and cells。

5、 Example of AMH antibody pairing selection

AMH has different forms. According to the antibody pairing of different regional sites, the detection of different forms of AMH is different. The following figure illustrates antibodies for different sites

A. B is located in the "pro" area, and C and D are located in the "maturity" area (A, B, C and D are only indicators of the area, not antibodies at specific locations)。

AMH is a symmetrically arranged dimer. In sandwich ELISA, the same antibody can be used either as a capture antibody or as a detection antibody. For example, C-C (unless otherwise specified, the former is used as a capture antibody, and the latter is used as a detection antibody) can be used as the match feature region of AMH. The detection of this pair is not affected by whether the sample is hydrolyzed or not because it is targeted at the same site. Similarly, A-A can be used as the pro pro region of AMH. C-D can be used as the measure feature area of AMH. A-C can be used as the pro feature area detection of AMH. D-B can be used as the measure pro area of AMH。

Use each pair of reagents to measure the AMH content in the same sample. The results are shown in the following table

	C-C	A-A	C-D	A-C	D-B
Sample	6.553	0.369	12.012	23.525	>19
Calibrators(OD value)
Blank	0.086	0.069	0.093	--	0.062
0.1ng/ml AMH	0.106	0.076	0.133	--	0.08
10ng/ml AMH	1.26	1.178	2.94	--	1.5

Different paired reagents are considered to measure different forms of AMH in the sample. C-C measures the AMH of the full length dimer and the dimer in the matrix region. C-D texture feature region detection, measuring the full-length dimer and monomer, and the monomer and dimer AMH in the texture region. Because this pairing can detect more forms of AMH, the measured value is higher than that of C-C. A-C's pro feature region detection measures the full-length dimer and monomer, and the full-length and monomer AMH fragments that cannot be recognized by C-D. Therefore, some subtypes can be detected by pro feature, but cannot be recognized by match feature。

6、 Site information of major antibodies of Tongren Heart